Reproduction, Locomotion and Skin

**53**

**Chapter 4**

Mare

**Abstract**

*David A. Trundell*

dealing with twinning in the mare.

**1. Introduction**

**2. Mare reproduction**

**2.1 Seasonality and photoperiod**

**Keywords:** mare, endometritis, photoperiod, twins, kisspeptin

Equine Reproduction: Seasonality,

Endometritis, and Twinning in the

This chapter reviews the seasonality and effect of photoperiod in mares and how, as clinicians, we can shorten the vernal transition period and improve our efficiency in getting mares in foal. Different protocols have been utilized to shorten the vernal transition, and each will be discussed. We will also examine endometritis in the mare. The role of biofilms in causing endometritis in our equine patients, and potential treatment plans, in particular breeding the dirty mare, will be reviewed. Finally, we will examine the effect of twin pregnancies in the mare, the most common cause of noninfectious abortion, and offer two management therapies for

Our knowledge of reproduction in the mare has expanded considerably in the last few decades and continues at pace. The scope of this chapter is to try to answer some of the most common questions an attending veterinarian may be asked to deal with, namely, shortening the vernal transition, dealing with postmating endometritis, and dealing with twin pregnancy, all of which can frustrate even the seasoned clinician. The aim of this chapter is to give the reader the background knowledge of why certain therapies may or may not work and to give the clinician workable solutions to

some of the more common aspects of clinical reproductive work in the mare.

The mare is a seasonal, long-day, polyestrous animal, meaning that her reproductive status is intrinsically linked to photoperiod. In the Northern Hemisphere, the normal physiologic breeding season starts in spring (April) and continues through to autumn (September). This corresponds to increasing photoperiod (increasing daylight length). The light signals are received by the retina, processed by melanopsin [1], which, as a pigment, is located in retinal ganglion cells, themselves being photosensitive. This information reaches the suprachiasmatic nucleus via the retino-hypothalamic tract [2]. Melatonin produced in the pineal

#### **Chapter 4**

## Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare

*David A. Trundell*

#### **Abstract**

This chapter reviews the seasonality and effect of photoperiod in mares and how, as clinicians, we can shorten the vernal transition period and improve our efficiency in getting mares in foal. Different protocols have been utilized to shorten the vernal transition, and each will be discussed. We will also examine endometritis in the mare. The role of biofilms in causing endometritis in our equine patients, and potential treatment plans, in particular breeding the dirty mare, will be reviewed. Finally, we will examine the effect of twin pregnancies in the mare, the most common cause of noninfectious abortion, and offer two management therapies for dealing with twinning in the mare.

**Keywords:** mare, endometritis, photoperiod, twins, kisspeptin

#### **1. Introduction**

Our knowledge of reproduction in the mare has expanded considerably in the last few decades and continues at pace. The scope of this chapter is to try to answer some of the most common questions an attending veterinarian may be asked to deal with, namely, shortening the vernal transition, dealing with postmating endometritis, and dealing with twin pregnancy, all of which can frustrate even the seasoned clinician. The aim of this chapter is to give the reader the background knowledge of why certain therapies may or may not work and to give the clinician workable solutions to some of the more common aspects of clinical reproductive work in the mare.

#### **2. Mare reproduction**

#### **2.1 Seasonality and photoperiod**

The mare is a seasonal, long-day, polyestrous animal, meaning that her reproductive status is intrinsically linked to photoperiod. In the Northern Hemisphere, the normal physiologic breeding season starts in spring (April) and continues through to autumn (September). This corresponds to increasing photoperiod (increasing daylight length). The light signals are received by the retina, processed by melanopsin [1], which, as a pigment, is located in retinal ganglion cells, themselves being photosensitive. This information reaches the suprachiasmatic nucleus via the retino-hypothalamic tract [2]. Melatonin produced in the pineal

gland is suppressed during hours of darkness. This fall in melatonin as photoperiod increases during the spring stimulates the mare to, reproductively, enter the spring or vernal transition. The classical hypothalamic–pituitary-ovarian axis, known to many clinicians, is oversimplified.

In the last decade or so, numerous authors have examined the role of kisspeptin neurons in the relation of cyclicity in many animal models, including the mare. It appears that increasing photoperiod stimulates the main kisspeptin neuron population located in the arcuate nucleus of the hypothalamus [3]. Distinctly, the horse does not appear to have a secondary population of kisspeptin neurons in the preoptic area, unlike cattle and sheep [4]. A small population of these receptors are located within the ventromedial nucleus of the hypothalamus [5]. The kisspeptin neuron fibers are found throughout the septo-preoptic region, an area that the majority of gonadotrophin-releasing hormone (GnRH) neurons are located [6]. In 2007, Smith et al. were the first to note that kisspeptin neurons may be influenced by photoperiod [7]. They saw an increase in KISS1 mRNA in the arcuate nucleus in sheep in their physiologic breeding season. Our understanding of the role of kisspeptin in the mare and effects on her reproductive status is derived mainly from studies on sheep models; there have been limited studies in the mare.

In the sheep model, it appears that in artificially decreasing photoperiod, thereby eliciting a stimulatory effect in sheep (sheep are short-day breeders), there is a corresponding increase in the number of kisspeptin neurons [8]. Kisspeptin neurons appear to form numerous synapses with other neurons that produce dopamine [9], melanocyte-stimulating hormone [10], and GnRH [11], among others. The regulation of kisspeptin is still not fully understood, but it appears that it may consist of a combination of negative feedback via estrogen in the sheep model [12] and via dopamine. The dopamine neurons in the retrochiasmatic area of the hypothalamus exerts an inhibitory effect on GnRH secretion during anestrous but not during the physiologic breeding season [13]. There is an upregulation of dopamine receptors in the kisspeptin neurons during breeding season [13]. There appears to be a seasonal difference in the number of kisspeptin neurons in the population found in the arcuate nucleus of the hypothalamus, but no seasonal difference in the preoptic population. This has been confirmed in the mare [4]. This seasonality difference appears to be driven, or at least modulated, by photoperiod. However, from sheep models, we know kisspeptin does not express melatonin receptors [14], and it is proposed that any effect of melatonin on the functionality of kisspeptin may be indirect [3]. It has also been postulated there is an indirect effect of photoperiod that is modulated via the thyroid hormones [3]. Nearly all preoptic kisspeptin neurons express thyroid receptors [15]. It has been shown that the thyrotropes (the cells secreting these hormones) located in the pars tuberalis of the rostral adenohypophysis are melatonin responsive [16, 17]. It appears these cells display dramatic melatonin-dependent photoperiodic changes; under short photoperiod, there is low level expression, while under long photoperiod, there is high level expression [18, 19].

GnRH is a 10 amino acid peptide secreted by the hypothalamus. Its secretion, regulated by decreasing melatonin during increased photoperiod, is modulated via kisspeptin neurons mentioned above. Secretion of this peptide enters the hypothalamic-hypophyseal blood portal system, which bathes over the gonadotrophs located in the anterior pituitary, cells that synthesize and secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH). During the vernal transition, there are ever-increasing circulating concentrations of FSH and LH. This increase is gradual, with LH in particular remaining low prior to the first ovulation. It is thought that the low circulating LH is due to the low storage of LH within the gonadotrophs [20]. Under the regulation described above, the increased GnRH stimulates FSH secretion and thus drives the growth of ovarian follicles. However during the vernal transition,

**55**

mares in February and March.

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare*

mares undergo several follicular waves. Under these waves, follicles seemingly grow (although they rarely reach pre-ovulatory size), only to regress, and be replaced by another follicular wave. In a report by Watson et al. [21], only 31% of all FSH surges during this transition period lead to the production of a follicular wave. Only one a follicle under the influence of FSH, produces sufficient estrogen, will a follicle ovulate under the LH surge. Estrogen appears to be involved in numerous components of the mare reproductive cycle. It appears that estrogen has a negative and a positive feedback mechanism on kisspeptin within the arcuate nucleus [7, 22], while the fall in circulating estrogen levels leads to increased LH secretion at the level of the anterior pituitary. During the peri-ovulatory surge, declining FSH under the influence of increasing estrogen and inhibin production from the growing follicle, LH reaches maximum concentrations 1 day post ovulation. LH promotes the maturation

Numerous issues can be seen related to this complex reproductive cycle. During

In the Northern Hemisphere, it has become standard industry practice to place mares under light regimes starting December 1 [23] in the breeding of thoroughbred racehorses. Nonetheless many breeders of other types of horses also utilize light manipulation to hasten time to first ovulation. A 200 watt incandescent light bulb in a 12 × 12 foot stall is sufficient to begin stimulating follicular activity. Typically light is added to the evening; mares are brought into their stalls from pasture before dusk and then exposed to artificial light until 23.00 hours. It is now known that light in the short wave spectrum (465–485 nm) is most effective at inhibiting melatonin production [24]. It is important to allow the mare to receive some hours of darkness and that exposing them constantly to light stimulation actually extends the anestrous period. On larger horse farms, the use of indoor schools, housing numerous barren, and/or maiden mares can be effective. However, in those large building, it is important to check the light intensity at all areas. A loose rule of thumb is that the light should cast no shadows and that you should be able to read a newspaper anywhere in the building. For those who want to be more scientific in their approach, a photometer can be utilized. Exposure to this light regimen should continue for at least 70 days. Within 60 days, most mares will show some follicular activity, with the majority expressing their first ovulation with 70 days of onset of light exposure. While exposure to artificial light does not eliminate the vernal transition, it simply moves it forward. In the natural physiologic breeding season, the mare will not display follicular activity until April, and many will not experience their first ovulation of the season until May. Moving the vernal transition forward several months allows the clinician to start breeding these

the vernal transition, mares may be presented with multiple small follicles evident on the ovaries via transrectal ultrasonography. Follicles may grow and then regress. The unpredictable nature of the folliculogenesis and the exact duration of the vernal transition are not only frustrating for the owner but a headache for the attending clinician. It is imperative that the clinician has a thorough understanding of the mechanisms at play during this period. Having this knowledge will allow the veterinarian to potentially manipulate these mechanisms and the hormones involved and shorten this phase. Understanding the systems at play allows for proper management by the veterinarian and will increase the productivity of the

*DOI: http://dx.doi.org/10.5772/intechopen.92999*

and subsequent ovulation of the follicle.

*2.1.1 Taking control of the vernal transition*

mares under their control.

*2.1.1.1 Light manipulation*

#### *Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare DOI: http://dx.doi.org/10.5772/intechopen.92999*

mares undergo several follicular waves. Under these waves, follicles seemingly grow (although they rarely reach pre-ovulatory size), only to regress, and be replaced by another follicular wave. In a report by Watson et al. [21], only 31% of all FSH surges during this transition period lead to the production of a follicular wave. Only one a follicle under the influence of FSH, produces sufficient estrogen, will a follicle ovulate under the LH surge. Estrogen appears to be involved in numerous components of the mare reproductive cycle. It appears that estrogen has a negative and a positive feedback mechanism on kisspeptin within the arcuate nucleus [7, 22], while the fall in circulating estrogen levels leads to increased LH secretion at the level of the anterior pituitary. During the peri-ovulatory surge, declining FSH under the influence of increasing estrogen and inhibin production from the growing follicle, LH reaches maximum concentrations 1 day post ovulation. LH promotes the maturation and subsequent ovulation of the follicle.

Numerous issues can be seen related to this complex reproductive cycle. During the vernal transition, mares may be presented with multiple small follicles evident on the ovaries via transrectal ultrasonography. Follicles may grow and then regress. The unpredictable nature of the folliculogenesis and the exact duration of the vernal transition are not only frustrating for the owner but a headache for the attending clinician. It is imperative that the clinician has a thorough understanding of the mechanisms at play during this period. Having this knowledge will allow the veterinarian to potentially manipulate these mechanisms and the hormones involved and shorten this phase. Understanding the systems at play allows for proper management by the veterinarian and will increase the productivity of the mares under their control.

#### *2.1.1 Taking control of the vernal transition*

#### *2.1.1.1 Light manipulation*

*Equine Science*

many clinicians, is oversimplified.

gland is suppressed during hours of darkness. This fall in melatonin as photoperiod increases during the spring stimulates the mare to, reproductively, enter the spring or vernal transition. The classical hypothalamic–pituitary-ovarian axis, known to

In the last decade or so, numerous authors have examined the role of kisspeptin neurons in the relation of cyclicity in many animal models, including the mare. It appears that increasing photoperiod stimulates the main kisspeptin neuron population located in the arcuate nucleus of the hypothalamus [3]. Distinctly, the horse does not appear to have a secondary population of kisspeptin neurons in the preoptic area, unlike cattle and sheep [4]. A small population of these receptors are located within the ventromedial nucleus of the hypothalamus [5]. The kisspeptin neuron fibers are found throughout the septo-preoptic region, an area that the majority of gonadotrophin-releasing hormone (GnRH) neurons are located [6]. In 2007, Smith et al. were the first to note that kisspeptin neurons may be influenced by photoperiod [7]. They saw an increase in KISS1 mRNA in the arcuate nucleus in sheep in their physiologic breeding season. Our understanding of the role of kisspeptin in the mare and effects on her reproductive status is derived mainly from

In the sheep model, it appears that in artificially decreasing photoperiod, thereby

eliciting a stimulatory effect in sheep (sheep are short-day breeders), there is a corresponding increase in the number of kisspeptin neurons [8]. Kisspeptin neurons appear to form numerous synapses with other neurons that produce dopamine [9], melanocyte-stimulating hormone [10], and GnRH [11], among others. The regulation of kisspeptin is still not fully understood, but it appears that it may consist of a combination of negative feedback via estrogen in the sheep model [12] and via dopamine. The dopamine neurons in the retrochiasmatic area of the hypothalamus exerts an inhibitory effect on GnRH secretion during anestrous but not during the physiologic breeding season [13]. There is an upregulation of dopamine receptors in the kisspeptin neurons during breeding season [13]. There appears to be a seasonal difference in the number of kisspeptin neurons in the population found in the arcuate nucleus of the hypothalamus, but no seasonal difference in the preoptic population. This has been confirmed in the mare [4]. This seasonality difference appears to be driven, or at least modulated, by photoperiod. However, from sheep models, we know kisspeptin does not express melatonin receptors [14], and it is proposed that any effect of melatonin on the functionality of kisspeptin may be indirect [3]. It has also been postulated there is an indirect effect of photoperiod that is modulated via the thyroid hormones [3]. Nearly all preoptic kisspeptin neurons express thyroid receptors [15]. It has been shown that the thyrotropes (the cells secreting these hormones) located in the pars tuberalis of the rostral adenohypophysis are melatonin responsive [16, 17]. It appears these cells display dramatic melatonin-dependent photoperiodic changes; under short photoperiod, there is low level expression, while

studies on sheep models; there have been limited studies in the mare.

under long photoperiod, there is high level expression [18, 19].

GnRH is a 10 amino acid peptide secreted by the hypothalamus. Its secretion, regulated by decreasing melatonin during increased photoperiod, is modulated via kisspeptin neurons mentioned above. Secretion of this peptide enters the hypothalamic-hypophyseal blood portal system, which bathes over the gonadotrophs located in the anterior pituitary, cells that synthesize and secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH). During the vernal transition, there are ever-increasing circulating concentrations of FSH and LH. This increase is gradual, with LH in particular remaining low prior to the first ovulation. It is thought that the low circulating LH is due to the low storage of LH within the gonadotrophs [20]. Under the regulation described above, the increased GnRH stimulates FSH secretion and thus drives the growth of ovarian follicles. However during the vernal transition,

**54**

In the Northern Hemisphere, it has become standard industry practice to place mares under light regimes starting December 1 [23] in the breeding of thoroughbred racehorses. Nonetheless many breeders of other types of horses also utilize light manipulation to hasten time to first ovulation. A 200 watt incandescent light bulb in a 12 × 12 foot stall is sufficient to begin stimulating follicular activity. Typically light is added to the evening; mares are brought into their stalls from pasture before dusk and then exposed to artificial light until 23.00 hours. It is now known that light in the short wave spectrum (465–485 nm) is most effective at inhibiting melatonin production [24]. It is important to allow the mare to receive some hours of darkness and that exposing them constantly to light stimulation actually extends the anestrous period. On larger horse farms, the use of indoor schools, housing numerous barren, and/or maiden mares can be effective. However, in those large building, it is important to check the light intensity at all areas. A loose rule of thumb is that the light should cast no shadows and that you should be able to read a newspaper anywhere in the building. For those who want to be more scientific in their approach, a photometer can be utilized. Exposure to this light regimen should continue for at least 70 days. Within 60 days, most mares will show some follicular activity, with the majority expressing their first ovulation with 70 days of onset of light exposure. While exposure to artificial light does not eliminate the vernal transition, it simply moves it forward. In the natural physiologic breeding season, the mare will not display follicular activity until April, and many will not experience their first ovulation of the season until May. Moving the vernal transition forward several months allows the clinician to start breeding these mares in February and March.

There are disadvantages to this regime. The mares must be housed either in stalls or a large barn, which intensifies their maintenance. Stalls need to be cleaned out regularly, adding to staffing responsibilities. In addition mares housed in groups in barns allow for opportune risk for injury especially if they are fed together—the lowly mare has nowhere to run from her aggressive barn mate.

#### *2.1.1.2 Equilume™ face mask*

To counter the problems of intensified housing of mares under light, researchers in Ireland have come up with a novel way to provide the mare with enough stimulatory light to advance the physiologic breeding season, while in their pasture. These masks provide blue light to one eye. It was concluded by Murphy et al. [23] that one light stimulation to one eye is sufficient to stimulate onset of follicular activity, is as effective as stall or barn light regimes, but also has added benefits of being more economic, especially to the small-scale breeder, while increasing horse welfare. Horses can remain out in their pastures, which reduces stress on these animals. However, these are currently one-time use items (as in for one season) and cost a few hundred dollars per mask, and occasionally inquisitive mares may pull off the face mask of another. On larger studs where the infrastructure for housing numerous animals under artificial lights, and with adequate staffing, it appears that the traditional light regimes remain the favor. Despite this, there is a place of the use for such masks. Mares seem to lose interest in the mask of other horses within a few days (the likelihood of a mask being pulled off is highest at the start). Providing there is nothing in the pasture on which the mare could hook the mask on (access to tree branches or fence posts above rails), and given that it is securely fastened, the mask should remain in place. Those that have only a handful of broodmares may prefer this method, as it reduces labor costs involved with stalling the mare.

#### *2.1.1.3 Kisspeptin supplementation*

As described above, kisspeptin appears to regulate GnRH secretion. As of yet, no commercial kisspeptin product is available. A recent report by Australian researchers found that although kisspeptin administered to mares as a constant rate infusion elevated circulating LH levels, it did not lead to an LH surge and therefore did not evoke ovulations within their group of mares during the vernal transition [4]. It remains to be seen whether the use of kisspeptin may shorten time to first ovulation, by potentially driving follicle maturation, under influence of LH, without necessarily causing ovulation.

#### *2.1.1.4 Use of dopamine antagonists (domperidone, sulpiride)*

As shown, dopamine plays an essential role in the stimulation of the reproductive axis in the mare. Dopamine has an inhibitory effect on GnRH release. For completeness it appears that dopamine antagonist acts via the stimulation of prolactin. For both domperidone and sulpiride, the dose is 1 mg/kg given PO and IM, respectively. Both are administered once daily for 25 days. The reports on the efficacy of the use of these preparations to shorten time to first ovulation in the mare are conflicting. A recent study by Mari et al. [25], comparing the two products, found that sulpiride significantly shortened time to pregnancy establishment (61 days) compared with domperidone-treated mares (83 days). That group concluded sulpiride is effective in advancing the vernal transition, whereas domperidone is only effective in some mares.

**57**

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare*

As mentioned the long transitional phase exhibited by mares is characterized by numerous follicular waves, unpredictable follicle growth, and follicle regression. Many protocols have examined the use of progesterone (P4) to dampen down these unpredictable features of the transitional phase and to drive a follicle to become dominant and one that will ovulate. The physiological effect of exogenous progesterone supplementation is relatively simple. P4 exerts an inhibitory mechanism with regard to LH but has minimal effect on FSH secretion. As described earlier, LH is required for maturation and final ovulation of the dominant follicle. While the mare is exposed to exogenous P4, LH is blocked at the level of the anterior pituitary, while FSH continues to be secreted. Therefore the follicles continue to grow under influence of FSH. Once the exogenous source of P4 is removed, this sudden fall in circulating P4 stimulates the LH surge, leading to final maturation and ovulation of a dominant follicle. The typical regimen is a dietary supplementation with altrenogest (Regumate®) at 0.044 mg/kg PO for 10 days. Injectable P4 products are becoming more routinely available. In the USA compounded products such as progesterone in oil can be utilized. Controlled release of P4 from these compounds last between 7 and 10 days. Daily application of oral altrenogest can be time-consuming. There also is a risk of noncompliance, should a mare be difficult to catch, not to mention potential side effect for the operator. The use of these long-acting P4 BioRelease products have been shown to be effective [26]. It appears that the use of exogenous P4 has maximal benefits when the mare exhibits a follicle of at least 20 mm in diameter and when administered in deep anestrous has little effect [27, 28]. An injectable altrenogest marketed via BOVA has recently become available in the UK for the first time, although no studies on its efficacy are cur-

Endometritis is a leading cause of subfertility in the mare [29] and is the third most reported condition seen in our equine patients [30]. Endometritis, simply, the inflammation of the lining of the uterus, has historically been attributed to bacterial colonization and infection of the uterus. However there are a subgroup of mares that will exhibit persistent mating-induced endometritis (PMIE), in the absence of bacterial isolation. Furthermore we will also examine in the chapter the role of

Post breeding, a normal, physiologic endometritis will be observed in all mares [31]. This normal, transient event, which peaks around 8 hours post insemination, occurs to eliminate excessive spermatozoa, seminal plasma, and contaminants from the uterus [32]. This physiologic response should be over by 48 hours post insemination [33]. The subgroup of mares that experience PMIE appear to have an altered inflammatory response to the presence of spermatozoa and seminal plasma within the uterus. These mares tend to be aged, have increased parity, may exhibit chronic inflammatory changes within the endometrium [34], and exhibit failure to clear intrauterine bacterial challenges [35]. Susceptibility rates among thoroughbred broodmares is 15% [36], and crucially the early embryonic death rate is three times higher in this group of mares [37]. A persistent inflammatory uterine environment

5 days post fertilization is incompatible with embryo survival [38].

It has long been proposed that mares are classified as either susceptible or resistant to PMIE. It has been shown that susceptible mares do have altered protein

*DOI: http://dx.doi.org/10.5772/intechopen.92999*

*2.1.1.5 Use of progesterone*

rently available.

**2.2 Endometritis**

*2.2.1 Endometritis in the mare*

biofilm formation and bacterial endometritis.

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare DOI: http://dx.doi.org/10.5772/intechopen.92999*

#### *2.1.1.5 Use of progesterone*

*Equine Science*

*2.1.1.2 Equilume™ face mask*

with stalling the mare.

*2.1.1.3 Kisspeptin supplementation*

necessarily causing ovulation.

only effective in some mares.

*2.1.1.4 Use of dopamine antagonists (domperidone, sulpiride)*

There are disadvantages to this regime. The mares must be housed either in stalls or a large barn, which intensifies their maintenance. Stalls need to be cleaned out regularly, adding to staffing responsibilities. In addition mares housed in groups in barns allow for opportune risk for injury especially if they are fed together—the

To counter the problems of intensified housing of mares under light, researchers in Ireland have come up with a novel way to provide the mare with enough stimulatory light to advance the physiologic breeding season, while in their pasture. These masks provide blue light to one eye. It was concluded by Murphy et al. [23] that one light stimulation to one eye is sufficient to stimulate onset of follicular activity, is as effective as stall or barn light regimes, but also has added benefits of being more economic, especially to the small-scale breeder, while increasing horse welfare. Horses can remain out in their pastures, which reduces stress on these animals. However, these are currently one-time use items (as in for one season) and cost a few hundred dollars per mask, and occasionally inquisitive mares may pull off the face mask of another. On larger studs where the infrastructure for housing numerous animals under artificial lights, and with adequate staffing, it appears that the traditional light regimes remain the favor. Despite this, there is a place of the use for such masks. Mares seem to lose interest in the mask of other horses within a few days (the likelihood of a mask being pulled off is highest at the start). Providing there is nothing in the pasture on which the mare could hook the mask on (access to tree branches or fence posts above rails), and given that it is securely fastened, the mask should remain in place. Those that have only a handful of broodmares may prefer this method, as it reduces labor costs involved

As described above, kisspeptin appears to regulate GnRH secretion. As of yet, no commercial kisspeptin product is available. A recent report by Australian researchers found that although kisspeptin administered to mares as a constant rate infusion elevated circulating LH levels, it did not lead to an LH surge and therefore did not evoke ovulations within their group of mares during the vernal transition [4]. It remains to be seen whether the use of kisspeptin may shorten time to first ovulation, by potentially driving follicle maturation, under influence of LH, without

As shown, dopamine plays an essential role in the stimulation of the reproductive axis in the mare. Dopamine has an inhibitory effect on GnRH release. For completeness it appears that dopamine antagonist acts via the stimulation of prolactin. For both domperidone and sulpiride, the dose is 1 mg/kg given PO and IM, respectively. Both are administered once daily for 25 days. The reports on the efficacy of the use of these preparations to shorten time to first ovulation in the mare are conflicting. A recent study by Mari et al. [25], comparing the two products, found that sulpiride significantly shortened time to pregnancy establishment (61 days) compared with domperidone-treated mares (83 days). That group concluded sulpiride is effective in advancing the vernal transition, whereas domperidone is

lowly mare has nowhere to run from her aggressive barn mate.

**56**

As mentioned the long transitional phase exhibited by mares is characterized by numerous follicular waves, unpredictable follicle growth, and follicle regression. Many protocols have examined the use of progesterone (P4) to dampen down these unpredictable features of the transitional phase and to drive a follicle to become dominant and one that will ovulate. The physiological effect of exogenous progesterone supplementation is relatively simple. P4 exerts an inhibitory mechanism with regard to LH but has minimal effect on FSH secretion. As described earlier, LH is required for maturation and final ovulation of the dominant follicle. While the mare is exposed to exogenous P4, LH is blocked at the level of the anterior pituitary, while FSH continues to be secreted. Therefore the follicles continue to grow under influence of FSH. Once the exogenous source of P4 is removed, this sudden fall in circulating P4 stimulates the LH surge, leading to final maturation and ovulation of a dominant follicle. The typical regimen is a dietary supplementation with altrenogest (Regumate®) at 0.044 mg/kg PO for 10 days. Injectable P4 products are becoming more routinely available. In the USA compounded products such as progesterone in oil can be utilized. Controlled release of P4 from these compounds last between 7 and 10 days. Daily application of oral altrenogest can be time-consuming. There also is a risk of noncompliance, should a mare be difficult to catch, not to mention potential side effect for the operator. The use of these long-acting P4 BioRelease products have been shown to be effective [26]. It appears that the use of exogenous P4 has maximal benefits when the mare exhibits a follicle of at least 20 mm in diameter and when administered in deep anestrous has little effect [27, 28]. An injectable altrenogest marketed via BOVA has recently become available in the UK for the first time, although no studies on its efficacy are currently available.

#### **2.2 Endometritis**

#### *2.2.1 Endometritis in the mare*

Endometritis is a leading cause of subfertility in the mare [29] and is the third most reported condition seen in our equine patients [30]. Endometritis, simply, the inflammation of the lining of the uterus, has historically been attributed to bacterial colonization and infection of the uterus. However there are a subgroup of mares that will exhibit persistent mating-induced endometritis (PMIE), in the absence of bacterial isolation. Furthermore we will also examine in the chapter the role of biofilm formation and bacterial endometritis.

Post breeding, a normal, physiologic endometritis will be observed in all mares [31]. This normal, transient event, which peaks around 8 hours post insemination, occurs to eliminate excessive spermatozoa, seminal plasma, and contaminants from the uterus [32]. This physiologic response should be over by 48 hours post insemination [33]. The subgroup of mares that experience PMIE appear to have an altered inflammatory response to the presence of spermatozoa and seminal plasma within the uterus. These mares tend to be aged, have increased parity, may exhibit chronic inflammatory changes within the endometrium [34], and exhibit failure to clear intrauterine bacterial challenges [35]. Susceptibility rates among thoroughbred broodmares is 15% [36], and crucially the early embryonic death rate is three times higher in this group of mares [37]. A persistent inflammatory uterine environment 5 days post fertilization is incompatible with embryo survival [38].

It has long been proposed that mares are classified as either susceptible or resistant to PMIE. It has been shown that susceptible mares do have altered protein composition of their endometrial fluid [39] and these mares also exhibit higher levels of pro-inflammatory cytokines [40, 41]. It has also been shown that these mares with a delayed uterine clearance have contractile defects of the endometrium, possibly contributing to this delay in uterine fluid clearance [42]. It has been proposed that nitric oxide mediates smooth muscle relaxation [43]. It also important that mares that fall into this subgroup tend to have poor perineal conformation and a forward tilt to the uterus, such that it sits over the pelvic brim. It is therefore paramount to be able to identify these mares and initiate appropriate therapy.

Bacterial endometritis in the mare is primarily caused by four pathogenic species: *Streptococcus equi* subspecies *zooepidemicus*, *Escherichia coli*, *Klebsiella pneumoniae*, and *Pseudomonas aeruginosa* [44–47]. By far the most commonly isolated are *Escherichia coli* and *Streptococcus equi* subspecies *zooepidemicus* (**Figure 1**). Diagnosis of bacterial endometritis is based on transrectal ultrasonography, uterine culture, and uterine cytology. In transrectal ultrasonography, these mares may show increased uterine edema and increased uterine luminal fluid, which may be echogenic in nature. Any mare that has uterine fluid with an accompanying corpus luteum (CL) should be highly suspected to have a uterine infection. There are now several reports of bacterial species becoming resistant to commonly used antimicrobials especially gram-negative species. It is therefore paramount that the attending veterinarian takes cultures to identify which bacterial species are present and then to select an appropriate antimicrobial based on antibiotic sensitivities.

A list of commonly used intrauterine antibiotics and dosages can be found in **Table 1** in the therapy section.

#### *2.2.2 Fungal endometritis*

Around 5 percent of infectious endometritis are attributed to fungal organisms [48], of which *Candida* spp., *Aspergillus* spp., and *Mucor* spp. are most frequently isolated. Again, mares that have anatomical defects are predisposed, and the use of previous intrauterine antimicrobial therapy is thought to increase the likelihood of fungal infections. Two schools of thought exist as to why this may be the case. Firstly, with repeated infusions, fungal organisms may be transplanted into the uterus (i.e., via contamination), and secondly, whether the antimicrobials may disrupt the normal bacterial flora of the caudal reproductive tract and subsequently

#### **Figure 1.**

Streptococcus equi *subspecies growing on blood agar showing distinct beta hemolysis. Image courtesy of BioTe veterinary laboratories, England.*

**59**

the mare.

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare*

**Product Dosage Notes**

Ceftiofur (1 g vial) 1 g in 20 ml sterile

Ciprofloxacin (10 mg/ml) 400–500 mg in

Gentamicin (100 mg/ml) 1–2 g, buffer with

500 mg in 50 mls sterile saline

100–250 mg in 50 ml sterile water

sterile saline

50 ml sterile saline

10 ml 8.4% sodium bicarbonate

15 ml, dilute to 50 ml in sterile saline

saline

Add 5 ml DMSO per 1 gram of fluconazole to aid in dissolving

Mainly effective against gram-

Mainly effective against gramnegative bacteria. Not first line; only utilize if strains are resistant to other antimicrobials

Mainly effective against gram-

Mainly effective against gram-

Broad-spectrum antibiotic

tablets

positive bacteria

negative bacteria

positive bacteria

allow colonization by the fungus. These sites (such as the clitoral fossa) can then

*Doses for commonly utilized antimicrobials for intrauterine administration (table reproduced courtesy of* 

Therapy for endometritis in the mare will vary depending on whether the attending veterinarian is dealing with a bacterial endometritis, fungal endometritis, or indeed PMIE. Nonetheless there are some therapies that will be necessary in all

It is imperative to correct any caudal reproductive tract anatomical anomalies, such as poor perineal conformation. Surgical correction, such as a vulvoplasty (also known as a Caslick procedure), should be performed on these mares prior to breeding. A temporary Caslick can aid in treatment during the few days intrauterine access is required. A permanent Caslick can then be placed after treatment has ceased or after breeding (and resolution of any post-breeding fluid). An alternative is to place a permanent Caslick and to administer the treatment via a speculum, giving access to the cervix. Fixing anatomical defects in this area will prevent recontamination of the caudal reproductive tract and helps to "pull" the uterus into a more caudal position, aiding the natural mechanical cleansing mechanism of

All mares that have excess fluid should undergo uterine lavages. In cases of infectious endometritis, these uterine lavages reduce the organism load, aid in removal of biofilms (see below for further treatments), and reduce particulate mat-

In any suspected fungal endometritis, it is imperative to send the sample swab to be tested for polymerase chain reaction (PCR). Fungal growth in routine laboratory cultures encompasses a long wait for results, whereas the turnaround for PCR is

*DOI: http://dx.doi.org/10.5772/intechopen.92999*

Antifungals Clotrimazole (100 mg/ tablet)

tablet)

Fluconazole (200 mg/

Antibacterials Ampicillin (1 g vial) 1–2 g in 50 ml

Penicillin (procaine) 300,000 units per ml

*equine reproduction laboratory, Colorado State University).*

serve as a nidus for uterine infection.

cases, and they are dealt with first.

ter that may interfere with the antimicrobials used.

relatively quick.

*2.2.3 Therapy*

**Table 1.**


*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare DOI: http://dx.doi.org/10.5772/intechopen.92999*

#### **Table 1.**

*Equine Science*

**Table 1** in the therapy section.

*2.2.2 Fungal endometritis*

composition of their endometrial fluid [39] and these mares also exhibit higher levels of pro-inflammatory cytokines [40, 41]. It has also been shown that these mares with a delayed uterine clearance have contractile defects of the endometrium, possibly contributing to this delay in uterine fluid clearance [42]. It has been proposed that nitric oxide mediates smooth muscle relaxation [43]. It also important that mares that fall into this subgroup tend to have poor perineal conformation and a forward tilt to the uterus, such that it sits over the pelvic brim. It is therefore paramount to be able to identify these mares and initiate appropriate therapy. Bacterial endometritis in the mare is primarily caused by four pathogenic species: *Streptococcus equi* subspecies *zooepidemicus*, *Escherichia coli*, *Klebsiella pneumoniae*, and *Pseudomonas aeruginosa* [44–47]. By far the most commonly isolated are *Escherichia coli* and *Streptococcus equi* subspecies *zooepidemicus* (**Figure 1**). Diagnosis of bacterial endometritis is based on transrectal ultrasonography, uterine culture, and uterine cytology. In transrectal ultrasonography, these mares may show increased uterine edema and increased uterine luminal fluid, which may be echogenic in nature. Any mare that has uterine fluid with an accompanying corpus luteum (CL) should be highly suspected to have a uterine infection. There are now several reports of bacterial species becoming resistant to commonly used antimicrobials especially gram-negative species. It is therefore paramount that the attending veterinarian takes cultures to identify which bacterial species are present and then to select an appropriate antimicrobial based on antibiotic sensitivities.

A list of commonly used intrauterine antibiotics and dosages can be found in

Around 5 percent of infectious endometritis are attributed to fungal organisms [48], of which *Candida* spp., *Aspergillus* spp., and *Mucor* spp. are most frequently isolated. Again, mares that have anatomical defects are predisposed, and the use of previous intrauterine antimicrobial therapy is thought to increase the likelihood of fungal infections. Two schools of thought exist as to why this may be the case. Firstly, with repeated infusions, fungal organisms may be transplanted into the uterus (i.e., via contamination), and secondly, whether the antimicrobials may disrupt the normal bacterial flora of the caudal reproductive tract and subsequently

Streptococcus equi *subspecies growing on blood agar showing distinct beta hemolysis. Image courtesy of BioTe* 

**58**

**Figure 1.**

*veterinary laboratories, England.*

*Doses for commonly utilized antimicrobials for intrauterine administration (table reproduced courtesy of equine reproduction laboratory, Colorado State University).*

allow colonization by the fungus. These sites (such as the clitoral fossa) can then serve as a nidus for uterine infection.

In any suspected fungal endometritis, it is imperative to send the sample swab to be tested for polymerase chain reaction (PCR). Fungal growth in routine laboratory cultures encompasses a long wait for results, whereas the turnaround for PCR is relatively quick.

#### *2.2.3 Therapy*

Therapy for endometritis in the mare will vary depending on whether the attending veterinarian is dealing with a bacterial endometritis, fungal endometritis, or indeed PMIE. Nonetheless there are some therapies that will be necessary in all cases, and they are dealt with first.

It is imperative to correct any caudal reproductive tract anatomical anomalies, such as poor perineal conformation. Surgical correction, such as a vulvoplasty (also known as a Caslick procedure), should be performed on these mares prior to breeding. A temporary Caslick can aid in treatment during the few days intrauterine access is required. A permanent Caslick can then be placed after treatment has ceased or after breeding (and resolution of any post-breeding fluid). An alternative is to place a permanent Caslick and to administer the treatment via a speculum, giving access to the cervix. Fixing anatomical defects in this area will prevent recontamination of the caudal reproductive tract and helps to "pull" the uterus into a more caudal position, aiding the natural mechanical cleansing mechanism of the mare.

All mares that have excess fluid should undergo uterine lavages. In cases of infectious endometritis, these uterine lavages reduce the organism load, aid in removal of biofilms (see below for further treatments), and reduce particulate matter that may interfere with the antimicrobials used.

#### *2.2.4 Ecbolics*

No attending veterinarian should underestimate the use of ecbolic when dealing with endometritis in the mare. The two commonly used preparations are oxytocin and prostaglandin F2α (PGF2α) in dealing with uterine fluid.

Oxytocin is by far the most commonly used of these two. Its ease of administration either given IM (intramuscular) or IV (intravenous) and its relatively short duration of approximately 30–45 minutes make it an essential product to have on standby when breeding mares. Side effects are minimal. However given its short duration of action, it does require multiple doses. Typically 1 ml either IV or IM of oxytocin given every 4 hours for 1 day, starting a minimum 4 hours post breeding, will be sufficient in treating most minor cases of uterine fluid retention.

The use of prostaglandins is not as straightforward as oxytocin. There are more side effects with the use of this preparation, and some are potentially quite serious. Prostaglandin is a known abortifacient. It is a good practice to always identify the mare in front of you for any reproductive treatment and, if in doubt, ultrasound the mare to confirm that she is indeed empty. Duration of action is approximately 4 hours. During this time, the mare may sweat, may act colicky, and may exhibit loose stools. It is recommended to monitor the mare during these 4 hours. Many clinicians are familiar with the use of PGF2α, as a luteolytic agent, and that is by far the most common use in equine reproduction. However, the veterinarian should not be afraid of its use when dealing with uterine fluid retention. Caution must be taken, however, when dosing PGF2α on the day of ovulation, as some studies have suggested that it can impact the formation of the corpus hemorrhagicum (which later becomes the CL, the source of progesterone required for maintenance of pregnancy). On the day of ovulation, it would steer the clinician away from use of prostaglandins, unless he or she is prepared to place that mare on an exogenous source of progesterone. The typical protocol initiated at my practice is that we would start with oxytocin for the first day and a half. If the mare has yet to respond satisfactorily to oxytocin therapy in that time, she is unlikely to respond. Throwing more oxytocin her way is futile. It is at this point we would consider the use of prostaglandin. In exceptional and severe cases, where there is significant fluid retention, it is not unknown to utilize both oxytocin and prostaglandin simultaneously on day 1.

#### *2.2.5 Bacterial endometritis*

Typically 3 days of intrauterine therapy is sufficient to see a positive outcome to therapy. It is bad practice to initiate intrauterine therapy for more than 3 days and predispose the bacterial inhabitants of the uterus to develop resistance to the antimicrobial utilized.

If the mare is presented with significant uterine fluid (in excess of 1 cm on ultrasonography), care must be taken to remove excessive uterine luminal fluid before commencement of the therapy. This is because we now know that certain antimicrobials may be affected by the fluid, but also there is a dilution factor to consider. Removal of fluid may include uterine lavages where 1–2 L of sterile fluid is distilled into the uterus and then allowed to flow back through the same giving set back into their original bags. Manual palpation of the uterus via the rectum at the same time the veterinarian is trying to remove the fluid may aid in evacuation of the uterine fluid. Ecbolics can be utilized concurrently, namely, oxytocin (see **Table 2**). For mares that present with minimal fluid, the use of ecbolics may be sufficient to remove the fluid. It is recommended to ultrasound the uterus prior to each intrauterine infusion.

**61**

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare*

**administration**

Oxytocin (20 units/ml) 20 units IV or IM q 6 hours

Estrumate® (cloprostenol) 250 μg IM once Synthetic prostaglandin F2α

*Doses and routes of administration for the commonly utilized ecbolic agents (table reproduced courtesy of* 

**Notes**

prostaglandin F2α

5–10 mg IM once Naturally occurring

Further to the treatments below (**Table 1**), it is indicated to lavage the uterus

The use of exercise postmating, whether pasture turnout on the use of a horse walker, is widespread, yet the efficacy and examination in control studies are lacking. It is hypothesized that increases in intra-abdominal pressure from exercise transfer pressure to the uterus to aid in evacuating the contents and improve the lymphatic drainage [49]. Others have suggested that exercise can tone the hindquarters and leads to an improvement of perineal conformation [50]. Swift et al. [51] demonstrated that exercise was an effective management technique to aid in evacuation of uterine contents post breeding in mares. In their study, they note the lack of control studies on the efficacy of exercise alone as a treatment for uterine

The use of IV dexamethasone at a dose of 50 mg at time of treatment has become widespread following the classic studies by Bucca's group in Ireland [52]. It has been shown that there is a negative correlation between elevated endometrial score at time of breeding and pregnancy rates [53]. Dexamethasone

A recent and growing addition to the treatment of endometritis in the mare is acupuncture. It has been suggested that electroacupuncture stimulates afferent nerve fibers, leading to modulation of hormone release through ascending pathways to the hypothalamus as well as reflex activation of the autonomic efferent pathways to the uterus [54]. The first control study examining the use of electroacupuncture in the mare as a treatment modality for endometritis found mare resistance to treatment was a major limitation in the use of this treatment, and that given the multiple acupuncture points, as of yet, does not appear to be an effective mechanism when

has been shown to modulate the inflammatory process, possessing antiinflammatory effects (decreasing IgG) while showing a stimulatory effect on α1-antitrypsin and transthyretin, which both enhance the defense mechanisms

*DOI: http://dx.doi.org/10.5772/intechopen.92999*

**Product Dose and route of** 

with dilute acetic acid or dilute povidone-iodine.

*equine reproduction laboratory, Colorado State University).*

fluid retention post breeding in the mare.

*2.2.8 Glucocorticoid treatment*

of the uterus.

*2.2.9 Acupuncture*

treating endometritis in the mare.

*2.2.6 Fungal endometritis*

Lutalyse® (dinoprost tromethamine)

*2.2.7 Exercise*

**Table 2.**

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare DOI: http://dx.doi.org/10.5772/intechopen.92999*


**Table 2.**

*Equine Science*

*2.2.4 Ecbolics*

No attending veterinarian should underestimate the use of ecbolic when dealing with endometritis in the mare. The two commonly used preparations are oxytocin

Oxytocin is by far the most commonly used of these two. Its ease of administration either given IM (intramuscular) or IV (intravenous) and its relatively short duration of approximately 30–45 minutes make it an essential product to have on standby when breeding mares. Side effects are minimal. However given its short duration of action, it does require multiple doses. Typically 1 ml either IV or IM of oxytocin given every 4 hours for 1 day, starting a minimum 4 hours post breeding,

The use of prostaglandins is not as straightforward as oxytocin. There are more side effects with the use of this preparation, and some are potentially quite serious. Prostaglandin is a known abortifacient. It is a good practice to always identify the mare in front of you for any reproductive treatment and, if in doubt, ultrasound the mare to confirm that she is indeed empty. Duration of action is approximately 4 hours. During this time, the mare may sweat, may act colicky, and may exhibit loose stools. It is recommended to monitor the mare during these 4 hours. Many clinicians are familiar with the use of PGF2α, as a luteolytic agent, and that is by far the most common use in equine reproduction. However, the veterinarian should not be afraid of its use when dealing with uterine fluid retention. Caution must be taken, however, when dosing PGF2α on the day of ovulation, as some studies have suggested that it can impact the formation of the corpus hemorrhagicum (which later becomes the CL, the source of progesterone required for maintenance of pregnancy). On the day of ovulation, it would steer the clinician away from use of prostaglandins, unless he or she is prepared to place that mare on an exogenous source of progesterone. The typical protocol initiated at my practice is that we would start with oxytocin for the first day and a half. If the mare has yet to respond satisfactorily to oxytocin therapy in that time, she is unlikely to respond. Throwing more oxytocin her way is futile. It is at this point we would consider the use of prostaglandin. In exceptional and severe cases, where there is significant fluid retention, it is not unknown to utilize both oxytocin and prostaglandin simultane-

Typically 3 days of intrauterine therapy is sufficient to see a positive outcome to therapy. It is bad practice to initiate intrauterine therapy for more than 3 days and predispose the bacterial inhabitants of the uterus to develop resistance to the

If the mare is presented with significant uterine fluid (in excess of 1 cm on ultrasonography), care must be taken to remove excessive uterine luminal fluid before commencement of the therapy. This is because we now know that certain antimicrobials may be affected by the fluid, but also there is a dilution factor to consider. Removal of fluid may include uterine lavages where 1–2 L of sterile fluid is distilled into the uterus and then allowed to flow back through the same giving set back into their original bags. Manual palpation of the uterus via the rectum at the same time the veterinarian is trying to remove the fluid may aid in evacuation of the uterine fluid. Ecbolics can be utilized concurrently, namely, oxytocin (see **Table 2**). For mares that present with minimal fluid, the use of ecbolics may be sufficient to remove the fluid. It is recommended to ultrasound the uterus prior to each intrauterine infusion.

and prostaglandin F2α (PGF2α) in dealing with uterine fluid.

will be sufficient in treating most minor cases of uterine fluid retention.

**60**

ously on day 1.

*2.2.5 Bacterial endometritis*

antimicrobial utilized.

*Doses and routes of administration for the commonly utilized ecbolic agents (table reproduced courtesy of equine reproduction laboratory, Colorado State University).*

#### *2.2.6 Fungal endometritis*

Further to the treatments below (**Table 1**), it is indicated to lavage the uterus with dilute acetic acid or dilute povidone-iodine.

#### *2.2.7 Exercise*

The use of exercise postmating, whether pasture turnout on the use of a horse walker, is widespread, yet the efficacy and examination in control studies are lacking. It is hypothesized that increases in intra-abdominal pressure from exercise transfer pressure to the uterus to aid in evacuating the contents and improve the lymphatic drainage [49]. Others have suggested that exercise can tone the hindquarters and leads to an improvement of perineal conformation [50]. Swift et al. [51] demonstrated that exercise was an effective management technique to aid in evacuation of uterine contents post breeding in mares. In their study, they note the lack of control studies on the efficacy of exercise alone as a treatment for uterine fluid retention post breeding in the mare.

#### *2.2.8 Glucocorticoid treatment*

The use of IV dexamethasone at a dose of 50 mg at time of treatment has become widespread following the classic studies by Bucca's group in Ireland [52]. It has been shown that there is a negative correlation between elevated endometrial score at time of breeding and pregnancy rates [53]. Dexamethasone has been shown to modulate the inflammatory process, possessing antiinflammatory effects (decreasing IgG) while showing a stimulatory effect on α1-antitrypsin and transthyretin, which both enhance the defense mechanisms of the uterus.

#### *2.2.9 Acupuncture*

A recent and growing addition to the treatment of endometritis in the mare is acupuncture. It has been suggested that electroacupuncture stimulates afferent nerve fibers, leading to modulation of hormone release through ascending pathways to the hypothalamus as well as reflex activation of the autonomic efferent pathways to the uterus [54]. The first control study examining the use of electroacupuncture in the mare as a treatment modality for endometritis found mare resistance to treatment was a major limitation in the use of this treatment, and that given the multiple acupuncture points, as of yet, does not appear to be an effective mechanism when treating endometritis in the mare.

#### *2.2.10 Breeding on a dirty cycle*

In an ideal world, we would swab the uterus, and if found to have an infection, we would "clean" her up and wait for the mare's next cycle. However in the time-pressured breeding season, and in particular when dealing with valuable thoroughbred racehorses, time is seldom something the attending veterinarian has. This author has had great success breeding mares on dirty cycles, as long as there is at least 3 days prior to cover, to allow 3 days of intrauterine therapy. It is well established that the optimum time to swab the mare's uterus is when there is presence of uterine edema; swabbing when there is no uterine edema raises the risk of a false-negative result. An assumption is that the mare is infection-free only to be found negative on her pregnancy scan. Moreover it was inappropriate for the attending clinician to swab the uterus of a mare in diestrus (i.e., that she has a CL present). For one, the cervix will be tightly closed, and you may damage the cervix while trying to force the culture instrument through. Additionally, as the cervix is tightly closed, if you have accidently tracked bacterial isolates from the external vulva, or indeed the vaginal vault into the uterus, thereby inoculating the uterus with an infectious agent, the infection will take hold as the mare will be unable to "cleanse" herself with a closed cervix.

#### *2.2.11 A frustrating scenario and the role of biofilm*

We have all been there, as attending clinicians. We swab the mare, she cultures negative, there are no ultrasonic changes to make us think there may be an infection, and she returns negative on multiple cycles. There is a caveat here, that reproduction is a complex beast, and many, many things must fall into place for successful fertilization and subsequent embryonic development to take place. As the saying goes, it takes two to tango. However as this part of the chapter is dedicated to endometritis and often we do not have access to the stallion, it is fair for the clinician to start with the mare, and indeed her uterus, when beginning to evaluate why a mare may not become pregnant.

In the short breeding season, the author recommends that any mare that is negative on two cycles (i.e., she has been inseminated twice) should undergo a full reproductive examination that includes swabbing the uterus for culture. If there is any suspicion that the mare maybe dirty, but has a negative culture, then the clinician should explore other diagnostic routes. This would, namely, be lowvolume lavage.

Nonetheless there are some mares that either routinely cultured negative but fail to conceive or conversely routinely cultured positive despite appropriate therapy based on sensitivities. In these cases, the attending clinician must consider the possibility of a biofilm. A biofilm as defined by Loncar et al. [55] is a community of bacteria that are attached to an interface or to one another, encased within an extrapolymeric matrix consisting of nucleic acids, lipids, proteins, and exopolysaccharides. These biofilm plaques are inherently resistance to both antimicrobial and innate immune defenses, which leads to a persistent, chronic infection, even in the face of prolonged antimicrobial therapy. The matrix reduces the penetration of antimicrobials. Gram-negative bacteria, such as *Escherichia coli*, *Pseudomonas aeruginosa*, and *Klebsiella pneumoniae* are all capable of producing biofilms. The workout of Colorado State University has given us incredible information on treating biofilms. The work of Loncar et al. [55] showed that no single treatment was effective against all three bacterial species named above and suggests appropriate identification of bacterial species is paramount for successful treatment. The administration of dimethyl sulfoxide to the uterus shows promise in treating biofilms caused by *E. coli* and *K. pneumoniae*.

**63**

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare*

Twin or multiple pregnancies in the mare is the most common noninfectious cause of pregnancy loss. Twin pregnancies have been reported to account for up to 30% of abortions in the mare [56, 57]. When twin pregnancies are established, the pregnancy will continue as normal to approximately 6 months, when one or both fetuses will die due to insufficient placental supply. Typically mares carrying twins will abort around 5–9 months of gestation [58]. Only 14% of twin pregnancies resulted in foals surviving into their second week of postnatal life [56]. Mares producing live twins will inevitably require intense (and therefore expensive) neonatal care. The majority of twin pregnancies are dizygotic and arise from multiple ovulations which can be either synchronous or asynchronous in nature. Ginther [59] has proposed that there are familial lines higher than normal incidences of multiple ovulations and thereby there may be a genetic predisposition. Thoroughbreds show the highest incidence of multiple ovulations, while it is low in native breeds and yet to be reported in native Shetland ponies. Older mares also seem to have high incidences of multiple ovulations [60]; however lactating mares appear to have lower multiple ovulation rates, presumably due to the suckling effect on the hypothalamic–pituitary-ovary axis. Not only do mare normally abort twin pregnancies, they also show high incidence of dystocia, damage to the reproductive tract (including the cervix), retained placenta, and delayed uterine involution. These have ramifications on the future reproductive health of these mares. In one study only 38% of mares that had a twin pregnancy in the previous breeding season produced a viable foal the following year [61]. Given the significant risks associated with twin pregnancies, detection of these is paramount. The attending veterinarian should make detailed notes of the presence of large follicles on the ovary and, at ovulation detection, note all ovulations. However do not be fooled, if only one large follicle has ovulated and another large follicle remains. If this follicle should subsequently ovulate, there is a chance of the establishment of asynchronous twins. It is advised to examine the mare in stocks and have the mare adequately restrained. Checking for twins in the field, where a mare may be fractious and/or not restrained correctly, will lead the clinician to potentially rush through examination. There is a danger element to ultrasounding mares not in stocks. Occasionally owners will state that they do not wish to transport the mare to facilities that have the required setup. If this is the case, get the mare restrained as best as possible, and advise the owner that this is not optimal. No ultrasound examination is foolproof. Begin in a systematic manner. The author starts with the left horn, runs the ultrasound probe laterally until the left ovary is seen, and then returns to the bifurcation. This is repeated twice. The same is then done for the right horn. Finally the body of the uterus is examined twice. During the examination, it is paramount to retain the uterus within the center of the screen at all times. If you feel as though a section of the uterus has been missed, repeat. As can been seen, to do this in the field without stocks in a fractious mare can be difficult. Natural reduction of unilateral twins

*DOI: http://dx.doi.org/10.5772/intechopen.92999*

before day 40 is reported at 85% [62, 63].

Given the limitations of this chapter, only two techniques for dealing with twin pregnancies in the mare will be described. There are numerous other techniques described and the readers are encouraged to examine these. At approximately 16 days post ovulation, the embryo (in this case the embryos) become fixed. Up until this point, the embryos are highly mobile and move throughout the uterine lumen. Typically a twin check using transrectal ultrasound takes place before this day 16. Identification of the small embryo takes place. If the pregnancies are adjacent to one another, the probe is gently oscillated to move them apart. The smaller embryo is then moved to the tip of the uterine horn, while a downward pressure

**2.3 Twinning events in the mare**

#### **2.3 Twinning events in the mare**

*Equine Science*

*2.2.10 Breeding on a dirty cycle*

"cleanse" herself with a closed cervix.

why a mare may not become pregnant.

volume lavage.

*2.2.11 A frustrating scenario and the role of biofilm*

In an ideal world, we would swab the uterus, and if found to have an infection, we would "clean" her up and wait for the mare's next cycle. However in the time-pressured breeding season, and in particular when dealing with valuable thoroughbred racehorses, time is seldom something the attending veterinarian has. This author has had great success breeding mares on dirty cycles, as long as there is at least 3 days prior to cover, to allow 3 days of intrauterine therapy. It is well established that the optimum time to swab the mare's uterus is when there is presence of uterine edema; swabbing when there is no uterine edema raises the risk of a false-negative result. An assumption is that the mare is infection-free only to be found negative on her pregnancy scan. Moreover it was inappropriate for the attending clinician to swab the uterus of a mare in diestrus (i.e., that she has a CL present). For one, the cervix will be tightly closed, and you may damage the cervix while trying to force the culture instrument through. Additionally, as the cervix is tightly closed, if you have accidently tracked bacterial isolates from the external vulva, or indeed the vaginal vault into the uterus, thereby inoculating the uterus with an infectious agent, the infection will take hold as the mare will be unable to

We have all been there, as attending clinicians. We swab the mare, she cultures negative, there are no ultrasonic changes to make us think there may be an infection, and she returns negative on multiple cycles. There is a caveat here, that reproduction is a complex beast, and many, many things must fall into place for successful fertilization and subsequent embryonic development to take place. As the saying goes, it takes two to tango. However as this part of the chapter is dedicated to endometritis and often we do not have access to the stallion, it is fair for the clinician to start with the mare, and indeed her uterus, when beginning to evaluate

In the short breeding season, the author recommends that any mare that is negative on two cycles (i.e., she has been inseminated twice) should undergo a full reproductive examination that includes swabbing the uterus for culture. If there is any suspicion that the mare maybe dirty, but has a negative culture, then the clinician should explore other diagnostic routes. This would, namely, be low-

Nonetheless there are some mares that either routinely cultured negative but fail to conceive or conversely routinely cultured positive despite appropriate therapy based on sensitivities. In these cases, the attending clinician must consider the possibility of a biofilm. A biofilm as defined by Loncar et al. [55] is a community of bacteria that are attached to an interface or to one another, encased within an extrapolymeric matrix consisting of nucleic acids, lipids, proteins, and exopolysaccharides. These biofilm plaques are inherently resistance to both antimicrobial and innate immune defenses, which leads to a persistent, chronic infection, even in the face of prolonged antimicrobial therapy. The matrix reduces the penetration of antimicrobials. Gram-negative bacteria, such as *Escherichia coli*, *Pseudomonas aeruginosa*, and *Klebsiella pneumoniae* are all capable of producing biofilms. The workout of Colorado State University has given us incredible information on treating biofilms. The work of Loncar et al. [55] showed that no single treatment was effective against all three bacterial species named above and suggests appropriate identification of bacterial species is paramount for successful treatment. The administration of dimethyl sulfoxide to the uterus shows

promise in treating biofilms caused by *E. coli* and *K. pneumoniae*.

**62**

Twin or multiple pregnancies in the mare is the most common noninfectious cause of pregnancy loss. Twin pregnancies have been reported to account for up to 30% of abortions in the mare [56, 57]. When twin pregnancies are established, the pregnancy will continue as normal to approximately 6 months, when one or both fetuses will die due to insufficient placental supply. Typically mares carrying twins will abort around 5–9 months of gestation [58]. Only 14% of twin pregnancies resulted in foals surviving into their second week of postnatal life [56]. Mares producing live twins will inevitably require intense (and therefore expensive) neonatal care. The majority of twin pregnancies are dizygotic and arise from multiple ovulations which can be either synchronous or asynchronous in nature. Ginther [59] has proposed that there are familial lines higher than normal incidences of multiple ovulations and thereby there may be a genetic predisposition. Thoroughbreds show the highest incidence of multiple ovulations, while it is low in native breeds and yet to be reported in native Shetland ponies. Older mares also seem to have high incidences of multiple ovulations [60]; however lactating mares appear to have lower multiple ovulation rates, presumably due to the suckling effect on the hypothalamic–pituitary-ovary axis. Not only do mare normally abort twin pregnancies, they also show high incidence of dystocia, damage to the reproductive tract (including the cervix), retained placenta, and delayed uterine involution. These have ramifications on the future reproductive health of these mares. In one study only 38% of mares that had a twin pregnancy in the previous breeding season produced a viable foal the following year [61]. Given the significant risks associated with twin pregnancies, detection of these is paramount. The attending veterinarian should make detailed notes of the presence of large follicles on the ovary and, at ovulation detection, note all ovulations. However do not be fooled, if only one large follicle has ovulated and another large follicle remains. If this follicle should subsequently ovulate, there is a chance of the establishment of asynchronous twins. It is advised to examine the mare in stocks and have the mare adequately restrained. Checking for twins in the field, where a mare may be fractious and/or not restrained correctly, will lead the clinician to potentially rush through examination. There is a danger element to ultrasounding mares not in stocks. Occasionally owners will state that they do not wish to transport the mare to facilities that have the required setup. If this is the case, get the mare restrained as best as possible, and advise the owner that this is not optimal. No ultrasound examination is foolproof. Begin in a systematic manner. The author starts with the left horn, runs the ultrasound probe laterally until the left ovary is seen, and then returns to the bifurcation. This is repeated twice. The same is then done for the right horn. Finally the body of the uterus is examined twice. During the examination, it is paramount to retain the uterus within the center of the screen at all times. If you feel as though a section of the uterus has been missed, repeat. As can been seen, to do this in the field without stocks in a fractious mare can be difficult. Natural reduction of unilateral twins before day 40 is reported at 85% [62, 63].

Given the limitations of this chapter, only two techniques for dealing with twin pregnancies in the mare will be described. There are numerous other techniques described and the readers are encouraged to examine these. At approximately 16 days post ovulation, the embryo (in this case the embryos) become fixed. Up until this point, the embryos are highly mobile and move throughout the uterine lumen. Typically a twin check using transrectal ultrasound takes place before this day 16. Identification of the small embryo takes place. If the pregnancies are adjacent to one another, the probe is gently oscillated to move them apart. The smaller embryo is then moved to the tip of the uterine horn, while a downward pressure

from the ultrasound probe on the selected embryo is performed. While keeping the embryo in focus on the ultrasound screen, rupture of the embryonic wall will be observed, and leakage of the embryonic fluid into the uterine lumen will also be observed. A quick check on the remaining embryo should also be performed, following this procedure. Adjunct therapy typically includes a single dose of flunixin meglumine (1 mg/kg IV) given prior to the elimination procedure. Typically these mares are placed on oral Regumate® (dose of 0.088 mg/kg SID PO), until a P4 sample is taken around the heartbeat ultrasound check (approximately day 25 post ovulation). Success rates of continued survival of the singleton pregnancy after a twin reduction around this time is in excess of 90% [64].

If you are presented with twin pregnancies beyond this stage, the clinician has a few options to choose from. After day 40, 63% of these pregnancies result in loss of both fetuses [65]. One of the authors preferred mechanism of twin reduction after day 40, which is cranio-cervical dislocation. Here the clinician is dislocating the first cervical vertebrae from the cranium along with disruption of the ligamentous attachments and severing the spinal cord via transrectal manipulation. This technique can be utilized between 60 and 110 days' gestation. The mare is sedated and placed in stocks. Buscopan (2 cc IV) can facilitate manipulation of the fetuses. Flunixin meglumine (1 mg/kg IV) is administered prior. The small fetus is selected and identification of the head performed, via identification of the mandible. Stabilize the head between the thumb and the finger and move the head side to side. Place the thumb at the base of the cranium and apply pressure proximally and dorsally; this will result in dislocation, whereby a "pop" is felt. Adjunct therapy included altrenogest (Regumate® at dose 0.088 mg/kg SID PO). Fetal death should be confirmed in 1 week post procedure via transrectal ultrasonography. Viability of the remaining conceptus should be evaluated (viz., by continued growth and the presence of a fetal heartbeat). If both pregnancies continue to be viable, then further intervention will be necessary.

#### **3. Conclusions**

With a thorough understanding on the physiologic events in the spring/vernal transition, the clinician can aid in hastening time to first ovulation. Most mares, if not all, will show some transient uterine fluid accumulation post breeding. Having the skills to note which mares are likely candidates to have excessive fluid accumulation, or which mares have a uterine infection, will greatly improve pregnancy rates. Identification of mares that may develop twin pregnancies is a key skill of the equine theriogenologist, but transrectal ultrasonography has its limitations if the mare is examined in the field. Twin pregnancies are easily dealt with if identified prior to fixation.

**65**

**Author details**

David A. Trundell

DT Veterinary Services, Salisbury, UK

provided the original work is properly cited.

\*Address all correspondence to: dt.vet2020@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare*

*DOI: http://dx.doi.org/10.5772/intechopen.92999*

#### **Conflict of interest**

The author declares no conflict of interest.

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare DOI: http://dx.doi.org/10.5772/intechopen.92999*

*Equine Science*

from the ultrasound probe on the selected embryo is performed. While keeping the embryo in focus on the ultrasound screen, rupture of the embryonic wall will be observed, and leakage of the embryonic fluid into the uterine lumen will also be observed. A quick check on the remaining embryo should also be performed, following this procedure. Adjunct therapy typically includes a single dose of flunixin meglumine (1 mg/kg IV) given prior to the elimination procedure. Typically these mares are placed on oral Regumate® (dose of 0.088 mg/kg SID PO), until a P4 sample is taken around the heartbeat ultrasound check (approximately day 25 post ovulation). Success rates of continued survival of the singleton pregnancy after a

If you are presented with twin pregnancies beyond this stage, the clinician has a few options to choose from. After day 40, 63% of these pregnancies result in loss of both fetuses [65]. One of the authors preferred mechanism of twin reduction after day 40, which is cranio-cervical dislocation. Here the clinician is dislocating the first cervical vertebrae from the cranium along with disruption of the ligamentous attachments and severing the spinal cord via transrectal manipulation. This technique can be utilized between 60 and 110 days' gestation. The mare is sedated and placed in stocks. Buscopan (2 cc IV) can facilitate manipulation of the fetuses. Flunixin meglumine (1 mg/kg IV) is administered prior. The small fetus is selected and identification of the head performed, via identification of the mandible. Stabilize the head between the thumb and the finger and move the head side to side. Place the thumb at the base of the cranium and apply pressure proximally and dorsally; this will result in dislocation, whereby a "pop" is felt. Adjunct therapy included altrenogest (Regumate® at dose 0.088 mg/kg SID PO). Fetal death should be confirmed in 1 week post procedure via transrectal ultrasonography. Viability of the remaining conceptus should be evaluated (viz., by continued growth and the presence of a fetal heartbeat). If both pregnancies continue to be viable, then

With a thorough understanding on the physiologic events in the spring/vernal transition, the clinician can aid in hastening time to first ovulation. Most mares, if not all, will show some transient uterine fluid accumulation post breeding. Having the skills to note which mares are likely candidates to have excessive fluid accumulation, or which mares have a uterine infection, will greatly improve pregnancy rates. Identification of mares that may develop twin pregnancies is a key skill of the equine theriogenologist, but transrectal ultrasonography has its limitations if the mare is examined in the field. Twin pregnancies are easily dealt with if identified

twin reduction around this time is in excess of 90% [64].

further intervention will be necessary.

**3. Conclusions**

prior to fixation.

**Conflict of interest**

The author declares no conflict of interest.

**64**

#### **Author details**

David A. Trundell DT Veterinary Services, Salisbury, UK

\*Address all correspondence to: dt.vet2020@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Provencio I, Rodriquez IR, Jiang G, Hayes WP, Moreira EF, Rollag MD. A novel human opsin in the inner retina. The Journal of Neuroscience. 2002;**20**:600-605. DOI: 0270-6474/00/200600-06\$15.00/0

[2] Lucas RJ, Freedman MS, Lupi D, Munoz M, David-Gray ZK, Foster RG. Identifying the photoreceptive inputs to the mammalian circadian system using transgenic and retinally degenerate mice. Behavioural Brain Research. 2001;**125**:97-102. DOI: 10.1016/50166-4328(01)00274-1

[3] Scott CJ, Rose JL, Allan JG, McGrath BM. Kisspeptin and the regulation of the reproductive axis in domestic animals. The Journal of Endocrinology. 2019;**240**(1):R1-R16. DOI: 10.1530/JOE-18-0485

[4] McGrath BM, Scott CJ, Wynn PC, Loy J, Norman ST. Kisspeptin stimulates LH secretion but not ovulation in mares during vernal transition. Theriogenology. 2016;**86**(6):1566-1572. DOI: 10.1016/j. theriogenology.2016.05.016

[5] Decourt C, Tilley Y, Franceschini I, Briant C. Kisspeptin immunoreactive neurons in the equine hypothalamus: Interactions with GnRH neuronal system. Journal of Chemical Neuroanatomy. 2008;**36**:131-137. DOI: 10.1016/j.chemneu.2008.07.008

[6] Lehman MN, Robinson JE, Karsch FJ, Silverman AJ. Immunocytochemical localization of luteinizing hormonereleasing hormone (LHRH) pathways in the sheep brain during anestrus and mid-luteal phase of the estrous cycle. The Journal of Comparative Neurology. 1986;**244**:19-35. DOI: 10.1002/ cne.902440103

[7] Smith JT, Clay CM, Caraty A, Clarke IJ. KiSS-1 messenger ribonucleic acid expression in the hypothalamus of the ewe is regulated by sex steroids and season. Endocrine. 2007;**148**:1150-1157. DOI: 10.1210/en.2006-1435

[8] Chalivoix S, Bagnolini A, Caraty A, Cognie J, Malpaux B, Dufourny L. Effects of photoperiod on kisspeptin neuronal populations of the ewe diencephalon in connection with reproductive function. Journal of Neuroendocrinology. 2010;**22**:110-118. DOI: 10.1111/j.1365-2826.2009.01939.x

[9] Goodman RL, Maltby MJ, Millar RP, Hileman SM, Nestor CC, Whited B, et al. Evidence that dopamine acts via kisspeptin to hold GnRH pulse frequency in check in anestrous ewes. Endocrine. 2012;**153**:5918-5927

[10] Cardoso RC, Alves BRC, Sharpton SM, Williams GL, Amstalden M. Nutritional programming of accelerated puberty in heifers; involvement of pro-opiomelanocortin neurons in the arcuate nucleus. Journal of Neuroendocrinology. 2015;**27**:647- 657. DOI: 10.111/jne.12291

[11] Rose JL. The Role of RFRP-3 and Kisspeptin on GnRH Secretion in the Merino Ram (Thesis). Australia: Charles Sturt University; 2017

[12] Merkey CM, Porter KL, Coolen LM, Hileman SM, Billings HJ, Drews S, et al. KNDy (Kisspeptin/Neurokinin B/Dynorphin) neurons are activated during both pulsatile and surge secretion of LH in the ewe. Endocrine. 2012;**153**:5406-5414. DOI: 10.1210/ en.2012-1357

[13] Goodman RL, Jansen HT, Billings HJ, Coolen LM, Lehman MN. Neural systems mediating seasonal breeding in the ewe. Journal of Neuroendocrinology. 2010;**22**:674-681. DOI: 10.1111/j.1365-2826.210.02014.x

**67**

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare*

artificial regulation by progesterone treatment. Journal of Reproduction and Fertility. Supplement. 1991;**44**:307-318

[21] Watson E, Heald M, Tsigos A, Leask R, Steele M, Groome N, et al. Plasma FSH, inhibin a and inhibin isoforms containing pro-and -αC during winter anoestrus, spring transition and the breeding season in mares. Reproduction. 2002;**123**:535-542. DOI:

10.1530/rep.0.1230535

10.1210/en.2005-0488

[23] Murphy BA, Walsh CM, Woodward EM, Prendergast RL, Ryle JP, Fallon LH, et al. Blue light from individual light mask directed at a single eye advances the breeding season in mares. Equine Veterinary Journal. 2013;**46**(5):601-605. DOI: 10.111/evj.12153

[24] Lockley SW, Brainard GC, Czeisler CA. High sensitivity of the human circadian melatonin rhythm to resetting by short wavelength light. The Journal of Clinical Endocrinology and Metabolism. 2003;**88**:4502-4505. DOI:

[25] Mari G, Morganti M, Merlo B, Castagnetti C, Parameggiani F, Govoni N, et al. Administration of sulpiride or domperidone for advancing the first ovulation in deep anestrous mares. Theriogenology. 2009;**71**(6):959-965. DOI: 10.1016/j.

10.1210/jc.2003-030570

theriogenology.2008.11.001

jevs.2011.06.016

[26] Staempfli S, Clavier S, Thompson D, Burns P, Lyle S, McKinnon AO. Effect of a single injection of long-acting progesterone on the first ovulation in early and late spring transitional mares. Journal of Equine Veterinary Science. 2011;**31**(12):744-748. DOI: 10.1016/j.

[22] Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA. Regulation of Kiss1 gene expression in the brain of the female mouse. Endocine. 2005;**146**:36-3692. DOI:

*DOI: http://dx.doi.org/10.5772/intechopen.92999*

[14] Li Q, Rao A, Pereira A, Clarke IJ, Smith JT. Kisspeptin cells in the ovine arcuate nucleus express prolactin receptor but not melatonin receptor. Journal of Neuroendocrinology.

10.1111/j.1365-2826.0211.02195.x

[16] Klosen P, Bienvenu C, Demarteau O, Dardente H, Guerrero H, Pevet P, et al. The mt1 melatonin receptor and RORbeta receptor are co-localized in specific TSH-immunoreactive cells in the pars tuberalis of the rat pituitary. The Journal of Histochemistry and Cytochemistry. 2002;**50**:1647-5710. DOI: 10.1177/002215540205001209

[17] Dardente H, Klosen P, Pevet P, Masson-Pevet M. MT1 melatonin receptor mRNA expressing cells in the pars tuberalis of the European hamster: Effect of photoperiod. Journal of Neuroendocrinology. 2003;**15**:778-861. DOI: 10.1046/j.1365-2826.2003.01060.x

[18] Wittkowski W, Hewing M, Hoffmann K, Bergmann M,

[19] Wittkowski W, Bergmann M, Hoffmann K, Pera F. Photoperioddependent changes in TSH-like immunoreactivity of cells in the hypophyseal pars tuberalis of the Djungarian hamster, *Phodopus sungorus*. Cell and Tissue Research. 1988;**251**:183-

710. DOI: 10.1007/bf00215463

[20] Alexander S, Irvine CHG. Control of breeding season in the mare and its

10.1007/bf00215166

Fechner J. Influence of photoperiod on the ultrastructure of the hypophyseal pars tuberalis of the Djungarian hamster, *Phodopus sungorus*. Cell and Tissue Research. 1984;**238**:213. DOI:

[15] Dufourny L, Gennetay D, Martinet S, Lomet D, Caraty A. The contents of thyroid hormone receptor alpha in ewe kisspeptin neurons is not season-dependent. Journal of Neuroendocrinology. 2015;**28**:12344.

2011;**23**:871-228. DOI:

DOI: 10.1111/jne.12344

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare DOI: http://dx.doi.org/10.5772/intechopen.92999*

[14] Li Q, Rao A, Pereira A, Clarke IJ, Smith JT. Kisspeptin cells in the ovine arcuate nucleus express prolactin receptor but not melatonin receptor. Journal of Neuroendocrinology. 2011;**23**:871-228. DOI: 10.1111/j.1365-2826.0211.02195.x

[15] Dufourny L, Gennetay D, Martinet S, Lomet D, Caraty A. The contents of thyroid hormone receptor alpha in ewe kisspeptin neurons is not season-dependent. Journal of Neuroendocrinology. 2015;**28**:12344. DOI: 10.1111/jne.12344

[16] Klosen P, Bienvenu C, Demarteau O, Dardente H, Guerrero H, Pevet P, et al. The mt1 melatonin receptor and RORbeta receptor are co-localized in specific TSH-immunoreactive cells in the pars tuberalis of the rat pituitary. The Journal of Histochemistry and Cytochemistry. 2002;**50**:1647-5710. DOI: 10.1177/002215540205001209

[17] Dardente H, Klosen P, Pevet P, Masson-Pevet M. MT1 melatonin receptor mRNA expressing cells in the pars tuberalis of the European hamster: Effect of photoperiod. Journal of Neuroendocrinology. 2003;**15**:778-861. DOI: 10.1046/j.1365-2826.2003.01060.x

[18] Wittkowski W, Hewing M, Hoffmann K, Bergmann M, Fechner J. Influence of photoperiod on the ultrastructure of the hypophyseal pars tuberalis of the Djungarian hamster, *Phodopus sungorus*. Cell and Tissue Research. 1984;**238**:213. DOI: 10.1007/bf00215166

[19] Wittkowski W, Bergmann M, Hoffmann K, Pera F. Photoperioddependent changes in TSH-like immunoreactivity of cells in the hypophyseal pars tuberalis of the Djungarian hamster, *Phodopus sungorus*. Cell and Tissue Research. 1988;**251**:183- 710. DOI: 10.1007/bf00215463

[20] Alexander S, Irvine CHG. Control of breeding season in the mare and its

artificial regulation by progesterone treatment. Journal of Reproduction and Fertility. Supplement. 1991;**44**:307-318

[21] Watson E, Heald M, Tsigos A, Leask R, Steele M, Groome N, et al. Plasma FSH, inhibin a and inhibin isoforms containing pro-and -αC during winter anoestrus, spring transition and the breeding season in mares. Reproduction. 2002;**123**:535-542. DOI: 10.1530/rep.0.1230535

[22] Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA. Regulation of Kiss1 gene expression in the brain of the female mouse. Endocine. 2005;**146**:36-3692. DOI: 10.1210/en.2005-0488

[23] Murphy BA, Walsh CM, Woodward EM, Prendergast RL, Ryle JP, Fallon LH, et al. Blue light from individual light mask directed at a single eye advances the breeding season in mares. Equine Veterinary Journal. 2013;**46**(5):601-605. DOI: 10.111/evj.12153

[24] Lockley SW, Brainard GC, Czeisler CA. High sensitivity of the human circadian melatonin rhythm to resetting by short wavelength light. The Journal of Clinical Endocrinology and Metabolism. 2003;**88**:4502-4505. DOI: 10.1210/jc.2003-030570

[25] Mari G, Morganti M, Merlo B, Castagnetti C, Parameggiani F, Govoni N, et al. Administration of sulpiride or domperidone for advancing the first ovulation in deep anestrous mares. Theriogenology. 2009;**71**(6):959-965. DOI: 10.1016/j. theriogenology.2008.11.001

[26] Staempfli S, Clavier S, Thompson D, Burns P, Lyle S, McKinnon AO. Effect of a single injection of long-acting progesterone on the first ovulation in early and late spring transitional mares. Journal of Equine Veterinary Science. 2011;**31**(12):744-748. DOI: 10.1016/j. jevs.2011.06.016

**66**

*Equine Science*

**References**

[1] Provencio I, Rodriquez IR, Jiang G, Hayes WP, Moreira EF, Rollag MD. A novel human opsin in the inner retina. The Journal of Neuroscience.

acid expression in the hypothalamus of the ewe is regulated by sex steroids and season. Endocrine. 2007;**148**:1150-1157.

[8] Chalivoix S, Bagnolini A, Caraty A, Cognie J, Malpaux B, Dufourny L. Effects of photoperiod on kisspeptin neuronal populations of the ewe diencephalon in connection with reproductive function. Journal of Neuroendocrinology. 2010;**22**:110-118. DOI: 10.1111/j.1365-2826.2009.01939.x

[9] Goodman RL, Maltby MJ, Millar RP, Hileman SM, Nestor CC, Whited B, et al. Evidence that dopamine acts via kisspeptin to hold GnRH pulse frequency in check in anestrous ewes. Endocrine. 2012;**153**:5918-5927

Amstalden M. Nutritional programming

[10] Cardoso RC, Alves BRC, Sharpton SM, Williams GL,

657. DOI: 10.111/jne.12291

Sturt University; 2017

en.2012-1357

[13] Goodman RL, Jansen HT,

Billings HJ, Coolen LM, Lehman MN. Neural systems mediating seasonal breeding in the ewe. Journal of

Neuroendocrinology. 2010;**22**:674-681. DOI: 10.1111/j.1365-2826.210.02014.x

of accelerated puberty in heifers; involvement of pro-opiomelanocortin neurons in the arcuate nucleus. Journal of Neuroendocrinology. 2015;**27**:647-

[11] Rose JL. The Role of RFRP-3 and Kisspeptin on GnRH Secretion in the Merino Ram (Thesis). Australia: Charles

[12] Merkey CM, Porter KL, Coolen LM, Hileman SM, Billings HJ, Drews S, et al. KNDy (Kisspeptin/Neurokinin B/Dynorphin) neurons are activated during both pulsatile and surge secretion of LH in the ewe. Endocrine. 2012;**153**:5406-5414. DOI: 10.1210/

DOI: 10.1210/en.2006-1435

0270-6474/00/200600-06\$15.00/0

Lupi D, Munoz M, David-Gray ZK,

photoreceptive inputs to the mammalian circadian system using transgenic and retinally degenerate mice. Behavioural Brain Research. 2001;**125**:97-102. DOI: 10.1016/50166-4328(01)00274-1

2002;**20**:600-605. DOI:

[2] Lucas RJ, Freedman MS,

Foster RG. Identifying the

[3] Scott CJ, Rose JL, Allan JG, McGrath BM. Kisspeptin and the regulation of the reproductive axis in domestic animals. The Journal of Endocrinology. 2019;**240**(1):R1-R16.

DOI: 10.1530/JOE-18-0485

Loy J, Norman ST. Kisspeptin stimulates LH secretion but not ovulation in mares during vernal transition. Theriogenology.

theriogenology.2016.05.016

[4] McGrath BM, Scott CJ, Wynn PC,

2016;**86**(6):1566-1572. DOI: 10.1016/j.

[5] Decourt C, Tilley Y, Franceschini I, Briant C. Kisspeptin immunoreactive neurons in the equine hypothalamus: Interactions with GnRH neuronal system. Journal of Chemical

Neuroanatomy. 2008;**36**:131-137. DOI: 10.1016/j.chemneu.2008.07.008

[6] Lehman MN, Robinson JE, Karsch FJ, Silverman AJ. Immunocytochemical localization of luteinizing hormonereleasing hormone (LHRH) pathways in the sheep brain during anestrus and mid-luteal phase of the estrous cycle. The Journal of Comparative Neurology.

1986;**244**:19-35. DOI: 10.1002/

[7] Smith JT, Clay CM, Caraty A, Clarke IJ. KiSS-1 messenger ribonucleic

cne.902440103

[27] van Niekerk CH, Coubrough RI, Doms HW. Progesterone treatment of mares with abnormal oestrous cycles early in the breeding season. Journal of the South African Veterinary Association. 1973;**44**:37-45

[28] Squires EL, Stevens WB, McGlothlin DE, Pickett BW. Effect of an oral progestin on the estrous cycle and fertility of mares. Journal of Animal Science. 1979;**49**:729. DOI: 10.2527/ jas/979.493729x

[29] Liu IK, Troedsson MH. The diagnosis and treatment of endometritis in the mare: Yesterday and today. Theriogenology. 2008;**70**:415-420. DOI: 10.1016.j.theriogenology.2008.05.040

[30] Traub-Dargatz JL, Salman MD, Voss JL. Medical problems of adult horses, as ranked by equine practitioners. Journal of the American Veterinary Medical Association. 1991;**198**:1745-1447

[31] Terttu K. Effect of the inseminate and the site of insemination on the uterus and pregnancy rates of mares. Animal Reproduction Science. 2005;**89**(1-4):31-38

[32] Kotilainen T, Hutinen M, Katila T. Sperm induced leucocytosis in the equine uterus. Theriogenology. 1994;**41**:629-636. DOI: 10.1016/0093-691x(94)90173-g

[33] Katila T. Onset and duration of uterine inflammatory response of mares after insemination with fresh semen. In: Biol Reprod Mono. 1. Equine Reproduction VI. Ann Abor, MI: Edward Brothers Inc; 1995. pp. 515-517

[34] Troedsson MHT, de Moraes MJ, Liu IKM. Correlation between histologic endometrial lesions in mares and clinical response to intrauterine exposure to streptococcus zooepidemicus. American Journal of Veterinary Research. 1993;**54**:570-572

[35] Troedsson MH, Liu IK, Ing M, Pascoe J, Thurmond M. Multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible and resistant to chronic uterine infection. Journal of Reproduction and Fertility. 1993;**99**:307-313. DOI: 10.1530/ jrf.0.0990307

[36] Zent WW, Troedsson MHT. Post breeding uterine fluid accumulation in a normal population of thoroughbred mares: A field study. Proceedings of the American Association of Equine Practitioners. 1998;**44**:64-65

[37] Malschitzky E, Schilela A, Mattos ALG, Garbade P, Gregory RM, Mattos RC. Intrauterine fluid accumulation during foal heat increases embryonic death. Pferdeheilkunde. 2003;**19**:246-249

[38] Troedsson MH. Uterine response to semen deposition in the mare. In: Proceedings: The Annual Meeting of the Society for Theriogenology. 1995. pp. 130-135

[39] Malschitzky E, Fiala S, Esmeraldino AT, Neves AP, Garbade P, Jobim MIM, et al. Persistent matinginduced endometritis susceptibility: The role of uterine secretion. Pferdeheilkunde. 2008;**24**:74-78

[40] Fumuso E, Giguere S, Wade R, Rogan D, Videla-Dorna I, Bowden RA. Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post mating induced endometritis. Effects of estrous cycle, artificial insemination and immunomodulation. Veterinary Immunology and Immunopathology. 2003;**96**:31-41. DOI: 10.1016/ s0165-2427(03)00137-5

[41] Fumuso E, Aguilar J, Giguere S, David O, Wade J, Rogan D. Interleukin-8 (IL-8) and 10 (IL-10) mRNA transcriptions in the endometrium of

**69**

*Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare*

Journal of the American Veterinary Medical Association. 2013;**242**(7):977- 983. DOI: 10.2460/javma.242.7.977

[48] Scott C. A review of fungal endometritis in the mare. Equine Veterinary Education. 2018. DOI:

[49] McKinnon AO, Squires EL, Vaala EV, et al. Equine Reproduction. Hoboken: John Wiley & Sons; 2011

[50] Scoggins CF. Endometritis: Nontraditional therapies. The Veterinary Clinics of North America. Equine Practice. 2016;**32**:499-511. DOI: 10.1016/j.cveq.2016.08.002

[51] Swift L, Christensen B, Samocha M,

[52] Bucca S, Carli A, Buckley T, Dolci G, Fogarty U. The use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced endometritis. Theriogenology. 2008;**70**(7):1093-1100. DOI: 10.1016/j.

theriogenology.2008.06.029

[53] Samper JC. How to interpret endometrial edema in brood mares. Proceeding of the American Association of Equine Practitioners.

[54] Dunn P, App M, Rogers D, et al. Transcutaneous electrical nerve stimulation at acupuncture points in the induction of uterine contractions.

Obstetrics and Gynecology.

[55] Loncar K, Ferris R, McCue P, Borlee G, Hennet M, Borlee B. In vitro biofilm disruption and bacterial

 le Jeune S, Millares-Ramirez E, Dujovne G. Randomized comparative trial of acupuncture and exercise versus uterine Ecbolics in the treatment of persistent Postbreeding Endometritis in mares. Journal of Equine Veterinary Science. 2020;**86**:102821. DOI: 10.1016/j.

jevs.2019.102821

2007;**53**:571-572

1989;**73**:286-290

10.1111/eve.13010

*DOI: http://dx.doi.org/10.5772/intechopen.92999*

normal mares and mares susceptible to persistent post-breeding endometritis.

[42] Rigby S, Barhoumi R, Burghardt R, Colleran P, Thompson J, Varner D, et al. Mares with delayed uterine clearance have an intrinsic defect in Myometrial function. Biology of Reproduction. 2001;**65**(3):740-747. DOI: 10.1095/

Animal Reproduction Science. 2006;**94**:282-285. DOI: 10.1016/

jvetimm.2007.04.009

biolreprod65.3.740

[43] Alghamdi AS, Foster DN, Carlson CS, Troedsson MH. Nitric oxide levels and nitric oxide synthase expression in the uterine samples from mares susceptible and resistance to persistent breeding-induced endometritis. American Journal of Reproductive Immunology. 2005;**53**:230-237. DOI: 10.1111/j.1600-0897.2005.00270.x

[44] LeBlanc MM, Causey RC. Clinical

[45] Frontoso R, De Carlo E, Pasolini M, van der Meulen K, Pagnini U, Iovane G, et al. Retrospective study of bacterial isolates and their antimicrobial susceptibilities in equine uteri during fertility problems. Research in Veterinary Science. 2008;**84**(1):1-6. DOI: 10.016/j.rvsc.2007.02.008

and subclinical endometritis in the mares: Both threats to fertility. Reproduction in Domestic Animals. 2009;**3**:10-22. DOI: 10.1111/j.1439-0531.2009.01485.x

[46] Riddle W, LeBlanc M,

practice. Theriogenology.

theriogenology.2007.05.050

Stromberg A. Relationships between uterine culture, cytology and pregnancy rates in a thoroughbred

2007;**68**(3):395-402. DOI: 10.1016/j.

[47] Davis H, Stanton M, Thungrat K, Boothe D. Uterine bacterial isolates from mares and their resistance to antimicrobials: 8,296 cases (2003-2008). *Equine Reproduction: Seasonality, Endometritis, and Twinning in the Mare DOI: http://dx.doi.org/10.5772/intechopen.92999*

normal mares and mares susceptible to persistent post-breeding endometritis. Animal Reproduction Science. 2006;**94**:282-285. DOI: 10.1016/ jvetimm.2007.04.009

*Equine Science*

[27] van Niekerk CH, Coubrough RI, Doms HW. Progesterone treatment of mares with abnormal oestrous cycles early in the breeding season. Journal of the South African Veterinary [35] Troedsson MH, Liu IK, Ing M, Pascoe J, Thurmond M. Multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible

and resistant to chronic uterine

jrf.0.0990307

pp. 130-135

infection. Journal of Reproduction and Fertility. 1993;**99**:307-313. DOI: 10.1530/

[36] Zent WW, Troedsson MHT. Post breeding uterine fluid accumulation in a normal population of thoroughbred mares: A field study. Proceedings of the American Association of Equine

Practitioners. 1998;**44**:64-65

[37] Malschitzky E, Schilela A, Mattos ALG, Garbade P,

Gregory RM, Mattos RC. Intrauterine fluid accumulation during foal heat increases embryonic death. Pferdeheilkunde. 2003;**19**:246-249

[38] Troedsson MH. Uterine response to semen deposition in the mare. In: Proceedings: The Annual Meeting of the Society for Theriogenology. 1995.

Esmeraldino AT, Neves AP, Garbade P, Jobim MIM, et al. Persistent matinginduced endometritis susceptibility: The role of uterine secretion. Pferdeheilkunde. 2008;**24**:74-78

[39] Malschitzky E, Fiala S,

[40] Fumuso E, Giguere S,

2003;**96**:31-41. DOI: 10.1016/ s0165-2427(03)00137-5

(IL-8) and 10 (IL-10) mRNA

[41] Fumuso E, Aguilar J, Giguere S, David O, Wade J, Rogan D. Interleukin-8

transcriptions in the endometrium of

Wade R, Rogan D, Videla-Dorna I, Bowden RA. Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post mating induced endometritis. Effects of estrous cycle, artificial insemination and immunomodulation. Veterinary Immunology and Immunopathology.

Association. 1973;**44**:37-45

[28] Squires EL, Stevens WB,

[29] Liu IK, Troedsson MH. The

in the mare: Yesterday and today. Theriogenology. 2008;**70**:415-420. DOI: 10.1016.j.theriogenology.2008.05.040

[30] Traub-Dargatz JL, Salman MD, Voss JL. Medical problems of adult horses, as ranked by equine practitioners. Journal of the American Veterinary Medical Association.

[31] Terttu K. Effect of the inseminate and the site of insemination on the uterus and pregnancy rates of mares. Animal Reproduction Science.

Katila T. Sperm induced leucocytosis in the equine uterus. Theriogenology.

diagnosis and treatment of endometritis

jas/979.493729x

1991;**198**:1745-1447

2005;**89**(1-4):31-38

1994;**41**:629-636. DOI:

[32] Kotilainen T, Hutinen M,

10.1016/0093-691x(94)90173-g

[33] Katila T. Onset and duration of uterine inflammatory response of mares after insemination with fresh semen. In: Biol Reprod Mono. 1. Equine Reproduction VI. Ann Abor, MI: Edward Brothers Inc; 1995. pp. 515-517

[34] Troedsson MHT, de Moraes MJ, Liu IKM. Correlation between histologic endometrial lesions in mares and clinical response to intrauterine exposure to streptococcus

zooepidemicus. American Journal of Veterinary Research. 1993;**54**:570-572

McGlothlin DE, Pickett BW. Effect of an oral progestin on the estrous cycle and fertility of mares. Journal of Animal Science. 1979;**49**:729. DOI: 10.2527/

**68**

[42] Rigby S, Barhoumi R, Burghardt R, Colleran P, Thompson J, Varner D, et al. Mares with delayed uterine clearance have an intrinsic defect in Myometrial function. Biology of Reproduction. 2001;**65**(3):740-747. DOI: 10.1095/ biolreprod65.3.740

[43] Alghamdi AS, Foster DN, Carlson CS, Troedsson MH. Nitric oxide levels and nitric oxide synthase expression in the uterine samples from mares susceptible and resistance to persistent breeding-induced endometritis. American Journal of Reproductive Immunology. 2005;**53**:230-237. DOI: 10.1111/j.1600-0897.2005.00270.x

[44] LeBlanc MM, Causey RC. Clinical and subclinical endometritis in the mares: Both threats to fertility. Reproduction in Domestic Animals. 2009;**3**:10-22. DOI: 10.1111/j.1439-0531.2009.01485.x

[45] Frontoso R, De Carlo E, Pasolini M, van der Meulen K, Pagnini U, Iovane G, et al. Retrospective study of bacterial isolates and their antimicrobial susceptibilities in equine uteri during fertility problems. Research in Veterinary Science. 2008;**84**(1):1-6. DOI: 10.016/j.rvsc.2007.02.008

[46] Riddle W, LeBlanc M, Stromberg A. Relationships between uterine culture, cytology and pregnancy rates in a thoroughbred practice. Theriogenology. 2007;**68**(3):395-402. DOI: 10.1016/j. theriogenology.2007.05.050

[47] Davis H, Stanton M, Thungrat K, Boothe D. Uterine bacterial isolates from mares and their resistance to antimicrobials: 8,296 cases (2003-2008). Journal of the American Veterinary Medical Association. 2013;**242**(7):977- 983. DOI: 10.2460/javma.242.7.977

[48] Scott C. A review of fungal endometritis in the mare. Equine Veterinary Education. 2018. DOI: 10.1111/eve.13010

[49] McKinnon AO, Squires EL, Vaala EV, et al. Equine Reproduction. Hoboken: John Wiley & Sons; 2011

[50] Scoggins CF. Endometritis: Nontraditional therapies. The Veterinary Clinics of North America. Equine Practice. 2016;**32**:499-511. DOI: 10.1016/j.cveq.2016.08.002

[51] Swift L, Christensen B, Samocha M, le Jeune S, Millares-Ramirez E, Dujovne G. Randomized comparative trial of acupuncture and exercise versus uterine Ecbolics in the treatment of persistent Postbreeding Endometritis in mares. Journal of Equine Veterinary Science. 2020;**86**:102821. DOI: 10.1016/j. jevs.2019.102821

[52] Bucca S, Carli A, Buckley T, Dolci G, Fogarty U. The use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced endometritis. Theriogenology. 2008;**70**(7):1093-1100. DOI: 10.1016/j. theriogenology.2008.06.029

[53] Samper JC. How to interpret endometrial edema in brood mares. Proceeding of the American Association of Equine Practitioners. 2007;**53**:571-572

[54] Dunn P, App M, Rogers D, et al. Transcutaneous electrical nerve stimulation at acupuncture points in the induction of uterine contractions. Obstetrics and Gynecology. 1989;**73**:286-290

[55] Loncar K, Ferris R, McCue P, Borlee G, Hennet M, Borlee B. In vitro biofilm disruption and bacterial

killing using nonantibiotic compounds against gram-negative equine uterine pathogens. Journal of Equine Veterinary Science. 2017;**53**:94-99

[56] Jeffcott LB, Whitwell KW. Twinning as a cause of neonatal loss in the thoroughbred. Journal of Comparative Pathology. 1973;**38**:91-105. DOI: 10.1016/j.jevs.2017.02.003

[57] Giles R, Donahue J, Hong C, et al. Causes of abortion, stillbirth, and prenatal death in horses. Journal of the American Veterinary Medical Association. 1993;**8**:1170-1175

[58] Roberts C. Termination of twin gestation by blastocyst crush in the broodmare. Journal of Reproduction and Fertility. Supplement. 1982;**32**:447-449

[59] Ginther OJ. Twins: Origin and development. In: Ultrasonic Imaging and Animal Reproduction. Crossplains: Equiservices; 1995. pp. 249-306

[60] Deskur S. Twinning in thoroughbred mares in Poland. Theriogenology. 1985;**23**(5):711-718. DOI: 10.1016/0093-691X(85)90146-3

[61] Pascoe RR, Pascoe DR, Wilson MC. Influence of follicular status on twinning rate in mares. Journal of Reproduction and Fertility. Supplement. 1987;**35**:183-189

[62] Ginther O. Twin embryos in mares I: From ovulation to fixation. Equine Veterinary Journal. 1989;**21**(3):166- 170. DOI: 10.1111/j.2042-3306.1989. tb02132.x

[63] Ginther O. Twin embryos in mares II: Post fixation embryo reduction. Equine Veterinary Journal. 1989;**21**(3):171-174. DOI: 10.1111/j.2042- 3306.1989.tb02134.x

[64] Schramme-Josson A. Diagnosis and management of twinning in mares. Practice. 2009;**31**(5):226-231. DOI: 10.1136/inpract.31.5.26

[65] Merkt H, Jungnickel S, Klug E. Reduction of early twin pregnancy to a single pregnancy in the mare by dietetic means. Journal of Reproduction and Fertility. Supplement. 1982;**32**:451-452

**71**

**Chapter 5**

**Abstract**

horses/equids to the other.

**1. Introduction**

equine, sacroiliac joint, interosseous, kinematics

Investigation into Whether

Horses to Sacroiliac Disease

*Anne Skivington, Milomir Kovac, Elena Zakirova,* 

*Albert A. Rizvanov and Catrin Sian Rutland*

Proximal Suspensory Desmitis of

Proximal suspensory desmopathy/desmitis (PSD) of the hindlimb is a well understood condition with widely accepted treatment protocols; however, there is little research demonstrating understanding or potential correlation between hindlimb PSD and sacroiliac disease (SID). Several studies have examined the co-existence of hindlimb PSD and SID each investigating unique predisposing factors. This has led to little direct correlation of cause and effect with no definitive conclusions drawn. The need to be objective is highlighted by the limited number of studies and that two studies used anecdotal evidence to support their hypothesis and thus creating the question does hindlimb proximal suspensory desmopathy predispose horses to sacroiliac disease? This review looks at the two conditions and compares the literature for each, including the incidence, biomechanics, anatomy, and treatment. The review further discusses whether one disorder predisposes

**Keywords:** hindlimb proximal suspensory desmitis, sacroiliac disease, lameness,

background and present theories behind hindlimb PSD and SID.

The objective of this review was to assess whether there is a correlation between hindlimb proximal suspensory ligament desmopathy (hindlimb PSD) and sacroiliac dysfunction (SID), and provide an understanding of the current thought process of examining these disorders. There are several studies examining the coexistence of back pain and poor performance, however for the most part, the discussion focusses on the efficacy of diagnostic techniques of the thoracolumbar region with some recognition of influencing factors [1–3]. Some authors have assumed a correlation between the two disorders in their treatment programmes [4, 5] but none quantified the association or correlation of the two conditions. There are limited studies that have looked at the structure of the sacroiliac region and applied those principles to locomotion [2] however there are many text books that describe the structure alone [6, 7]. This chapter explores the two conditions and explores the

the Hindlimb Could Predispose

#### **Chapter 5**

*Equine Science*

Science. 2017;**53**:94-99

killing using nonantibiotic compounds against gram-negative equine uterine pathogens. Journal of Equine Veterinary Practice. 2009;**31**(5):226-231. DOI:

[65] Merkt H, Jungnickel S, Klug E. Reduction of early twin pregnancy to a single pregnancy in the mare by dietetic means. Journal of Reproduction and Fertility. Supplement. 1982;**32**:451-452

10.1136/inpract.31.5.26

[56] Jeffcott LB, Whitwell KW. Twinning

as a cause of neonatal loss in the thoroughbred. Journal of Comparative Pathology. 1973;**38**:91-105. DOI: 10.1016/j.jevs.2017.02.003

[57] Giles R, Donahue J, Hong C, et al. Causes of abortion, stillbirth, and prenatal death in horses. Journal of the American Veterinary Medical Association. 1993;**8**:1170-1175

[58] Roberts C. Termination of twin gestation by blastocyst crush in the broodmare. Journal of Reproduction

[59] Ginther OJ. Twins: Origin and development. In: Ultrasonic Imaging and Animal Reproduction. Crossplains:

Equiservices; 1995. pp. 249-306

[60] Deskur S. Twinning in thoroughbred mares in Poland. Theriogenology. 1985;**23**(5):711-718. DOI: 10.1016/0093-691X(85)90146-3

[61] Pascoe RR, Pascoe DR,

1987;**35**:183-189

tb02132.x

Wilson MC. Influence of follicular status on twinning rate in mares. Journal of Reproduction and Fertility. Supplement.

[62] Ginther O. Twin embryos in mares I: From ovulation to fixation. Equine Veterinary Journal. 1989;**21**(3):166- 170. DOI: 10.1111/j.2042-3306.1989.

[63] Ginther O. Twin embryos in mares II: Post fixation embryo reduction. Equine Veterinary Journal. 1989;**21**(3):171-174. DOI: 10.1111/j.2042-

[64] Schramme-Josson A. Diagnosis and management of twinning in mares.

3306.1989.tb02134.x

and Fertility. Supplement.

1982;**32**:447-449

**70**

## Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose Horses to Sacroiliac Disease

*Anne Skivington, Milomir Kovac, Elena Zakirova, Albert A. Rizvanov and Catrin Sian Rutland*

#### **Abstract**

Proximal suspensory desmopathy/desmitis (PSD) of the hindlimb is a well understood condition with widely accepted treatment protocols; however, there is little research demonstrating understanding or potential correlation between hindlimb PSD and sacroiliac disease (SID). Several studies have examined the co-existence of hindlimb PSD and SID each investigating unique predisposing factors. This has led to little direct correlation of cause and effect with no definitive conclusions drawn. The need to be objective is highlighted by the limited number of studies and that two studies used anecdotal evidence to support their hypothesis and thus creating the question does hindlimb proximal suspensory desmopathy predispose horses to sacroiliac disease? This review looks at the two conditions and compares the literature for each, including the incidence, biomechanics, anatomy, and treatment. The review further discusses whether one disorder predisposes horses/equids to the other.

**Keywords:** hindlimb proximal suspensory desmitis, sacroiliac disease, lameness, equine, sacroiliac joint, interosseous, kinematics

#### **1. Introduction**

The objective of this review was to assess whether there is a correlation between hindlimb proximal suspensory ligament desmopathy (hindlimb PSD) and sacroiliac dysfunction (SID), and provide an understanding of the current thought process of examining these disorders. There are several studies examining the coexistence of back pain and poor performance, however for the most part, the discussion focusses on the efficacy of diagnostic techniques of the thoracolumbar region with some recognition of influencing factors [1–3]. Some authors have assumed a correlation between the two disorders in their treatment programmes [4, 5] but none quantified the association or correlation of the two conditions. There are limited studies that have looked at the structure of the sacroiliac region and applied those principles to locomotion [2] however there are many text books that describe the structure alone [6, 7]. This chapter explores the two conditions and explores the background and present theories behind hindlimb PSD and SID.

#### **1.1 Sacroiliac joint structure and function**

The sacroiliac joint lies deep within the pelvis of the horse, made up of the sacrum (five vertebrae fused together) and the surrounding ligaments. It is known as an atypical synovial joint [2] and a cartilaginous joint [7]. The iliac surface has fibrocartilage coverage, with the sacral surface lined with hyaline cartilage, thus creating a modified symphysis [8]. There is great variation in the joint form from L shaped to C shaped either being relatively flat or concaved, although most are at an angle of 30° [2].

The sacroiliac joint lies between the ilium wings, forming a synchondrosis that is held in place by a multitude of ligaments. These ligaments are called the dorsal and ventral sacrosciatic ligaments and the broad sacrotuberous ligament [7]. The dorsal sacrosciatic ligament has two elements, a band that runs from the dorsal tuber sacrale to the apex of the sacral spinous processes; with the lateral dorsal sacrosciatic ligament running from the tuber sacrale and ilial wing to the sacral crest on the lateral aspect. The broad sacrotuberous ligament runs from the sacrum and transverse processes of the 1st and 2nd caudal vertebrae to the ischiatic spine and tuber ischium [2, 7]. The function of this joint is to provide a relatively inelastic structure that is capable of asymmetric pelvic deformation during movement [2, 9]. The muscle structure of the back plays significant influential roles in both anatomy and biomechanics.

The movement of the horses back differs depending on the location and mediolateral swing of body mass; dorsoventral movement is seen with the greatest being middle of the back (40–47 mm per peak per stride) with a reduction cranially and caudally [10–12]. The natural movement of the lumbosacral area and the hindlimb produce a sinusoidal movement of no more than 4° within each stride cycle. Extension within this sinusoidal curve starts just moments before ground contact with the hoof, with the hindlimb at maximal protraction. In the sound horse this means that movement of the sacroiliac joint is minimal as longissimus dorsi is inactive in the impact and support phase of the flight arc of the hoof, in theory resulting in a stable joint [12–14]. Having said that linear regression revealed a significant deviation in movement over Lumbar 1 and Sacral 3 correlated to increasing speed [12]. This indicated that the movement of the back and sacroiliac joint is complex [2] and changes with every change in pace (**Figure 1**) [11].

The movement within the joint is assumed to be little [15] due to the middle gluteal and surrounding ligaments holding it in place. Despite this, a series of studies of the human sacroiliac joint revealed adaptations to forces transmitted through the joint;

#### **Figure 1.**

*Schematic of the right lateral view of the pelvis showing the position of the sacroiliac joint between the wings of the ilium and wing of the sacrum and the sacrotuberous ligament (adapted from [2]).*

**73**

**Figure 2.**

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose…*

which was seen as roughened areas on the contrasting surfaces [16]. Comparable studies of the equine sacrum have looked at nutational forces to determine the degree of movement and suggested there is limited movement [2]. However, another investigation raised the interesting point that when the sacrotuberous ligament was cut there was a marked increase in movement [2]. This would seem obvious, as its function is to reduce movement but does suggest that ligament damage or laxity could cause increased asymmetrical movement which in itself could have an adverse effect on the

The structure of the third interosseous muscle, also known as the suspensory ligament, the middle interosseous muscle or the interosseous ligament, is relatively straight forward. It originates from the proximal palmer surface of the metacarpal bones, running distally where just proximal to the sesamoid bones it bifurcates inserting on to each of the two sesamoid bones. From here it travels as the extensor branch joining the common digital extensor tendon. Even though it is termed a muscle, it is believed that once the horse matures it becomes completely collagenous in nature [7]. However, this is an over simplification as others describe the ligament as having a reduction of muscle fibres [17], while still retaining some which reduce with increased age [18, 19]. Muscle fibres quantitation showed a difference of 40% between the Thoroughbreds and Standardbreds with the Thoroughbred having less muscle fibres than its counterpart, with more muscle content being found in the hindlimb suspensory ligament than the forelimb [20]. It was also noted that the proximal region of the suspensory ligament contained less muscular tissue [19, 21]. This work also showed that the number of muscle fibres reduced with increased work intensity, thus suggesting that the suspensory ligament becomes less elastic

*DOI: http://dx.doi.org/10.5772/intechopen.92353*

soft tissue structures of the distal limb.

**1.2 The interosseous muscle structure and function**

and more susceptible to strain with increased work load (**Figure 2**).

The composition of the interosseous muscle is something of a hybrid, with the majority being collagen fibres but approximately 10% being type I muscle fibres and less than 5% type II muscle fibres. The suspensory ligament is defined by the infrequent fibroblasts embedded in the collagen matrix. These fibres are dispersed differently throughout the length of the ligament. Proximally, they are grouped as loose fascicles medially and laterally with the greater concentration just below the

*Schematic left lateral view showing the interosseous ligament of the hindlimb (adapted from Budras et al. [6]).*

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose… DOI: http://dx.doi.org/10.5772/intechopen.92353*

which was seen as roughened areas on the contrasting surfaces [16]. Comparable studies of the equine sacrum have looked at nutational forces to determine the degree of movement and suggested there is limited movement [2]. However, another investigation raised the interesting point that when the sacrotuberous ligament was cut there was a marked increase in movement [2]. This would seem obvious, as its function is to reduce movement but does suggest that ligament damage or laxity could cause increased asymmetrical movement which in itself could have an adverse effect on the soft tissue structures of the distal limb.

#### **1.2 The interosseous muscle structure and function**

The structure of the third interosseous muscle, also known as the suspensory ligament, the middle interosseous muscle or the interosseous ligament, is relatively straight forward. It originates from the proximal palmer surface of the metacarpal bones, running distally where just proximal to the sesamoid bones it bifurcates inserting on to each of the two sesamoid bones. From here it travels as the extensor branch joining the common digital extensor tendon. Even though it is termed a muscle, it is believed that once the horse matures it becomes completely collagenous in nature [7]. However, this is an over simplification as others describe the ligament as having a reduction of muscle fibres [17], while still retaining some which reduce with increased age [18, 19]. Muscle fibres quantitation showed a difference of 40% between the Thoroughbreds and Standardbreds with the Thoroughbred having less muscle fibres than its counterpart, with more muscle content being found in the hindlimb suspensory ligament than the forelimb [20]. It was also noted that the proximal region of the suspensory ligament contained less muscular tissue [19, 21]. This work also showed that the number of muscle fibres reduced with increased work intensity, thus suggesting that the suspensory ligament becomes less elastic and more susceptible to strain with increased work load (**Figure 2**).

The composition of the interosseous muscle is something of a hybrid, with the majority being collagen fibres but approximately 10% being type I muscle fibres and less than 5% type II muscle fibres. The suspensory ligament is defined by the infrequent fibroblasts embedded in the collagen matrix. These fibres are dispersed differently throughout the length of the ligament. Proximally, they are grouped as loose fascicles medially and laterally with the greater concentration just below the

*Equine Science*

angle of 30° [2].

and biomechanics.

**1.1 Sacroiliac joint structure and function**

changes with every change in pace (**Figure 1**) [11].

The sacroiliac joint lies deep within the pelvis of the horse, made up of the sacrum (five vertebrae fused together) and the surrounding ligaments. It is known as an atypical synovial joint [2] and a cartilaginous joint [7]. The iliac surface has fibrocartilage coverage, with the sacral surface lined with hyaline cartilage, thus creating a modified symphysis [8]. There is great variation in the joint form from L shaped to C shaped either being relatively flat or concaved, although most are at an

The sacroiliac joint lies between the ilium wings, forming a synchondrosis that is held in place by a multitude of ligaments. These ligaments are called the dorsal and ventral sacrosciatic ligaments and the broad sacrotuberous ligament [7]. The dorsal sacrosciatic ligament has two elements, a band that runs from the dorsal tuber sacrale to the apex of the sacral spinous processes; with the lateral dorsal sacrosciatic ligament running from the tuber sacrale and ilial wing to the sacral crest on the lateral aspect. The broad sacrotuberous ligament runs from the sacrum and transverse processes of the 1st and 2nd caudal vertebrae to the ischiatic spine and tuber ischium [2, 7]. The function of this joint is to provide a relatively inelastic structure that is capable of asymmetric pelvic deformation during movement [2, 9]. The muscle structure of the back plays significant influential roles in both anatomy

The movement of the horses back differs depending on the location and mediolateral swing of body mass; dorsoventral movement is seen with the greatest being middle of the back (40–47 mm per peak per stride) with a reduction cranially and caudally [10–12]. The natural movement of the lumbosacral area and the hindlimb produce a sinusoidal movement of no more than 4° within each stride cycle. Extension within this sinusoidal curve starts just moments before ground contact with the hoof, with the hindlimb at maximal protraction. In the sound horse this means that movement of the sacroiliac joint is minimal as longissimus dorsi is inactive in the impact and support phase of the flight arc of the hoof, in theory resulting in a stable joint [12–14]. Having said that linear regression revealed a significant deviation in movement over Lumbar 1 and Sacral 3 correlated to increasing speed [12]. This indicated that the movement of the back and sacroiliac joint is complex [2] and

The movement within the joint is assumed to be little [15] due to the middle gluteal and surrounding ligaments holding it in place. Despite this, a series of studies of the human sacroiliac joint revealed adaptations to forces transmitted through the joint;

*Schematic of the right lateral view of the pelvis showing the position of the sacroiliac joint between the wings of* 

*the ilium and wing of the sacrum and the sacrotuberous ligament (adapted from [2]).*

**72**

**Figure 1.**

surface. As it reaches the three quarter mark they become less distinct, fewer in number with reduced striations. Interestingly these fibres are arranged pinnately between 45 and 80° [17, 22, 23] leading to theories that high forces are created because of the greater pinnate angle in order to stabilise the joint and indications that its purpose is anti-fatigue and postural support [24]. This was supported further by the suggestion that the elasticity of the lower limb, creating a vibration of 30–40 Hz, needs damping to reduce the likelihood of damage to tendons or bones and that this is achieved through these short muscle fibres [25, 26]. Due to the elastic nature of the suspensory ligament, it is unable to cope with sudden surges in force and is not built to deal with increased amounts of fatigue [27, 28]. It has also been noted that as the age of the horse increases so does the stiffness of a tendon unit which in turn could induce a change in kinematics [29].

#### **1.3 Elastic strain energy**

It is commonly understood that tendons and ligaments play an important role in elastic strain energy during locomotion. Humans and ungulates have evolved to have more efficient locomotory systems; with equine evolution determining the distal limb muscle mass would not only be challenging to manoeuvre but very costly in terms of energy expenditure. Thus we see tendons and ligaments in the distal limb as a means of storing elastic energy [25, 28, 30–33]. In order for the horse to utilise this mechanism within the suspensory ligament the energy from the ground reaction force is stored as strain energy to retract the limb [27, 32] helping to produce the break over point [34].

The function of the suspensory ligament is to stabilise the metacarpophalangeal joint and hindlimb in preventing hyper flexion in locomotion but also to act as part of the stay apparatus in preventing collapse of the fetlock joint when immobile [35, 36] effectively acting as passive control [17, 28]. However, the suspensory ligament differs slightly in its role compared to the other tendons of the distal limb. For example, the maximal stress the superficial digital flexor tendon (SDFT) and deep digital flexor tendon (DDFT) functions at is 40–50 MPa (mega-pascal units) compared to the suspensory ligament functioning at 18–25 MPa when in gallop; of course this is maximal output and decreases with decreased speeds. To gain a relative perspective, muscles work at 200–240 MPa. By comparison this seems quite small but provides an elastic energy saving of 25% for the suspensory ligament and 40% for the SDFT and DDFT which translates into an energy saving of 1.23 J/Kg at trot and 6 J/Kg at the gallop [33]; thus reducing metabolic expenditure [25, 31].

Biewener [31] calculated the peak activity stress mean standard deviation on the fore and hindlimb suspensory ligament with 53 ± 14% from walk to trot and 23 ± 19% into gallop. When ground reaction forces are considered and coupled with an increase in pace, the change in stress has an astonishingly small mean of 4%. This could be due to the kinematic calculation methods or potentially due to the biomechanical nature of the suspensory ligament. As the hoof makes contact with the ground, the suspensory ligament briefly stretches as a reaction to the ground reaction force and the sinking action of the metacarpal-phalangeal joint. The ligament then shortens to create an anti-hyperextension force. This elastic strain energy depends greatly upon the tendon shape and volume. These are varied as the suspensory ligament bifurcates distally resulting in a greatly reduced cross sectional area, leaving it under greater stress and strain [25, 31]. The elastic property of the lower limb is also heavily influenced by the individual gait pattern of each horse.

**75**

force [14].

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose…*

In order to understand the process of veterinary examination and its resultant observations; it is imperative to fully understand the kinematics and kinetics of the locomotion of the horse. The structure and function of the cursorial musculoskeletal systems have evolved to provide structures and patterns of movement that favour acceleration, manoeuvrability speed and endurance [30, 37, 38] which has been harnessed over centuries for various disciplines such as racing and dressage. It is also important to note the influence central pattern generators (CPG) and proprioception have on the biomechanics of the horse. The regulated rhythm of a pace is created by the CPG neurons which are capable of generating the stimuli and therefore a rhythmic motor behaviour. Even though some believe that the CPG neurons are capable of producing this regulatory rhythm without stimulus, sensory feedback is still required [39, 40]. Minute differences the timings or intensity of these impulses of the right and left central pattern generators cause asymmetrical movement [41]. Horses that have modified their locomotory movement in an attempt to compensate for discomfort or pain of either hindlimb PSD or SID will in effect cause the CPG neurons to adapt their "pacemaker" like outputs; thus creating

Locomotion occurs as a result of torque at the hip joint [42, 43] and ground reaction forces exerted on the hoof which in gallop can be as much as 2.5 times the horses body weight [44, 45], with equal magnitude working in the opposite direction providing propulsion [46]. Therefore, it is worth considering the kinematic pattern of hoof placement, to determine how the pathology of SID and hindlimb PSD may occur. The structure and function of the cursorial musculoskeletal systems have evolved to provide structures and patterns of movement that favour

The hoof does not hit the ground with a total sole impact, but instead, as a measure of control, impacts the ground with the lateral edge. This reduces the concussive effect of the initial ground contact [47, 48]. It is important to remember that the hoof at ground contact is moving forward and downward during the initial loading phase [38]. The degree of impact when the hoof hits the ground is determined by several factors; the 57:43% split of vertical impulse for fore and hindlimb respectively [23, 38], the hoof mass, size and shape of the hoof, contact surface, type of shoe i.e. racing plate or hunter with or without grips or studs. These all influence the vertical and horizontal hoof velocity, and degree of slip [37, 38, 49]. The degree of lameness also has a large influence on interplay between hoof and ground reaction

Several studies have analysed hoof velocity [38, 44, 50], two of which have considered horizontal hoof velocity of fore and hindlimbs; one demonstrating the greatest being in the non-leading limb [49] and other the leading limb [51]. The hoof velocity and leading limb has important implications to the structures in the hindlimbs; if it is the forelimb the majority of the velocity will be absorbed by the thoracic sling, if it is the hindlimb the velocity can only end at the sacroiliac joint, although this is greatly simplified. Having said that, longitudinal velocity reduces (regardless of limb) as the horse starts to break in early stance phase. In this early phase the hindlimb suspensory ligament (third interosseous muscle) is at its peak inertial capacity to prevent hyper extension, while at the same time the pitch avoidance movement of raising the head and neck backwards increases forces on the pelvic limb, as the weight is shifted backwards in the late stance phase. This increases propulsion of the moment arms of the hindlimbs, creating oscillating forces though the hindlimb [28, 52]. These oscillating forces are created with

*DOI: http://dx.doi.org/10.5772/intechopen.92353*

**1.4 Kinematics and kinetics of locomotion**

a new norm for the horses locomotion [38].

acceleration, manoeuvrability speed and endurance [30, 37].

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose… DOI: http://dx.doi.org/10.5772/intechopen.92353*

#### **1.4 Kinematics and kinetics of locomotion**

*Equine Science*

**1.3 Elastic strain energy**

duce the break over point [34].

surface. As it reaches the three quarter mark they become less distinct, fewer in number with reduced striations. Interestingly these fibres are arranged pinnately between 45 and 80° [17, 22, 23] leading to theories that high forces are created because of the greater pinnate angle in order to stabilise the joint and indications that its purpose is anti-fatigue and postural support [24]. This was supported further by the suggestion that the elasticity of the lower limb, creating a vibration of 30–40 Hz, needs damping to reduce the likelihood of damage to tendons or bones and that this is achieved through these short muscle fibres [25, 26]. Due to the elastic nature of the suspensory ligament, it is unable to cope with sudden surges in force and is not built to deal with increased amounts of fatigue [27, 28]. It has also been noted that as the age of the horse increases so does the stiffness of a tendon

It is commonly understood that tendons and ligaments play an important role in elastic strain energy during locomotion. Humans and ungulates have evolved to have more efficient locomotory systems; with equine evolution determining the distal limb muscle mass would not only be challenging to manoeuvre but very costly in terms of energy expenditure. Thus we see tendons and ligaments in the distal limb as a means of storing elastic energy [25, 28, 30–33]. In order for the horse to utilise this mechanism within the suspensory ligament the energy from the ground reaction force is stored as strain energy to retract the limb [27, 32] helping to pro-

The function of the suspensory ligament is to stabilise the metacarpophalangeal joint and hindlimb in preventing hyper flexion in locomotion but also to act as part of the stay apparatus in preventing collapse of the fetlock joint when immobile [35, 36] effectively acting as passive control [17, 28]. However, the suspensory ligament differs slightly in its role compared to the other tendons of the distal limb. For example, the maximal stress the superficial digital flexor tendon (SDFT) and deep digital flexor tendon (DDFT) functions at is 40–50 MPa (mega-pascal units) compared to the suspensory ligament functioning at 18–25 MPa when in gallop; of course this is maximal output and decreases with decreased speeds. To gain a relative perspective, muscles work at 200–240 MPa. By comparison this seems quite small but provides an elastic energy saving of 25% for the suspensory ligament and 40% for the SDFT and DDFT which translates into an energy saving of 1.23 J/Kg at trot and 6 J/Kg at the gallop [33]; thus reducing metabolic expendi-

Biewener [31] calculated the peak activity stress mean standard deviation on the fore and hindlimb suspensory ligament with 53 ± 14% from walk to trot and 23 ± 19% into gallop. When ground reaction forces are considered and coupled with an increase in pace, the change in stress has an astonishingly small mean of 4%. This could be due to the kinematic calculation methods or potentially due to the biomechanical nature of the suspensory ligament. As the hoof makes contact with the ground, the suspensory ligament briefly stretches as a reaction to the ground reaction force and the sinking action of the metacarpal-phalangeal joint. The ligament then shortens to create an anti-hyperextension force. This elastic strain energy depends greatly upon the tendon shape and volume. These are varied as the suspensory ligament bifurcates distally resulting in a greatly reduced cross sectional area, leaving it under greater stress and strain [25, 31]. The elastic property of the lower limb is also heavily influenced by the individual gait pattern of

unit which in turn could induce a change in kinematics [29].

**74**

each horse.

ture [25, 31].

In order to understand the process of veterinary examination and its resultant observations; it is imperative to fully understand the kinematics and kinetics of the locomotion of the horse. The structure and function of the cursorial musculoskeletal systems have evolved to provide structures and patterns of movement that favour acceleration, manoeuvrability speed and endurance [30, 37, 38] which has been harnessed over centuries for various disciplines such as racing and dressage.

It is also important to note the influence central pattern generators (CPG) and proprioception have on the biomechanics of the horse. The regulated rhythm of a pace is created by the CPG neurons which are capable of generating the stimuli and therefore a rhythmic motor behaviour. Even though some believe that the CPG neurons are capable of producing this regulatory rhythm without stimulus, sensory feedback is still required [39, 40]. Minute differences the timings or intensity of these impulses of the right and left central pattern generators cause asymmetrical movement [41]. Horses that have modified their locomotory movement in an attempt to compensate for discomfort or pain of either hindlimb PSD or SID will in effect cause the CPG neurons to adapt their "pacemaker" like outputs; thus creating a new norm for the horses locomotion [38].

Locomotion occurs as a result of torque at the hip joint [42, 43] and ground reaction forces exerted on the hoof which in gallop can be as much as 2.5 times the horses body weight [44, 45], with equal magnitude working in the opposite direction providing propulsion [46]. Therefore, it is worth considering the kinematic pattern of hoof placement, to determine how the pathology of SID and hindlimb PSD may occur. The structure and function of the cursorial musculoskeletal systems have evolved to provide structures and patterns of movement that favour acceleration, manoeuvrability speed and endurance [30, 37].

The hoof does not hit the ground with a total sole impact, but instead, as a measure of control, impacts the ground with the lateral edge. This reduces the concussive effect of the initial ground contact [47, 48]. It is important to remember that the hoof at ground contact is moving forward and downward during the initial loading phase [38]. The degree of impact when the hoof hits the ground is determined by several factors; the 57:43% split of vertical impulse for fore and hindlimb respectively [23, 38], the hoof mass, size and shape of the hoof, contact surface, type of shoe i.e. racing plate or hunter with or without grips or studs. These all influence the vertical and horizontal hoof velocity, and degree of slip [37, 38, 49]. The degree of lameness also has a large influence on interplay between hoof and ground reaction force [14].

Several studies have analysed hoof velocity [38, 44, 50], two of which have considered horizontal hoof velocity of fore and hindlimbs; one demonstrating the greatest being in the non-leading limb [49] and other the leading limb [51]. The hoof velocity and leading limb has important implications to the structures in the hindlimbs; if it is the forelimb the majority of the velocity will be absorbed by the thoracic sling, if it is the hindlimb the velocity can only end at the sacroiliac joint, although this is greatly simplified. Having said that, longitudinal velocity reduces (regardless of limb) as the horse starts to break in early stance phase. In this early phase the hindlimb suspensory ligament (third interosseous muscle) is at its peak inertial capacity to prevent hyper extension, while at the same time the pitch avoidance movement of raising the head and neck backwards increases forces on the pelvic limb, as the weight is shifted backwards in the late stance phase. This increases propulsion of the moment arms of the hindlimbs, creating oscillating forces though the hindlimb [28, 52]. These oscillating forces are created with

hoof-ground impact causing the limb to vibrate in a craniocaudal movement at 30–40 Hz, the greatest impact being distal in the limb. The muscles of the hindlimb act as adequate shock absorbers however risk of soft tissue damage increases with the increase in loading cycles [26]. This suggests that the greater the work load and discipline level of the horse, the more likely they are to sustain an injury. One method of removing force is slipping or sliding. The hoof is designed to allow an element of slip as a natural method of dissipating energy [53] however if sliding continues in the right conditions this can increase the risk of damage to soft tissue structures. Coupled with the ground reaction forces, this means that there are two opposing forces meeting at the horizontal axis, namely the sacroiliac joint [51].

#### **1.5 Conformation of the horse**

There are many variable factors when considering the relationship between hindlimb PSD and SID; one of which is the natural biological variation in every horse, in that no two are exactly the same in conformation which ultimately enhances or impedes function. Discipline desirable traits have been documented for enhancing performance, such as the warmblood breeds for dressage, with greater hock angle reducing the incidences of injury compare to those with smaller hock angles [54, 55]. However, this was refuted in a later study of 66 warmblood horses that had the supposedly undesirable tarsal joint angle of <155.50° [56]. This was agreed with in another study examining the hock angles of 194 Warmblood horses with hindlimb PSD [57]. Hobbs et al. [54] described a selection of horses that had variations between contralateral limbs conformation and those with bone morphology variance in contralateral limbs [58]. The results of these differences may induce compensatory movements in an attempt to redistribute the weight through the stride cycle. In an attempt to counter this, and stabilise the gait, the hindlimbs may start to load in a pattern similar to a lame horse. Having said that this load distribution pattern may come from the horses' handedness. This raises the question, if the horse is not physiologically capable of creating vertical impulsion (due to straight hocks), how and where will this affect the soft tissue structures in the hindlimb?

Asymmetries come in many forms, however each will have a marked effect on the biomechanics of the horse and more importantly the ground reaction forces; in the horses attempt to maintain equilibrium [54]. Of course, this need to maintain stability has different ground reaction forces depending on breed. Elite dressage Lusitano horses had lower vertical impulses compared to their Dutch Warmblood counterparts in collected trot with a range of 1.64 ± 0.02 N/Kg and 1.90 ± 0.08 N/ Kg respectively. However this evened out with a change from collected trot to passage, with minimal difference being seen. Nevertheless, the key point in this is that the centre of mass is moved closer to the hindlimbs in the higher movements. Heim and co-authors [11] demonstrated a significant difference between Franches-Montagnes stallions (n = 27) and a general populous of horses (n = 6) in the dorsoventral movement (p < 0.02) and mediolateral movement (p < 0.01) for the spine, although to say this is a generalisation of differing anatomical parts and their role in locomotion. There is also the influence of the rider to consider here; not only as their body mass is part of the calculation but as the elite rider is capable of re-balancing even the most uneducated of horses to maintain the uphill longitudinal balance that is required of a dressage horse [59]. Dyson and colleagues [60] refuted this in their pilot study of rider weight, in that the weight of the rider had a greater significance than body mass index. Although this situation is not definitive, as there are many influencing factors in this scenario. For example, the balance of the rider and the dynamics between saddle and rider, both of which have a role in distribution of forces. In essence if the rider is displaced by an ill-fitting saddle or the rider

**77**

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose…*

is inexperienced the horse has to re-balance itself in order to compensate [10, 44], which in itself produces compensatory locomotion. Another interesting factor relating to distribution of forces, body movement and rider interaction was demonstrated during the heavy and very heavy rider trials, as the horse demonstrated 3/8 lameness (based on the 0–8 grade lameness scale where 0 is sound and 8 is nonweightbearing) with these heavier riders [60]. The thoracolumbar width changed with weight of rider, from 3.9% with a light rider to 2.8% with a heavy rider. Heim et al. [11] noted that there was less mediolateral movement in the vertebrae when under saddle, with a difference of approximately 10 mm in the 3rd lumbar vertebrae as compared to an 8 mm difference in the movement of the tuber sacrale. This suggested that the horses may be bracing themselves against the movement of the heavier rider. However this was an observation and not a direct conclusion. It was also suggested that the interactive surface between horse and rider, the saddle, if not fitted correctly increased the mediolateral movement of the rider, which led to their conclusion that the closer contact the rider has with the horse the more likely

The conformation of the hoof capsule and the angle of the internal structures have a role to play in suspensory ligament desmopathy and limb kinematics. A significant level of research focusses on the correlation between the navicular bone angle and force applied to the deep digital flexor tendon [44, 61]. Although the research was not directed at the hindlimb suspensory ligament; their findings still shed light on this area due to the anatomical angle of bordering structure and limb kinematics. The shape of the hoof has been reported to change the kinetics and kinematics of the distal limb. Dyson et al. [61] reported that the distal phalanx to hoof wall angle and distal phalanx to horizontal angle were smallest for deep digital flexor tendon injuries at 52.27° ± 3.29 and 50.32° ± 3.70 (mean ± SD) respectively. However, it would seem there was no direct correlation between that and the angles of the hoof wall. Research suggests that optimal hoof angles for both front and back feet should be 50–55° [62]. In addition, minimal correlation between the dorsal aspect of the distal phalanx angle and deep digital flexor tendon injury has been found and the hoof wall angle was not the same as the distal phalanx angle [61],

The deviation of distal phalanx angle affects the orientation of the structures above it and subsequently the metacarpophalangeal joint; which in turn has the potential to cause soft tissue injuries [63, 64]. This is because the ground reaction forces are reduced delaying break-over to latter breaking phase [64] whereas the horse should have increased loading at this point [62, 65]. This has the potential to reduce the strain on the interosseous muscle but could also inhibit the elastic strain

Kane et al. [63] identified 43 race horses with ruptured suspensory ligaments with lower heel and toe angles; for example the difference between the toe heel angle control group and those with suspensory apparatus failure was 1.3° less, a relatively small number in terms of angles but quite significant over the lifetime of a horse. In real terms this means that an increase in angle of 10° increases the chance

Shoeing has been used since domestication of the horse as a means to improve performance and help maintain hoof balance. The combination of farriery techniques like rolled toes, plus different types of shoe have a significant effect on the horse's feet and their movement [34, 45, 66]. It could be assumed that the application of the shoe would only affect the gait pattern of the horse but an 11% vertical

*DOI: http://dx.doi.org/10.5772/intechopen.92353*

they are to be working in equilibrium with them [10].

**1.6 Conformation of the hoof and influence of shoeing**

which could account for natural variation in hoof pastern axis.

energy needed to create its passive force.

of suspensory ligament failure by 6.75 times [63].

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose… DOI: http://dx.doi.org/10.5772/intechopen.92353*

is inexperienced the horse has to re-balance itself in order to compensate [10, 44], which in itself produces compensatory locomotion. Another interesting factor relating to distribution of forces, body movement and rider interaction was demonstrated during the heavy and very heavy rider trials, as the horse demonstrated 3/8 lameness (based on the 0–8 grade lameness scale where 0 is sound and 8 is nonweightbearing) with these heavier riders [60]. The thoracolumbar width changed with weight of rider, from 3.9% with a light rider to 2.8% with a heavy rider. Heim et al. [11] noted that there was less mediolateral movement in the vertebrae when under saddle, with a difference of approximately 10 mm in the 3rd lumbar vertebrae as compared to an 8 mm difference in the movement of the tuber sacrale. This suggested that the horses may be bracing themselves against the movement of the heavier rider. However this was an observation and not a direct conclusion. It was also suggested that the interactive surface between horse and rider, the saddle, if not fitted correctly increased the mediolateral movement of the rider, which led to their conclusion that the closer contact the rider has with the horse the more likely they are to be working in equilibrium with them [10].

#### **1.6 Conformation of the hoof and influence of shoeing**

The conformation of the hoof capsule and the angle of the internal structures have a role to play in suspensory ligament desmopathy and limb kinematics. A significant level of research focusses on the correlation between the navicular bone angle and force applied to the deep digital flexor tendon [44, 61]. Although the research was not directed at the hindlimb suspensory ligament; their findings still shed light on this area due to the anatomical angle of bordering structure and limb kinematics. The shape of the hoof has been reported to change the kinetics and kinematics of the distal limb. Dyson et al. [61] reported that the distal phalanx to hoof wall angle and distal phalanx to horizontal angle were smallest for deep digital flexor tendon injuries at 52.27° ± 3.29 and 50.32° ± 3.70 (mean ± SD) respectively. However, it would seem there was no direct correlation between that and the angles of the hoof wall. Research suggests that optimal hoof angles for both front and back feet should be 50–55° [62]. In addition, minimal correlation between the dorsal aspect of the distal phalanx angle and deep digital flexor tendon injury has been found and the hoof wall angle was not the same as the distal phalanx angle [61], which could account for natural variation in hoof pastern axis.

The deviation of distal phalanx angle affects the orientation of the structures above it and subsequently the metacarpophalangeal joint; which in turn has the potential to cause soft tissue injuries [63, 64]. This is because the ground reaction forces are reduced delaying break-over to latter breaking phase [64] whereas the horse should have increased loading at this point [62, 65]. This has the potential to reduce the strain on the interosseous muscle but could also inhibit the elastic strain energy needed to create its passive force.

Kane et al. [63] identified 43 race horses with ruptured suspensory ligaments with lower heel and toe angles; for example the difference between the toe heel angle control group and those with suspensory apparatus failure was 1.3° less, a relatively small number in terms of angles but quite significant over the lifetime of a horse. In real terms this means that an increase in angle of 10° increases the chance of suspensory ligament failure by 6.75 times [63].

Shoeing has been used since domestication of the horse as a means to improve performance and help maintain hoof balance. The combination of farriery techniques like rolled toes, plus different types of shoe have a significant effect on the horse's feet and their movement [34, 45, 66]. It could be assumed that the application of the shoe would only affect the gait pattern of the horse but an 11% vertical

*Equine Science*

**1.5 Conformation of the horse**

hoof-ground impact causing the limb to vibrate in a craniocaudal movement at 30–40 Hz, the greatest impact being distal in the limb. The muscles of the hindlimb act as adequate shock absorbers however risk of soft tissue damage increases with the increase in loading cycles [26]. This suggests that the greater the work load and discipline level of the horse, the more likely they are to sustain an injury. One method of removing force is slipping or sliding. The hoof is designed to allow an element of slip as a natural method of dissipating energy [53] however if sliding continues in the right conditions this can increase the risk of damage to soft tissue structures. Coupled with the ground reaction forces, this means that there are two opposing forces meeting at the horizontal axis, namely the sacroiliac joint [51].

There are many variable factors when considering the relationship between hindlimb PSD and SID; one of which is the natural biological variation in every horse, in that no two are exactly the same in conformation which ultimately

enhances or impedes function. Discipline desirable traits have been documented for enhancing performance, such as the warmblood breeds for dressage, with greater hock angle reducing the incidences of injury compare to those with smaller hock angles [54, 55]. However, this was refuted in a later study of 66 warmblood horses that had the supposedly undesirable tarsal joint angle of <155.50° [56]. This was agreed with in another study examining the hock angles of 194 Warmblood horses with hindlimb PSD [57]. Hobbs et al. [54] described a selection of horses that had variations between contralateral limbs conformation and those with bone morphology variance in contralateral limbs [58]. The results of these differences may induce compensatory movements in an attempt to redistribute the weight through the stride cycle. In an attempt to counter this, and stabilise the gait, the hindlimbs may start to load in a pattern similar to a lame horse. Having said that this load distribution pattern may come from the horses' handedness. This raises the question, if the horse is not physiologically capable of creating vertical impulsion (due to straight hocks), how and where will this affect the soft tissue structures in the hindlimb? Asymmetries come in many forms, however each will have a marked effect on the biomechanics of the horse and more importantly the ground reaction forces; in the horses attempt to maintain equilibrium [54]. Of course, this need to maintain stability has different ground reaction forces depending on breed. Elite dressage Lusitano horses had lower vertical impulses compared to their Dutch Warmblood counterparts in collected trot with a range of 1.64 ± 0.02 N/Kg and 1.90 ± 0.08 N/ Kg respectively. However this evened out with a change from collected trot to passage, with minimal difference being seen. Nevertheless, the key point in this is that the centre of mass is moved closer to the hindlimbs in the higher movements. Heim and co-authors [11] demonstrated a significant difference between Franches-Montagnes stallions (n = 27) and a general populous of horses (n = 6) in the dorsoventral movement (p < 0.02) and mediolateral movement (p < 0.01) for the spine, although to say this is a generalisation of differing anatomical parts and their role in locomotion. There is also the influence of the rider to consider here; not only as their body mass is part of the calculation but as the elite rider is capable of re-balancing even the most uneducated of horses to maintain the uphill longitudinal balance that is required of a dressage horse [59]. Dyson and colleagues [60] refuted this in their pilot study of rider weight, in that the weight of the rider had a greater significance than body mass index. Although this situation is not definitive, as there are many influencing factors in this scenario. For example, the balance of the rider and the dynamics between saddle and rider, both of which have a role in distribution of forces. In essence if the rider is displaced by an ill-fitting saddle or the rider

**76**

displacement of the trunk has been observed [66], which implies a physiological effect of the structures of the back over a lifetime of a horse. Different types of shoe also have varying effects on the horse [67]. The glue on heart bar increased strain of the suspensory ligament while the racing plate alone increased strain in the superficial digital flexor tendon, interestingly when packing was added to the racing plate the increased strain was seen in the suspensory ligament. Others demonstrated an increase force of 101 N between the unshod and the steel shod foot [45, 66]. However, when looking at this in greater detail it can be seen that there is a difference in kinetics between the two states. By comparison the shod foot remains medial throughout the entire stance phase putting greater strain on the medial aspect of the limb structures. This is due to the gripping nature of the steel shoe which effectively shortens the natural slip effect of the bare foot and increases musculoskeletal forces after impact, altering the dampening effect of the suspensory ligament and preventing hoof and frog expansion on impact [34]. The stride duration also increased with the application of a shoe from (mean) 694 to 706 ms as did the stride length from 2.78 to 2.82 m; with the stride protraction and retraction decreasing after the application of shoes. This was seen as the carpal joint extending later in the swing phase and the foot being behind the movement at impact [66]. The unshod foot lands medially to then shift laterally at mid stance to then move back again medially. The application of a metal shoe removed the hoofs natural cycle of wear from the equation, which proved to be beneficial for the horse when assessing the morphology of 100 feral Brumbies [68]. Increased substrate hardness and distance travelled reduced the likelihood of hoof wall flare, however a possible negative of this is the loading of the peripheral sole in locomotion as well as the expected loading of the hoof wall [68].

#### **1.7 Influence of discipline**

There are many influencing factors when taking into consideration the relationship between horse and rider; the riders ability to control their balance, the weight of the rider and the fit of the saddle, all of these factors can have an effect on the equilibrium and the physiology of the horse. The influence of rider weight on horse movement has also been investigated. Riders were classified as light, medium, heavy and very heavy; all of which were classified as experienced riders [69]. Horses were subjectively and objectively observed with inertial sensors to determine movement at the poll and pelvis, each horse was then assessed with each rider. All heavy and very heavy rider assessments were abandoned due to temporary lameness inducement, suggesting a biomechanical change with the introduction of a dynamic load. In a study that used a lead weight added to the saddle they found the addition of weight extended the spine [70]. Thoracolumbar width changes have also been observed in another study, differing by 7.3% from the lightest to heaviest riders [71]. Variables such as saddle fit were accounted for by Master Saddlers checking prior to the tests being ridden and on the days of the test being ridden. However oscillation of the saddle in trot was reported with all rider weight groups; very heavy 14.0%, heavy 50.0%, medium 76.9% and light 84.6%, although there was no depth of discussion as to the occurrence of this except to say not all saddles fitted perfectly. Saddle bounce also occurred with the very heavy rider on 4 out of 6 horses, although this was associated with the horse being crooked in canter. Having said that, in the objective gait analysis a pelvic minimal difference of 2.2 ± 4.8 (mean ± SD) was observed [72].

Influential factors also include rider height and leg length, as this affects the fit of the saddle for both horse and rider, plus the rider's core strength for which it is assumed that an increase in core strength would reduce rider movement in the

**79**

**1.8 Surface variables**

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose…*

saddle. One of the stark conclusions drawn from this study was that lameness was observed in most of the horses when being ridden regardless of rider weight (that was not apparent in hand) and that the heavier riders consistently induced severe lameness [71, 72]. This research did not answer the question of rider weight ratio but it highlighted the importance of a well-fitting saddle and the role that it plays in

An important consideration is also the discipline of the horse and the movements they are required to perfect. An example of this was elite dressage horses which are required to produce collection; "maintaining impulsion from behind to allow a lighter shoulder", to carry out higher level movements thus distinguishing the important factor of higher proportion of bodyweight carried by the pelvic limb [73]. Although this was recognised there was no appreciation that the movement must originate in the sacroiliac joint. Furthermore the link between tarsal joint compressions was made but not associated to orthopaedic injury. However this point was contradicted by the description that the greatest movement of the SIJ to be on the transverse plane [2]. This allowed for a wider overall viewpoint comparing the likelihood of SID by disciplines; with dressage horses and show jumpers being more susceptible [2]. This suggested that SID is induced by the greater degree of collection required of each discipline and increased angles of the moment arms of

Data analysis primarily segregates elite and non-elite horses in order to classify gross morphology [73], demonstrating the understanding that each discipline has a differing physiological impact. This is then subdivided to location or type of injury. Conversely, they did not make the distinction in forelimb and hindlimb suspensory ligament injuries, and although there were a significant number of classifications observed, it was not stated whether these were distinct individual injuries or if the horses had sustained more than one [73]. However Barstow and Dyson [1] went a step further and subdivided their cohort into sacroiliac pain only and sacroiliac pain with hindlimb lameness; thus starting to demonstrate a correlation between the two. In comparison, others recognised the presence of other abnormalities but mainly focussed on osseous changes [74]. Dyson [61] considered an alternative perspective of tarsal conformation predisposing horses to PSD and acknowledged biomechanics as a possible influencing factor but again with no correlation to SID.

The surface that horses work on have to be taken into consideration as they directly influence the impact on hoof loading (hoof sliding and the declarative longitudinal forces) and therefore the reaction of the limb structures [38]. Surfaces vary based on their composition, a ménage situation will have a hard under layer with surface applied to a specific depth, while some race tracks will run on turf. The most important element here is the cushion depth as this has the potential to absorb some of the concussion [75, 76]. Having said that, a softer surface encourages the toe to pivot causing a rotational force on the distal limb structures [38]. In a human based assessment it was found that peak forces reduced with an increase in compliant surfaces [76]. The compliance of track surfaces has also been examined, each type of surface had a distinct effect on the hoof velocity and swing phase, with the greatest deformation coming from the most compliant surface [75]. Even though it was noted that this surface caused significant increases in stance time and angle of hoof on landing, they did not draw any conclusions from this or discuss the soft tissue implications for the horse. However, it does imply that the suspensory ligament would have to sustain its force for a prolonged period and thus potentially fatigue if longer stance time occurred. This concept was looked at in greater detail with the use

*DOI: http://dx.doi.org/10.5772/intechopen.92353*

maintaining normal gait patterns for that horse.

the hindlimbs, in effect reducing stability of the joint.

#### *Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose… DOI: http://dx.doi.org/10.5772/intechopen.92353*

saddle. One of the stark conclusions drawn from this study was that lameness was observed in most of the horses when being ridden regardless of rider weight (that was not apparent in hand) and that the heavier riders consistently induced severe lameness [71, 72]. This research did not answer the question of rider weight ratio but it highlighted the importance of a well-fitting saddle and the role that it plays in maintaining normal gait patterns for that horse.

An important consideration is also the discipline of the horse and the movements they are required to perfect. An example of this was elite dressage horses which are required to produce collection; "maintaining impulsion from behind to allow a lighter shoulder", to carry out higher level movements thus distinguishing the important factor of higher proportion of bodyweight carried by the pelvic limb [73]. Although this was recognised there was no appreciation that the movement must originate in the sacroiliac joint. Furthermore the link between tarsal joint compressions was made but not associated to orthopaedic injury. However this point was contradicted by the description that the greatest movement of the SIJ to be on the transverse plane [2]. This allowed for a wider overall viewpoint comparing the likelihood of SID by disciplines; with dressage horses and show jumpers being more susceptible [2]. This suggested that SID is induced by the greater degree of collection required of each discipline and increased angles of the moment arms of the hindlimbs, in effect reducing stability of the joint.

Data analysis primarily segregates elite and non-elite horses in order to classify gross morphology [73], demonstrating the understanding that each discipline has a differing physiological impact. This is then subdivided to location or type of injury. Conversely, they did not make the distinction in forelimb and hindlimb suspensory ligament injuries, and although there were a significant number of classifications observed, it was not stated whether these were distinct individual injuries or if the horses had sustained more than one [73]. However Barstow and Dyson [1] went a step further and subdivided their cohort into sacroiliac pain only and sacroiliac pain with hindlimb lameness; thus starting to demonstrate a correlation between the two. In comparison, others recognised the presence of other abnormalities but mainly focussed on osseous changes [74]. Dyson [61] considered an alternative perspective of tarsal conformation predisposing horses to PSD and acknowledged biomechanics as a possible influencing factor but again with no correlation to SID.

#### **1.8 Surface variables**

*Equine Science*

expected loading of the hoof wall [68].

**1.7 Influence of discipline**

(mean ± SD) was observed [72].

displacement of the trunk has been observed [66], which implies a physiological effect of the structures of the back over a lifetime of a horse. Different types of shoe also have varying effects on the horse [67]. The glue on heart bar increased strain of the suspensory ligament while the racing plate alone increased strain in the superficial digital flexor tendon, interestingly when packing was added to the racing plate the increased strain was seen in the suspensory ligament. Others demonstrated an increase force of 101 N between the unshod and the steel shod foot [45, 66]. However, when looking at this in greater detail it can be seen that there is a difference in kinetics between the two states. By comparison the shod foot remains medial throughout the entire stance phase putting greater strain on the medial aspect of the limb structures. This is due to the gripping nature of the steel shoe which effectively shortens the natural slip effect of the bare foot and increases musculoskeletal forces after impact, altering the dampening effect of the suspensory ligament and preventing hoof and frog expansion on impact [34]. The stride duration also increased with the application of a shoe from (mean) 694 to 706 ms as did the stride length from 2.78 to 2.82 m; with the stride protraction and retraction decreasing after the application of shoes. This was seen as the carpal joint extending later in the swing phase and the foot being behind the movement at impact [66]. The unshod foot lands medially to then shift laterally at mid stance to then move back again medially. The application of a metal shoe removed the hoofs natural cycle of wear from the equation, which proved to be beneficial for the horse when assessing the morphology of 100 feral Brumbies [68]. Increased substrate hardness and distance travelled reduced the likelihood of hoof wall flare, however a possible negative of this is the loading of the peripheral sole in locomotion as well as the

There are many influencing factors when taking into consideration the relationship between horse and rider; the riders ability to control their balance, the weight of the rider and the fit of the saddle, all of these factors can have an effect on the equilibrium and the physiology of the horse. The influence of rider weight on horse movement has also been investigated. Riders were classified as light, medium, heavy and very heavy; all of which were classified as experienced riders [69]. Horses were subjectively and objectively observed with inertial sensors to determine movement at the poll and pelvis, each horse was then assessed with each rider. All heavy and very heavy rider assessments were abandoned due to temporary lameness inducement, suggesting a biomechanical change with the introduction of a dynamic load. In a study that used a lead weight added to the saddle they found the addition of weight extended the spine [70]. Thoracolumbar width changes have also been observed in another study, differing by 7.3% from the lightest to heaviest riders [71]. Variables such as saddle fit were accounted for by Master Saddlers checking prior to the tests being ridden and on the days of the test being ridden. However oscillation of the saddle in trot was reported with all rider weight groups; very heavy 14.0%, heavy 50.0%, medium 76.9% and light 84.6%, although there was no depth of discussion as to the occurrence of this except to say not all saddles fitted perfectly. Saddle bounce also occurred with the very heavy rider on 4 out of 6 horses, although this was associated with the horse being crooked in canter. Having said that, in the objective gait analysis a pelvic minimal difference of 2.2 ± 4.8

Influential factors also include rider height and leg length, as this affects the fit of the saddle for both horse and rider, plus the rider's core strength for which it is assumed that an increase in core strength would reduce rider movement in the

**78**

The surface that horses work on have to be taken into consideration as they directly influence the impact on hoof loading (hoof sliding and the declarative longitudinal forces) and therefore the reaction of the limb structures [38]. Surfaces vary based on their composition, a ménage situation will have a hard under layer with surface applied to a specific depth, while some race tracks will run on turf. The most important element here is the cushion depth as this has the potential to absorb some of the concussion [75, 76]. Having said that, a softer surface encourages the toe to pivot causing a rotational force on the distal limb structures [38]. In a human based assessment it was found that peak forces reduced with an increase in compliant surfaces [76]. The compliance of track surfaces has also been examined, each type of surface had a distinct effect on the hoof velocity and swing phase, with the greatest deformation coming from the most compliant surface [75]. Even though it was noted that this surface caused significant increases in stance time and angle of hoof on landing, they did not draw any conclusions from this or discuss the soft tissue implications for the horse. However, it does imply that the suspensory ligament would have to sustain its force for a prolonged period and thus potentially fatigue if longer stance time occurred. This concept was looked at in greater detail with the use of a dynamometric shoe applied to three race horses which showed that turf surfaces had a greater ground reaction force (42.9 ± 3.8 g; mean ± SEM) compared to synthetic surfaces which reduced the ground reaction forces significantly (28.5 ± 2.9 g; mean ± SEM) [77]. This implies that there will be less impact on the soft tissue structures of the hindlimb and subsequently the sacroiliac joint.

#### **1.9 Lameness and evaluation**

In order to gain a full understanding of the relationship between hindlimb PSD and SID, the way in which the horse works, its discipline and level, plus the rider influence and ability must be considered [73, 78, 82]. Barstow and Dyson [1] used rider colloquialisms to aid quantification of lameness; this is very subjective even when well versed in this terminology [12]. This highlights the need to be objective and specific in pinpointing lameness. Similarly another study used anecdotal evidence to support their hypothesis of sports performance level and orthopaedic injury diagnosis, suggesting that this is frequently seen in practice but not yet documented [73]. Having said that, some studies [4, 5] have noted that some horses may suffer concurrent injuries of the sacroiliac joint or proximal suspensory (respectively) but did not draw conclusions from this regarding cause and effect or relationship.

As already stated, it is difficult, if not impossible to ascertain where the pain is coming from within the sacroiliac joint; one of the possibilities is the articular surface. As the horse ages there is an increased likelihood of cartilaginous deterioration irrespective of breed type or discipline. This deterioration and possible changes may be the result of long term laxity of the surrounding ligaments [83] which in itself could cause instability of the sacroiliac joint or degenerative suspensory desmitis which would alter the gait pattern of the horse permanently [84]. Another factor, of course, could be the ground reaction forces and the impact of hard work on hard ground for sustained periods.

It is recognised that lameness of the hindlimb creates compensatory movements within the lumbosacral region [74, 85]. Signs of subtle discomfort or pain are not so easily detected. A reduction in equine motivation to work or refusing jumps or bolting with their rider can be seen [4]. However, use of inertial measurement units can make the process of assessing asymmetry objective. The assessment of 60 horses used for polo showed 36 horses (60%) demonstrated an asymmetrical movement in the head, pelvic or both [86]. Statistical analysis linear regression revealed none of these measures had a slope greater in difference than zero. This tells us two things; that inertial measures are able to quantify small asymmetries in the horse but the value of this in a lameness evaluation must be left with the veterinary professionals to interpret. In reality this technology is not commonly used in practice and the standardised approach is to use diagnostic nerve blocks to determine the area of pain. However, this is not straight forward as they need to be used in conjunction with clinical examination and imaging modalities. In fact Pilsworth and Dyson [87] described clinically sound horses receiving a palmer nerve block to have a change in gait. This was echoed by Denoix and co-authors [88] when describing the pitfalls of sacroiliac nerve blocks, in that potential error could cause a false positive. In contrast others focussed on the biomechanics of the entire vertebral column [11, 82] but limited the discussion of the limbs to kinematics. This was echoed following assessment of the dynamic asymmetry of polo ponies, which again reverberated the question of correlation and cause [89].

The need to be more specific was demonstrated by Murray et al. [73] in their results making reference to thoracolumbar and pelvis but not specifically the SIJ. Goff and co-authors [90] advanced this to identify degenerative changes of the SIJ causing poor performance. However there is no correlation to unilateral or bilateral distal

**81**

sacroiliac dysfunction and hindlimb PSD.

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose…*

limb lameness. To emphasise the need to be unambiguous Murray et al. [73] used a large sample size (1069 horses), which potentially could be representative of the equine population. However, as the study was conducted at a referral hospital it would not represent primary veterinarians seeing acute injuries or stages of disease; emphasising the need for a retrospective study of primary veterinary practices. In a study by Barstow and Dyson [1] 296 horses were assessed for SIJ pain, of which 203 (80%) showed hindlimb lameness with 181 specifically identified with proximal suspensory desmitis (89% [94% bilateral, 6% unilateral]). Although this represents relatively small numbers by comparison to sports performance studies [73] its findings are significant and showed a direct correlation. Furthermore, the work up of the horses was carried out by the same veterinarian reducing the likeli-

In a similar study the prevalence of orthopaedic injuries was examined, classifying the horse by injury alone [91]. Having said that, discipline was acknowledged but no relationship established; although the kinematics of the show jumper's pelvic limb were noted. A limitation of this study was that the information was extracted from yard records rather than from veterinarian's records. Furthermore the initial assessments were made by several veterinarians potentially providing greater diversity in objectivity of lameness detection. In contrast, a unique perspective examining the likelihood of heritable degenerative suspensory ligament desmitis in the Peruvian Paso was published [92]. Dyson [61] demonstrated an understanding

All of this begs the question as to how a horse with sacroiliac dysfunction and hindlimb PSD can be identified? Generalised pain detection using facial expressions has been used for many years with infants. Langford et al. [93] took this principle and adapted it to form the mouse grimace scale for those used in biomedical research, this was hailed as a great success as a pain indicator. Miller et al. [94] developed this further to include pain behaviours. The assessment of pain has always been subjective and relative to the experience of the practitioner, formalising a grimace scale for horses [95] has made this an objective process for the equine veterinarian. There are general indicators of pain as seen in the horse grimace scale whereby an assessment of the horses facial postures are calculated on an ethogram to determine general level of pain. For example, a horse with tension above the eye alone may not be indicative of pain, but coupled with ears stiffly backwards and prominent chewing muscles, it may indicate a level of pain [95]. The facial grimace scale alone has been identified as limiting an ethogram for equine pain behaviours both ridden and in hand has been developed [60]. Importantly this study ensured its efficacy by refining its use with a "within observer repeatability study" to confirm this as a suitable tool for quantifying pain behaviours. This concept was taken a step forward in order to develop a scale for the ridden horse, for example the horse moving on three tracks in trot or canter could be an indicator of sacroiliac pain [69–96]. Some other indicators are a direct reflexion of the location of pain such as bucking going into canter demonstrating pain in the sacroiliac region; however, a horse at the very start of its education may resist the rider and buck out of frustration. Having said that, persistent displays of these behaviours are a direct indicator of pain [69]. There are many more subtle signs including asymmetry of the tuber coxae and the tuber ischii that can be visually assessed by the practitioner, asymmetrical muscle mass of the superficial gluteal and holding the tail to one side can also be seen as pain indicators [97]. Saddle slip has also been identified as an indicator of hindlimb lameness with a direct correlation between bilateral and unilateral lameness (p = 0.344 and p = 0.286 respectively) [98]. This advancement could improve criteria in determining the subtle variations in lameness between

of this but also questioned conformation as a predisposing factor.

*DOI: http://dx.doi.org/10.5772/intechopen.92353*

hood of subjectivity in gait analysis.

#### *Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose… DOI: http://dx.doi.org/10.5772/intechopen.92353*

limb lameness. To emphasise the need to be unambiguous Murray et al. [73] used a large sample size (1069 horses), which potentially could be representative of the equine population. However, as the study was conducted at a referral hospital it would not represent primary veterinarians seeing acute injuries or stages of disease; emphasising the need for a retrospective study of primary veterinary practices.

In a study by Barstow and Dyson [1] 296 horses were assessed for SIJ pain, of which 203 (80%) showed hindlimb lameness with 181 specifically identified with proximal suspensory desmitis (89% [94% bilateral, 6% unilateral]). Although this represents relatively small numbers by comparison to sports performance studies [73] its findings are significant and showed a direct correlation. Furthermore, the work up of the horses was carried out by the same veterinarian reducing the likelihood of subjectivity in gait analysis.

In a similar study the prevalence of orthopaedic injuries was examined, classifying the horse by injury alone [91]. Having said that, discipline was acknowledged but no relationship established; although the kinematics of the show jumper's pelvic limb were noted. A limitation of this study was that the information was extracted from yard records rather than from veterinarian's records. Furthermore the initial assessments were made by several veterinarians potentially providing greater diversity in objectivity of lameness detection. In contrast, a unique perspective examining the likelihood of heritable degenerative suspensory ligament desmitis in the Peruvian Paso was published [92]. Dyson [61] demonstrated an understanding of this but also questioned conformation as a predisposing factor.

All of this begs the question as to how a horse with sacroiliac dysfunction and hindlimb PSD can be identified? Generalised pain detection using facial expressions has been used for many years with infants. Langford et al. [93] took this principle and adapted it to form the mouse grimace scale for those used in biomedical research, this was hailed as a great success as a pain indicator. Miller et al. [94] developed this further to include pain behaviours. The assessment of pain has always been subjective and relative to the experience of the practitioner, formalising a grimace scale for horses [95] has made this an objective process for the equine veterinarian. There are general indicators of pain as seen in the horse grimace scale whereby an assessment of the horses facial postures are calculated on an ethogram to determine general level of pain. For example, a horse with tension above the eye alone may not be indicative of pain, but coupled with ears stiffly backwards and prominent chewing muscles, it may indicate a level of pain [95]. The facial grimace scale alone has been identified as limiting an ethogram for equine pain behaviours both ridden and in hand has been developed [60]. Importantly this study ensured its efficacy by refining its use with a "within observer repeatability study" to confirm this as a suitable tool for quantifying pain behaviours. This concept was taken a step forward in order to develop a scale for the ridden horse, for example the horse moving on three tracks in trot or canter could be an indicator of sacroiliac pain [69–96]. Some other indicators are a direct reflexion of the location of pain such as bucking going into canter demonstrating pain in the sacroiliac region; however, a horse at the very start of its education may resist the rider and buck out of frustration. Having said that, persistent displays of these behaviours are a direct indicator of pain [69]. There are many more subtle signs including asymmetry of the tuber coxae and the tuber ischii that can be visually assessed by the practitioner, asymmetrical muscle mass of the superficial gluteal and holding the tail to one side can also be seen as pain indicators [97]. Saddle slip has also been identified as an indicator of hindlimb lameness with a direct correlation between bilateral and unilateral lameness (p = 0.344 and p = 0.286 respectively) [98]. This advancement could improve criteria in determining the subtle variations in lameness between sacroiliac dysfunction and hindlimb PSD.

*Equine Science*

**1.9 Lameness and evaluation**

ground for sustained periods.

of a dynamometric shoe applied to three race horses which showed that turf surfaces had a greater ground reaction force (42.9 ± 3.8 g; mean ± SEM) compared to synthetic surfaces which reduced the ground reaction forces significantly (28.5 ± 2.9 g; mean ± SEM) [77]. This implies that there will be less impact on the soft tissue

In order to gain a full understanding of the relationship between hindlimb PSD and SID, the way in which the horse works, its discipline and level, plus the rider influence and ability must be considered [73, 78, 82]. Barstow and Dyson [1] used rider colloquialisms to aid quantification of lameness; this is very subjective even when well versed in this terminology [12]. This highlights the need to be objective and specific in pinpointing lameness. Similarly another study used anecdotal evidence to support their hypothesis of sports performance level and orthopaedic injury diagnosis, suggesting that this is frequently seen in practice but not yet documented [73]. Having said that, some studies [4, 5] have noted that some horses may suffer concurrent injuries of the sacroiliac joint or proximal suspensory (respectively) but did not draw conclusions from this regarding cause and effect or relationship.

As already stated, it is difficult, if not impossible to ascertain where the pain is coming from within the sacroiliac joint; one of the possibilities is the articular surface. As the horse ages there is an increased likelihood of cartilaginous deterioration irrespective of breed type or discipline. This deterioration and possible changes may be the result of long term laxity of the surrounding ligaments [83] which in itself could cause instability of the sacroiliac joint or degenerative suspensory desmitis which would alter the gait pattern of the horse permanently [84]. Another factor, of course, could be the ground reaction forces and the impact of hard work on hard

It is recognised that lameness of the hindlimb creates compensatory movements within the lumbosacral region [74, 85]. Signs of subtle discomfort or pain are not so easily detected. A reduction in equine motivation to work or refusing jumps or bolting with their rider can be seen [4]. However, use of inertial measurement units can make the process of assessing asymmetry objective. The assessment of 60 horses used for polo showed 36 horses (60%) demonstrated an asymmetrical movement in the head, pelvic or both [86]. Statistical analysis linear regression revealed none of these measures had a slope greater in difference than zero. This tells us two things; that inertial measures are able to quantify small asymmetries in the horse but the value of this in a lameness evaluation must be left with the veterinary professionals to interpret. In reality this technology is not commonly used in practice and the standardised approach is to use diagnostic nerve blocks to determine the area of pain. However, this is not straight forward as they need to be used in conjunction with clinical examination and imaging modalities. In fact Pilsworth and Dyson [87] described clinically sound horses receiving a palmer nerve block to have a change in gait. This was echoed by Denoix and co-authors [88] when describing the pitfalls of sacroiliac nerve blocks, in that potential error could cause a false positive. In contrast others focussed on the biomechanics of the entire vertebral column [11, 82] but limited the discussion of the limbs to kinematics. This was echoed following assessment of the dynamic asymmetry of polo ponies, which again reverberated the question of correlation and cause [89]. The need to be more specific was demonstrated by Murray et al. [73] in their results making reference to thoracolumbar and pelvis but not specifically the SIJ. Goff and co-authors [90] advanced this to identify degenerative changes of the SIJ causing poor performance. However there is no correlation to unilateral or bilateral distal

structures of the hindlimb and subsequently the sacroiliac joint.

**80**

### **2. Conclusions**

Research in the last 10 years has focussed on poor performance and diagnostic techniques, back pain and biomechanics or suspensory ligament disease. The correlation of information to demonstrate that lameness may be from one or more sites in the horse is limited. This indicates the necessity for further studies to determine whether there are correlations between hindlimb proximal suspensory desmopathy and sacroiliac disease. Understanding whether correlations are present between the two disorders could have an impact on evaluation and diagnosis, treatment and recovery, prognostics and welfare.

### **Acknowledgements**

Albert A. Rizvanov (https://orcid.org/0000-0002-9427-5739) was supported by state assignments 20.5175.2017/6.7 and 17.9783.2017/8.9 of the Ministry of Science and Higher Education of Russian Federation. Kazan Federal University was supported by the Russian Government Program of Competitive Growth. Catrin S. Rutland (https://orcid.org/0000-0002-2009-4898) was funded by the University of Nottingham.

### **Conflicts of interest**

The authors declare no conflicts of interest.

### **Author details**

Anne Skivington1 , Milomir Kovac2 , Elena Zakirova<sup>3</sup> , Albert A. Rizvanov1,3 and Catrin Sian Rutland1 \*

1 School of Veterinary Medicine and Science, Faculty of Medicine**,** University of Nottingham**,** UK

2 Veterinary Clinic "New Century", Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia

3 Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia

\*Address all correspondence to: catrin.rutland@nottingham.ac.uk

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**83**

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose…*

3D sacroiliac joint loading. Equine Veterinary Journal. 2009;**41**(3):207-212. DOI: 10.2746/042516409x395697

journal.pone.0196960

evj.12455

tb05219.x

[11] Heim C, Pfau T, Gerber V, Schweizer C, Doherr M, Schupbach-Regula G, et al. Determination of vertebral range of motion using inertial measurement units in 27 Franches-Montagnes stallions and comparison between conditions and with a mixed population. Equine Veterinary Journal. 2016;**48**(4):509-516. DOI: 10.1111/

[12] Warner SM, Koch TO, Pfau T. Inertial sensors for assessment of back movement in horses during locomotion

over ground. Equine Veterinary Journal. 2010;**42**:417-424. DOI: 10.1111/j.2042-3306.2010.00200.x

[14] Greve L, Pfau T, Dyson S. Thoracolumbar movement in sound horses trotting in straight lines in hand and on the lunge and the relationship with hind limb symmetry or asymmetry. Veterinary Journal. 2017;**220**:95-104. DOI: 10.1016/j.tvjl.2017.01.003

[15] Degueurce C, Chateau H, Denoix JM. In vitro assessment of movements of the sacroiliac joint in the horse. Equine Veterinary Journal. 2004;**36**(8):694-698. DOI: 10.2746/0425164044848064

[13] Audigie F, Pourcelot P, Degueurce C, Denoix JM, Geiger D. Kinematics of the equine back: Flexion-extension movements in sound trotting horses. Equine Veterinary Journal. 1999;**31**: 210-213. DOI: 10.1111/j.2042-3306.1999.

[10] Clayton HM, Hampson A, Fraser P, White A, Egenvall A. Comparison of rider stability in a flapless saddle versus a conventional saddle. PLoS One. 2018;**13**(6):e0196960. DOI: 10.1371/

*DOI: http://dx.doi.org/10.5772/intechopen.92353*

**References**

[1] Barstow A, Dyson S. Clinical features and diagnosis of sacroiliac joint region pain in 296 horses: 2004-2014. Equine Veterinary Education. 2015;**27**(12): 637-647. DOI: 10.1111/eve.12377

[2] Goff LM, Jeffcott LB, Jasiewicz J, McGowan CM. Structural and biomechanical aspects of equine sacroiliac joint function and their relationship to clinical disease. Veterinary Journal. 2008;**176**(3):281- 293. DOI: 10.1016/j.tvjl.2007.03.005

[3] Zimmerman M, Dyson S, Murray R. Close, impinging and overriding spinous processes in the thoracolumbar

spine: The relationship between radiological and scintigraphic findings and clinical signs. Equine Veterinary Journal. 2012;**44**(2):178-184. DOI: 10.1111/j.2042-3306.2011.00373.x

[4] Dyson S. Hindlimb lameness associated with proximal suspensory desmopathy and injury of the accessory ligament of the suspensory ligament in five horses. Equine Veterinary Education. 2014;**26**(10):538-542. DOI:

[5] Bathe A. Outcome of the treatment of sacroiliac region pain. In: BEVA Congress. London, UK: British Equine

Hannover, Germany: Schlutersche; 2009

[7] Pasquini C, Spurgeon T, Pasquini S. Anatomy of Domestic Animals. 11th ed. Texas, USA: Sudz Publishing; 2007

[8] Reese WO. Functional Anatomy and Physiology of Domestic Animals. 4th ed. Iowa, USA: Wiley-Blackwell; 2009

[9] Haussiler KK, McGilvray KC, Ayturk UM, Puttlitz CM, Hill AE, McIlwraith CW. Deformation of the equine pelvis in response to in vitro

Veterinary Association; 2012

[6] Budras K, Sack WO, Rock S. Anatomy of the Horse. 6th ed.

10.1111/eve.12217

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose… DOI: http://dx.doi.org/10.5772/intechopen.92353*

#### **References**

*Equine Science*

**2. Conclusions**

recovery, prognostics and welfare.

**Acknowledgements**

**82**

Russia

**Author details**

of Nottingham.

**Conflicts of interest**

Anne Skivington1

Nottingham**,** UK

and Catrin Sian Rutland1

and Biotechnology, Moscow, Russia

provided the original work is properly cited.

, Milomir Kovac2

\*

The authors declare no conflicts of interest.

, Elena Zakirova<sup>3</sup>

Research in the last 10 years has focussed on poor performance and diagnostic techniques, back pain and biomechanics or suspensory ligament disease. The correlation of information to demonstrate that lameness may be from one or more sites in the horse is limited. This indicates the necessity for further studies to determine whether there are correlations between hindlimb proximal suspensory desmopathy and sacroiliac disease. Understanding whether correlations are present between the two disorders could have an impact on evaluation and diagnosis, treatment and

Albert A. Rizvanov (https://orcid.org/0000-0002-9427-5739) was supported

by state assignments 20.5175.2017/6.7 and 17.9783.2017/8.9 of the Ministry of Science and Higher Education of Russian Federation. Kazan Federal University was supported by the Russian Government Program of Competitive Growth. Catrin S. Rutland (https://orcid.org/0000-0002-2009-4898) was funded by the University

1 School of Veterinary Medicine and Science, Faculty of Medicine**,** University of

2 Veterinary Clinic "New Century", Moscow State Academy of Veterinary Medicine

3 Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: catrin.rutland@nottingham.ac.uk

, Albert A. Rizvanov1,3

[1] Barstow A, Dyson S. Clinical features and diagnosis of sacroiliac joint region pain in 296 horses: 2004-2014. Equine Veterinary Education. 2015;**27**(12): 637-647. DOI: 10.1111/eve.12377

[2] Goff LM, Jeffcott LB, Jasiewicz J, McGowan CM. Structural and biomechanical aspects of equine sacroiliac joint function and their relationship to clinical disease. Veterinary Journal. 2008;**176**(3):281- 293. DOI: 10.1016/j.tvjl.2007.03.005

[3] Zimmerman M, Dyson S, Murray R. Close, impinging and overriding spinous processes in the thoracolumbar spine: The relationship between radiological and scintigraphic findings and clinical signs. Equine Veterinary Journal. 2012;**44**(2):178-184. DOI: 10.1111/j.2042-3306.2011.00373.x

[4] Dyson S. Hindlimb lameness associated with proximal suspensory desmopathy and injury of the accessory ligament of the suspensory ligament in five horses. Equine Veterinary Education. 2014;**26**(10):538-542. DOI: 10.1111/eve.12217

[5] Bathe A. Outcome of the treatment of sacroiliac region pain. In: BEVA Congress. London, UK: British Equine Veterinary Association; 2012

[6] Budras K, Sack WO, Rock S. Anatomy of the Horse. 6th ed. Hannover, Germany: Schlutersche; 2009

[7] Pasquini C, Spurgeon T, Pasquini S. Anatomy of Domestic Animals. 11th ed. Texas, USA: Sudz Publishing; 2007

[8] Reese WO. Functional Anatomy and Physiology of Domestic Animals. 4th ed. Iowa, USA: Wiley-Blackwell; 2009

[9] Haussiler KK, McGilvray KC, Ayturk UM, Puttlitz CM, Hill AE, McIlwraith CW. Deformation of the equine pelvis in response to in vitro

3D sacroiliac joint loading. Equine Veterinary Journal. 2009;**41**(3):207-212. DOI: 10.2746/042516409x395697

[10] Clayton HM, Hampson A, Fraser P, White A, Egenvall A. Comparison of rider stability in a flapless saddle versus a conventional saddle. PLoS One. 2018;**13**(6):e0196960. DOI: 10.1371/ journal.pone.0196960

[11] Heim C, Pfau T, Gerber V, Schweizer C, Doherr M, Schupbach-Regula G, et al. Determination of vertebral range of motion using inertial measurement units in 27 Franches-Montagnes stallions and comparison between conditions and with a mixed population. Equine Veterinary Journal. 2016;**48**(4):509-516. DOI: 10.1111/ evj.12455

[12] Warner SM, Koch TO, Pfau T. Inertial sensors for assessment of back movement in horses during locomotion over ground. Equine Veterinary Journal. 2010;**42**:417-424. DOI: 10.1111/j.2042-3306.2010.00200.x

[13] Audigie F, Pourcelot P, Degueurce C, Denoix JM, Geiger D. Kinematics of the equine back: Flexion-extension movements in sound trotting horses. Equine Veterinary Journal. 1999;**31**: 210-213. DOI: 10.1111/j.2042-3306.1999. tb05219.x

[14] Greve L, Pfau T, Dyson S. Thoracolumbar movement in sound horses trotting in straight lines in hand and on the lunge and the relationship with hind limb symmetry or asymmetry. Veterinary Journal. 2017;**220**:95-104. DOI: 10.1016/j.tvjl.2017.01.003

[15] Degueurce C, Chateau H, Denoix JM. In vitro assessment of movements of the sacroiliac joint in the horse. Equine Veterinary Journal. 2004;**36**(8):694-698. DOI: 10.2746/0425164044848064

[16] Vleeming A. The Sacroiliac Joint: A Clinical-Anatomical, Biomechanical and Radiological Study. Netherlands: University of Rotterdam; 1990

[17] Soffler C, Hermanson JW. Muscular design in the equine interosseous muscle. Journal of Morphology. 2006;**267**:696-704

[18] Dyce KM, Sack WO, Wensing CJ. The Forelimb of the Horse. USA: Saunders Elsevier; 2010

[19] Spinella G, Loprete G, Castagnetti C, Musella V, Antonelli C, Vilar JM, et al. Evaluation of mean echogenicity of tendons and ligaments of the metacarpal region in neonatal foals: A preliminary study. Research in Veterinary Science. 2015;**101**:11-14. DOI: 10.1016/j.rvsc.2015.05.011

[20] Wilson DA, Baker GJ, Pijanowski GJ, Boero MJ, Badertscher RR II. Composition and morphologic features of the interosseous muscle in Standardbreds and thoroughbreds. American Journal of Veterinary Research. 1991;**52**(1):133-139

[21] Nagy A, Dyson S. Magnetic resonance imaging and histological findings in the proximal aspect of the suspensory ligament of forelimbs in nonlame horses. Equine Veterinary Journal. 2012;**44**(1):43-50. DOI: 10.1111/j.2042-3306.2011.00365.x

[22] Clayton HM. Horse Species Symposium: Biomechanics of the exercising horse. Journal of Animal Science. 2016;**94**(10):4076-4086. DOI: 10.2527/jas.2015-9990

[23] Payne RC, Hutchinson JR, Robilliard JJ, Smith NC, Wilson AM. Functional specialisation of pelvic limb anatomy in horses (*Equus caballus*). Journal of Anatomy. 2005;**206**(6):557-574. DOI: 10.1111/j.1469-7580.2005.00420.x

[24] Crook TC, Cruickshank SE, McGowan CM, Stubbs N, Wakeling JM, Wilson AM, et al. Comparative anatomy and muscle architecture of selected hind limb muscles in the Quarter Horse and Arab. Journal of Anatomy. 2008;**212**(2):144-152. DOI: 10.1111/j.1469-7580.2007.00848.x

[25] Biewener AA, Roberts TJ. Muscle and tendon contributions to force, work, and elastic energy savings: A comparative perspective. Exercise and Sport Sciences Reviews. 2000;**28**(3):99-107

[26] Wilson AM, McGuigan MP, Su A, van den Bogert AJ. Horses damp the spring in their step. Nature. 2001;**414**(6866):895-899. DOI: 10.1038/414895a

[27] Chavers JC, Allen AK, Ahmed W, Fuglsang-Damgaard LH, Harrison AP. The equine hindlimb proximal suspensory ligament: An assessment of health and function by means of its damping harmonic oscillator properties, measured using an acoustic myography system: A new modality study. Journal of Equine Veterinary Science. 2018;**71**:21-26. DOI: 10.1016/j. jevs.2018.09.006

[28] Harrison SM, Whitton RC, Kawcak CE, Stover SM, Pandy MG. Evaluation of a subject-specific finite-element model of the equine metacarpophalangeal joint under physiological load. Journal of Biomechanics. 2014;**47**(1):65-73. DOI: 10.1016/j.jbiomech.2013.10.001

[29] Addis PR, Lawson SE. The role of tendon stiffness in development of equine locomotion with age. Equine Veterinary Journal. 2010;(38):556-560. DOI: 10.1111/j.2042-3306.2010.00296.x

[30] Alexander MR, Alexander RM, Alex MR. Principles of Animal Locomotion. 2nd ed. USA: Princeton University Press; 2006

**85**

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose…*

determination of claw pain and its relationship to limb locomotion score in dairy cattle. Journal of Dairy Science. 2007;**90**(10):4592-4602. DOI: 10.3168/

[40] Guertin PA. Central pattern generator for locomotion: Anatomical, physiological, and pathophysiological

Neurology. 2013;**3**:183. DOI: 10.3389/

[41] Dyson S. Science-in-brief: Horse, rider, saddlery interactions: Welfare and performance. In: Saddle Research Trust 3rd International Conference. Nottingham, UK: Saddle Research

[42] Hobbs SJ, Licka T, Polman R. The difference in kinematics of horses walking, trotting and cantering on a flat and banked 10 m circle. Equine Veterinary Journal. 2011;**43**(6):686-694. DOI:

10.1111/j.2042-3306.2010.00334.x

Functional specialisation of the pelvic limb of the hare (*Lepus europeus*). Journal of Anatomy. 2007;**210**(4):472-490. DOI: 10.1111/j.1469-7580.2007.00704.x

[44] Eliashar E. The biomechanics of the equine foot as it pertains to farriery. Veterinary Clinics of North America: Equine Practice. 2012;**28**(2):283-291. DOI: 10.1016/j.cveq.2012.06.001

[45] Panagiotopoulou O, Rankin JW, Gatesy SM, Hutchinson JR. A

preliminary case study of the effect of shoe-wearing on the biomechanics of a horse's foot. PeerJ. 2016;**4**:e2164. DOI:

[46] Clayton HM. The Dynamic Horse: A Biomechanical Guide to Equine Movement and Performance. USA: Sport Horse Publications; 2004

10.7717/peerj.2164

[43] Williams SB, Payne RC, Wilson AM.

considerations. Frontiers in

jds.2007-0006

fneur.2012.00183

Trust; 2018

*DOI: http://dx.doi.org/10.5772/intechopen.92353*

[31] Biewener AA. Muscle-tendon stresses and elastic energy storage during locomotion in the horse. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology. 1998;**120**(1):73-87. DOI: 10.1016/s0305-0491(98)00024-8

[32] Harrison SM, Whitton RC, Kawcak CE, Stover SM, Pandy MG. Relationship between muscle forces, joint loading and utilization of elastic strain energy in equine locomotion. The Journal of Experimental Biology. 2010;**213**(23):3998-4009. DOI: 10.1242/

[33] Minetti AE, Ardigo LP, Reinach E, Saibene F. The relationship between mechanical work and energy

expenditure of locomotion in horses. The Journal of Experimental Biology.

[34] Aoki O. Biomechanical analysis of horse shoeing. Equine Veterinary Journal. 1999;**31**:629-630. DOI: 10.1111/

[35] Symons J, Hawkins D, Fyhrie D, Upadhyaya S, Stover S. Modelling the interaction between racehorse limb and race surface. Procedia Engineer. 2016;**147**:175-180. DOI: 10.1016/j.

[36] Back W, Clayton H. Equine Locomotion. 2nd ed. London, UK:

[37] Clayton HM, Dyson S, Harris P, Bondi A. Horses, saddles and riders: Applying the science. Equine Veterinary Education. 2015;**27**(9):447-452. DOI:

[38] Clayton HM, Hobbs SJ. Ground reaction forces: The sine qua non of legged locomotion. Journal of Equine Veterinary Science. 2019;**76**:25-35. DOI:

[39] Dyer RM, Neerchal NK, Tasch U, Wu Y, Dyer P, Rajkondawar PG. Objective

1999;**202**(17):2329-2338

j.2042-3306.1999.tb05298.x

proeng.2016.06.209

Saunders Elsevier; 2013

10.1111/eve.12407

10.1016/j.jevs.2019.02.022

jeb.044545

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose… DOI: http://dx.doi.org/10.5772/intechopen.92353*

[31] Biewener AA. Muscle-tendon stresses and elastic energy storage during locomotion in the horse. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology. 1998;**120**(1):73-87. DOI: 10.1016/s0305-0491(98)00024-8

*Equine Science*

2006;**267**:696-704

Saunders Elsevier; 2010

[19] Spinella G, Loprete G,

10.1016/j.rvsc.2015.05.011

[20] Wilson DA, Baker GJ,

[16] Vleeming A. The Sacroiliac Joint: A Clinical-Anatomical, Biomechanical and Radiological Study. Netherlands: University of Rotterdam; 1990

[24] Crook TC, Cruickshank SE,

2008;**212**(2):144-152. DOI: 10.1111/j.1469-7580.2007.00848.x

2000;**28**(3):99-107

10.1038/414895a

jevs.2018.09.006

McGowan CM, Stubbs N, Wakeling JM, Wilson AM, et al. Comparative anatomy and muscle architecture of selected hind limb muscles in the Quarter Horse and Arab. Journal of Anatomy.

[25] Biewener AA, Roberts TJ. Muscle and tendon contributions to force, work, and elastic energy savings: A comparative perspective. Exercise and Sport Sciences Reviews.

[26] Wilson AM, McGuigan MP, Su A, van den Bogert AJ. Horses damp the spring in their step. Nature. 2001;**414**(6866):895-899. DOI:

[27] Chavers JC, Allen AK, Ahmed W, Fuglsang-Damgaard LH, Harrison AP.

The equine hindlimb proximal suspensory ligament: An assessment of health and function by means of its damping harmonic oscillator properties, measured using an acoustic myography system: A new modality study. Journal of Equine Veterinary Science. 2018;**71**:21-26. DOI: 10.1016/j.

[28] Harrison SM, Whitton RC, Kawcak CE, Stover SM, Pandy MG. Evaluation of a subject-specific finite-element model of the equine metacarpophalangeal joint under physiological load. Journal of

10.1016/j.jbiomech.2013.10.001

[29] Addis PR, Lawson SE. The role of tendon stiffness in development of equine locomotion with age. Equine Veterinary Journal. 2010;(38):556-560. DOI: 10.1111/j.2042-3306.2010.00296.x

[30] Alexander MR, Alexander RM, Alex MR. Principles of Animal Locomotion. 2nd ed. USA: Princeton

University Press; 2006

Biomechanics. 2014;**47**(1):65-73. DOI:

[17] Soffler C, Hermanson JW. Muscular design in the equine interosseous muscle. Journal of Morphology.

[18] Dyce KM, Sack WO, Wensing CJ. The Forelimb of the Horse. USA:

Castagnetti C, Musella V, Antonelli C, Vilar JM, et al. Evaluation of mean echogenicity of tendons and ligaments of the metacarpal region in neonatal foals: A preliminary study. Research in Veterinary Science. 2015;**101**:11-14. DOI:

Pijanowski GJ, Boero MJ, Badertscher RR II. Composition and morphologic features of the interosseous muscle in Standardbreds and thoroughbreds. American Journal of Veterinary Research. 1991;**52**(1):133-139

[21] Nagy A, Dyson S. Magnetic resonance imaging and histological findings in the proximal aspect of the suspensory ligament of forelimbs in nonlame horses. Equine Veterinary Journal. 2012;**44**(1):43-50. DOI: 10.1111/j.2042-3306.2011.00365.x

[22] Clayton HM. Horse Species Symposium: Biomechanics of the exercising horse. Journal of Animal Science. 2016;**94**(10):4076-4086. DOI:

[23] Payne RC, Hutchinson JR, Robilliard JJ, Smith NC, Wilson AM. Functional specialisation of pelvic limb anatomy in horses (*Equus caballus*). Journal of Anatomy. 2005;**206**(6):557-574. DOI: 10.1111/j.1469-7580.2005.00420.x

10.2527/jas.2015-9990

**84**

[32] Harrison SM, Whitton RC, Kawcak CE, Stover SM, Pandy MG. Relationship between muscle forces, joint loading and utilization of elastic strain energy in equine locomotion. The Journal of Experimental Biology. 2010;**213**(23):3998-4009. DOI: 10.1242/ jeb.044545

[33] Minetti AE, Ardigo LP, Reinach E, Saibene F. The relationship between mechanical work and energy expenditure of locomotion in horses. The Journal of Experimental Biology. 1999;**202**(17):2329-2338

[34] Aoki O. Biomechanical analysis of horse shoeing. Equine Veterinary Journal. 1999;**31**:629-630. DOI: 10.1111/ j.2042-3306.1999.tb05298.x

[35] Symons J, Hawkins D, Fyhrie D, Upadhyaya S, Stover S. Modelling the interaction between racehorse limb and race surface. Procedia Engineer. 2016;**147**:175-180. DOI: 10.1016/j. proeng.2016.06.209

[36] Back W, Clayton H. Equine Locomotion. 2nd ed. London, UK: Saunders Elsevier; 2013

[37] Clayton HM, Dyson S, Harris P, Bondi A. Horses, saddles and riders: Applying the science. Equine Veterinary Education. 2015;**27**(9):447-452. DOI: 10.1111/eve.12407

[38] Clayton HM, Hobbs SJ. Ground reaction forces: The sine qua non of legged locomotion. Journal of Equine Veterinary Science. 2019;**76**:25-35. DOI: 10.1016/j.jevs.2019.02.022

[39] Dyer RM, Neerchal NK, Tasch U, Wu Y, Dyer P, Rajkondawar PG. Objective determination of claw pain and its relationship to limb locomotion score in dairy cattle. Journal of Dairy Science. 2007;**90**(10):4592-4602. DOI: 10.3168/ jds.2007-0006

[40] Guertin PA. Central pattern generator for locomotion: Anatomical, physiological, and pathophysiological considerations. Frontiers in Neurology. 2013;**3**:183. DOI: 10.3389/ fneur.2012.00183

[41] Dyson S. Science-in-brief: Horse, rider, saddlery interactions: Welfare and performance. In: Saddle Research Trust 3rd International Conference. Nottingham, UK: Saddle Research Trust; 2018

[42] Hobbs SJ, Licka T, Polman R. The difference in kinematics of horses walking, trotting and cantering on a flat and banked 10 m circle. Equine Veterinary Journal. 2011;**43**(6):686-694. DOI: 10.1111/j.2042-3306.2010.00334.x

[43] Williams SB, Payne RC, Wilson AM. Functional specialisation of the pelvic limb of the hare (*Lepus europeus*). Journal of Anatomy. 2007;**210**(4):472-490. DOI: 10.1111/j.1469-7580.2007.00704.x

[44] Eliashar E. The biomechanics of the equine foot as it pertains to farriery. Veterinary Clinics of North America: Equine Practice. 2012;**28**(2):283-291. DOI: 10.1016/j.cveq.2012.06.001

[45] Panagiotopoulou O, Rankin JW, Gatesy SM, Hutchinson JR. A preliminary case study of the effect of shoe-wearing on the biomechanics of a horse's foot. PeerJ. 2016;**4**:e2164. DOI: 10.7717/peerj.2164

[46] Clayton HM. The Dynamic Horse: A Biomechanical Guide to Equine Movement and Performance. USA: Sport Horse Publications; 2004

[47] Agass RF, Wilson AM, Weller R, Pfau T. The relationship between foot conformation, foot placement and motion symmetry in the equine hind limb. Equine Veterinary Journal. 2014;**46**(47):19-20

[48] Johnston C, Back W. Hoof ground interaction: When biomechanical stimuli challenge the tissues of the distal limb. Equine Veterinary Journal. 2006;**38**(7):634-641. DOI: 10.2746/042516406x158341

[49] Parsons KJ, Spence AJ, Morgan R, Thompson JA, Wilson AM. High speed field kinematics of foot contact in elite galloping horses in training. Equine Veterinary Journal. 2011;**43**(2):216-222. DOI: 10.1111/j.2042-3306.2010.00149.x

[50] Boye JK, Thomsen MH, Pfau T, Olsen E. Accuracy and precision of gait events derived from motion capture in horses during walk and trot. Journal of Biomechanics. 2014;**47**(5):1220-1224. DOI: 10.1016/j.jbiomech.2013.12.018

[51] Holden-Douilly L, Pourcelot P, Desquilbet L, Falala S, Crevier-Denoix N, Chateau H. Equine hoof slip distance during trot at training speed: Comparison between kinematic and accelerometric measurement techniques. Veterinary Journal. 2013;**197**(2):198-204. DOI: 10.1016/j. tvjl.2013.02.004

[52] Channon AJ, Walker AM, Pfau T, Sheldon IM, Wilson AM. Variability of Manson and Leaver locomotion scores assigned to dairy cows by different observers. The Veterinary Record. 2009;**164**(13):388-392. DOI: 10.1136/ vr.164.13.388

[53] Harvey AM, Williams SB, Singer ER. The effect of lateral heel studs on the kinematics of the equine digit while cantering on grass. Veterinary Journal. 2012;**192**(2):217-221. DOI: 10.1016/j. tvjl.2011.06.003

[54] Hobbs SJ, Nauwelaerts S, Sinclair J, Clayton HM, Back W. Sagittal plane fore hoof unevenness is associated with fore and hindlimb asymmetrical force vectors in the sagittal and frontal planes. PLoS One. 2018;**13**(8):e0203134. DOI: 10.1371/journal.pone.0203134

[55] Holmstrom M, Magnusson LE, Philipsson J. Variation in conformation of Swedish Warmblood horses and conformational characteristics of elite sport horses. Equine Veterinary Journal. 1990;**22**(3):186-193. DOI: 10.1111/ j.2042-3306.1990.tb04245.x

[56] Sole M, Santos R, Gomez MD, Galisteo AM, Valera M. Evaluation of conformation against traits associated with dressage ability in unridden Iberian horses at the trot. Research in Veterinary Science. 2013;**95**(2):660-666. DOI: 10.1016/j.rvsc.2013.06.017

[57] Routh J, Gilligan S, Strang C, Dyson S. Is there an association between straight tarsus (hock) conformation and hindlimb proximal suspensory desmopathy? Equine Veterinary Journal. 2017;**49**(51):17

[58] Watson KM, Stitson DJ, Davies HM. Third metacarpal bone length and skeletal asymmetry in the thoroughbred racehorse. Equine Veterinary Journal. 2003;**35**(7):712-714. DOI: 10.2746/042516403775696348

[59] Clayton HM, Schamhardt HC, Hobbs SJ. Ground reaction forces of elite dressage horses in collected trot and passage. Veterinary Journal. 2017;**221**:30-33. DOI: 10.1016/j. tvjl.2017.01.016

[60] Dyson S, Berger J, Ellis AD, Mullard J. Development of an ethogram for a pain scoring system in ridden horses and its application to determine the presence of musculoskeletal pain. Journal of Veterinary Behavior. 2018;**23**:47-57. DOI: 10.1016/j. jveb.2017.10.008

**87**

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose…*

de Laat MA, Pollitt CC. Morphometry and abnormalities of the feet of Kaimanawa feral horses in New Zealand. Australian Veterinary Journal. 2010;**88**(4):124-131. DOI: 10.1111/j.1751-0813.2010.00554.x

[69] Dyson S, editor. The Influence of Rider to Horse Body Weight Ratios on Equine Gait and Behaviour: A Pilot Study. London, UK: National Equine

[70] de Cocq P, van Weeren PR, Back W. Effects of girth, saddle and weight on movements of the horse. Equine Veterinary Journal. 2004;**36**(8):758-763. DOI: 10.2746/0425164044848000

[71] Quiney LE, Ellis A, Dyson SJ. The influence of rider weight on exercise induced changes in thoracolumbar dimensions and epaxial muscle tension and pain. Equine Veterinary Journal.

[72] Dyson S, Ellis AD, Mackechnie-Guire R, Douglas J, Bondi A,

Harris P. The influence of rider: Horse body weight ratio and rider horse saddle fit on equine gait and behaviour: A pilot study. Equine Veterinary Education.

[73] Murray RC, Dyson SJ, Tranquille C, Adams V. Association of type of sport and performance level with anatomical site of orthopaedic injury diagnosis.

2006;(36):411-416. DOI: 10.1111/j.2042-

[74] Girodroux M, Dyson S, Murray R. Osteoarthritis of the thoracolumbar synovial intervertebral articulations: Clinical and radiographic features in 77 horses with poor performance and back pain. Equine Veterinary Journal. 2009;**41**(2):130-138. DOI: 10.2746/042516408x345099

[75] Burn JF, Usmar SJ. Hoof landing velocity is related to track surface properties in trotting horses. Equine

Equine Veterinary Journal.

3306.2006.tb05578.x

Forum; 2018

2018;**50**(52):3-35

2019;**2**:1-13

*DOI: http://dx.doi.org/10.5772/intechopen.92353*

[61] Dyson S. Clinical commentary. Is degenerative change within hindlimb suspensory ligaments a prelude to all types of injury. Equine Veterinary Education. 2010;**22**(6):271-274

[62] Lesniak K, Williams J, Kuznik K, Douglas P. Does a 4-6 week shoeing interval promote optimal foot balance in the working equine? Animals. 2017;**7**(4):29. DOI: 10.3390/ani7040029

[63] Kane AJ, Stover SM, Gardner IA, Bock KB, Case JT, Johnson BJ, et al. Hoof size, shape, and balance as possible risk factors for catastrophic musculoskeletal injury of thoroughbred racehorses. American Journal of Veterinary Research. 1998;**59**(12):1545-1552

[64] Ramsey GD, Hunter PJ, Nash MP. The effect of hoof angle variations on dorsal lamellar load in the equine hoof. Equine Veterinary Journal. 2011;**43**(5):536-542. DOI: 10.1111/j.2042-3306.2010.00319.x

[65] McCarty CA, Thomason JJ, Gordon K, Hurtig M, Bignell W. Effect of hoof angle on joint contact area in the equine metacarpophalangeal joint following simulated impact loading ex vivo. Equine Veterinary Journal. 2015;**47**(6):715-720. DOI: 10.1111/

[66] Willemen MA, Savelberg HHCM, Barneveld A. The improvement of the gait quality of sound trotting warmblood horses by normal shoeing and its effect on the load on the lower forelimb. Livestock Production Science. 1997;**52**(2):145-153. DOI: 10.1016/

[67] Ault B, Starling G, Parkes R, Pfau T, Pardoe C, Day P, et al. The effects of three different shoeing conditions on tendon strain in the thoroughbred forelimb. Equine Veterinary Journal.

S0301-6226(97)00130-9

[68] Hampson BA, Ramsey G, Macintosh AMH, Mills PC,

2015;**47**(48):17

evj.12354

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose… DOI: http://dx.doi.org/10.5772/intechopen.92353*

[61] Dyson S. Clinical commentary. Is degenerative change within hindlimb suspensory ligaments a prelude to all types of injury. Equine Veterinary Education. 2010;**22**(6):271-274

*Equine Science*

2014;**46**(47):19-20

[47] Agass RF, Wilson AM, Weller R, Pfau T. The relationship between foot conformation, foot placement and motion symmetry in the equine hind limb. Equine Veterinary Journal.

[54] Hobbs SJ, Nauwelaerts S, Sinclair J, Clayton HM, Back W. Sagittal plane fore hoof unevenness is associated with fore and hindlimb asymmetrical force vectors in the sagittal and frontal planes. PLoS One. 2018;**13**(8):e0203134. DOI:

10.1371/journal.pone.0203134

[55] Holmstrom M, Magnusson LE, Philipsson J. Variation in conformation of Swedish Warmblood horses and conformational characteristics of elite sport horses. Equine Veterinary Journal. 1990;**22**(3):186-193. DOI: 10.1111/ j.2042-3306.1990.tb04245.x

[56] Sole M, Santos R, Gomez MD, Galisteo AM, Valera M. Evaluation of conformation against traits associated with dressage ability in unridden Iberian horses at the trot. Research in Veterinary Science. 2013;**95**(2):660-666. DOI:

10.1016/j.rvsc.2013.06.017

[58] Watson KM, Stitson DJ,

Davies HM. Third metacarpal bone length and skeletal asymmetry in the thoroughbred racehorse. Equine Veterinary Journal. 2003;**35**(7):712-714. DOI: 10.2746/042516403775696348

[59] Clayton HM, Schamhardt HC, Hobbs SJ. Ground reaction forces of elite dressage horses in collected trot and passage. Veterinary Journal. 2017;**221**:30-33. DOI: 10.1016/j.

[60] Dyson S, Berger J, Ellis AD,

2018;**23**:47-57. DOI: 10.1016/j.

Mullard J. Development of an ethogram for a pain scoring system in ridden horses and its application to determine the presence of musculoskeletal pain. Journal of Veterinary Behavior.

2017;**49**(51):17

tvjl.2017.01.016

jveb.2017.10.008

[57] Routh J, Gilligan S, Strang C, Dyson S. Is there an association between straight tarsus (hock) conformation and hindlimb proximal suspensory desmopathy? Equine Veterinary Journal.

[48] Johnston C, Back W. Hoof ground interaction: When biomechanical stimuli challenge the tissues of the distal limb. Equine Veterinary Journal. 2006;**38**(7):634-641. DOI:

[49] Parsons KJ, Spence AJ, Morgan R, Thompson JA, Wilson AM. High speed

field kinematics of foot contact in elite galloping horses in training. Equine Veterinary Journal. 2011;**43**(2):216-222. DOI: 10.1111/j.2042-3306.2010.00149.x

[50] Boye JK, Thomsen MH, Pfau T, Olsen E. Accuracy and precision of gait events derived from motion capture in horses during walk and trot. Journal of Biomechanics. 2014;**47**(5):1220-1224. DOI: 10.1016/j.jbiomech.2013.12.018

[51] Holden-Douilly L, Pourcelot P, Desquilbet L, Falala S, Crevier-Denoix N, Chateau H. Equine hoof slip distance during trot at training speed: Comparison between kinematic and accelerometric measurement techniques. Veterinary Journal. 2013;**197**(2):198-204. DOI: 10.1016/j.

[52] Channon AJ, Walker AM, Pfau T, Sheldon IM, Wilson AM. Variability of Manson and Leaver locomotion scores assigned to dairy cows by different observers. The Veterinary Record. 2009;**164**(13):388-392. DOI: 10.1136/

[53] Harvey AM, Williams SB, Singer ER. The effect of lateral heel studs on the kinematics of the equine digit while cantering on grass. Veterinary Journal. 2012;**192**(2):217-221. DOI: 10.1016/j.

tvjl.2013.02.004

vr.164.13.388

tvjl.2011.06.003

10.2746/042516406x158341

**86**

[62] Lesniak K, Williams J, Kuznik K, Douglas P. Does a 4-6 week shoeing interval promote optimal foot balance in the working equine? Animals. 2017;**7**(4):29. DOI: 10.3390/ani7040029

[63] Kane AJ, Stover SM, Gardner IA, Bock KB, Case JT, Johnson BJ, et al. Hoof size, shape, and balance as possible risk factors for catastrophic musculoskeletal injury of thoroughbred racehorses. American Journal of Veterinary Research. 1998;**59**(12):1545-1552

[64] Ramsey GD, Hunter PJ, Nash MP. The effect of hoof angle variations on dorsal lamellar load in the equine hoof. Equine Veterinary Journal. 2011;**43**(5):536-542. DOI: 10.1111/j.2042-3306.2010.00319.x

[65] McCarty CA, Thomason JJ, Gordon K, Hurtig M, Bignell W. Effect of hoof angle on joint contact area in the equine metacarpophalangeal joint following simulated impact loading ex vivo. Equine Veterinary Journal. 2015;**47**(6):715-720. DOI: 10.1111/ evj.12354

[66] Willemen MA, Savelberg HHCM, Barneveld A. The improvement of the gait quality of sound trotting warmblood horses by normal shoeing and its effect on the load on the lower forelimb. Livestock Production Science. 1997;**52**(2):145-153. DOI: 10.1016/ S0301-6226(97)00130-9

[67] Ault B, Starling G, Parkes R, Pfau T, Pardoe C, Day P, et al. The effects of three different shoeing conditions on tendon strain in the thoroughbred forelimb. Equine Veterinary Journal. 2015;**47**(48):17

[68] Hampson BA, Ramsey G, Macintosh AMH, Mills PC,

de Laat MA, Pollitt CC. Morphometry and abnormalities of the feet of Kaimanawa feral horses in New Zealand. Australian Veterinary Journal. 2010;**88**(4):124-131. DOI: 10.1111/j.1751-0813.2010.00554.x

[69] Dyson S, editor. The Influence of Rider to Horse Body Weight Ratios on Equine Gait and Behaviour: A Pilot Study. London, UK: National Equine Forum; 2018

[70] de Cocq P, van Weeren PR, Back W. Effects of girth, saddle and weight on movements of the horse. Equine Veterinary Journal. 2004;**36**(8):758-763. DOI: 10.2746/0425164044848000

[71] Quiney LE, Ellis A, Dyson SJ. The influence of rider weight on exercise induced changes in thoracolumbar dimensions and epaxial muscle tension and pain. Equine Veterinary Journal. 2018;**50**(52):3-35

[72] Dyson S, Ellis AD, Mackechnie-Guire R, Douglas J, Bondi A, Harris P. The influence of rider: Horse body weight ratio and rider horse saddle fit on equine gait and behaviour: A pilot study. Equine Veterinary Education. 2019;**2**:1-13

[73] Murray RC, Dyson SJ, Tranquille C, Adams V. Association of type of sport and performance level with anatomical site of orthopaedic injury diagnosis. Equine Veterinary Journal. 2006;(36):411-416. DOI: 10.1111/j.2042- 3306.2006.tb05578.x

[74] Girodroux M, Dyson S, Murray R. Osteoarthritis of the thoracolumbar synovial intervertebral articulations: Clinical and radiographic features in 77 horses with poor performance and back pain. Equine Veterinary Journal. 2009;**41**(2):130-138. DOI: 10.2746/042516408x345099

[75] Burn JF, Usmar SJ. Hoof landing velocity is related to track surface properties in trotting horses. Equine and Comparative Equine Physiology. 2005;**2**(1):37-41

[76] McMahon TA, Greene PR. The influence of track compliance on running. Journal of Biomechanics. 1979;**12**(12):893-904. DOI: 10.1016/0021-9290(79)90057-5

[77] Setterbo JJ, Garcia TC, Campbell IP, Reese JL, Morgan JM, Kim SY, et al. Hoof accelerations and ground reaction forces of thoroughbred racehorses measured on dirt, synthetic, and turf track surfaces. American Journal of Veterinary Research. 2009;**70**(10):1220-1229. DOI: 10.2460/ ajvr.70.10.1220

[78] Dyson S. Evaluation of poor performance in competition horses: A musculoskeletal perspective. Part 2: Further investigation. Equine Veterinary Education. 2016;**28**(7):379-387. DOI: 10.1111/eve.12498

[79] Dyson S, Murray R. Pain associated with the sacroiliac joint region: A clinical study of 74 horses. Equine Veterinary Journal. 2003;**35**(3):240-245. DOI: 10.2746/042516403776148255

[80] Dyson S, Murray R. Management of hindlimb proximal suspensory desmopathy by neurectomy of the deep branch of the lateral plantar nerve and plantar fasciotomy: 155 horses (2003-2008). Equine Veterinary Journal. 2012;**44**(3):361-367. DOI: 10.1111/j.2042-3306.2011.00445.x

[81] Orsini JA. Equine biomechanics. Journal of Equine Veterinary Science. 2010;**30**(7):344. DOI: 10.1016/j. jevs.2010.06.001

[82] van Weeren PR, McGowan C, Haussler KK. Science overview: Development of a structural and functional understanding of the equine back. Equine Veterinary Journal. 2010;**42**:393-400. DOI: 10.1111/j.2042-3306.2010.00207.x

[83] Jeffcott LB. Disorders of the thoracolumbar spine of the horse—A survey of 443 cases. Equine Veterinary Journal. 1980;**12**(4):197-210. DOI: 10.1111/j.2042-3306.1980.tb03427.x

[84] Halper J, Mueller POE. Dystrophic mineralisation in degenerative suspensory ligament desmitis. Equine Veterinary Education. 2018;**30**(8):424- 426. DOI: 10.1111/eve.12732

[85] Alvarez CBG, Bobbert MF, Lamers L, Johnston C, Back W, van Weeren PR. The effect of induced hindlimb lameness on thoracolumbar kinematics during treadmill locomotion. Equine Veterinary Journal. 2008;**40**(2):147-152. DOI: 10.2746/042516408x250184

[86] Pfau T, Parkes RS, Burden ER, Bell N, Fairhurst H, Witte TH. Movement asymmetry in working polo horses. Equine Veterinary Journal. 2016;**48**(4):517-522. DOI: 10.1111/ evj.12467

[87] Pilsworth R, Dyson S. Where does it hurt? Problems with interpretation of regional and intra-synovial diagnostic analgesia. Equine Veterinary Education. 2015;**27**(11):595-603. DOI: 10.1111/ eve.12392

[88] Denoix JM, Jacquet S. Ultrasoundguided injections of the sacroiliac area in horses. Equine Veterinary Education. 2008;**20**(4):203-207. DOI: 10.2746/095777308x292128

[89] Burden ER, Pfau T, Witte TH. Objective assessment of gait asymmetry in polo ponies. Equine Veterinary Journal. 2013;**45**(44):9

[90] Goff L, Jeffcott L, Riggs C, McGowan C. Sacroiliac joint morphology: Influence of age, body weight and previous back pain. In: International Conference on Equine Exercise Physiology. Chester, UK: Equine Veterinary Journal; 2014

**89**

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose…*

International Congress of World Equine Veterinary Association (WEVA). Marrakech, Morocco: World Equine Veterinary Association; 2009

[98] Greve L, Dyson SJ. An investigation of the relationship between hindlimb lameness and saddle slip. Equine Veterinary Journal. 2013;**45**(5):570-577.

DOI: 10.1111/evj.12029

*DOI: http://dx.doi.org/10.5772/intechopen.92353*

[91] de Sousa NR, Luna SPL, Pizzigatti D, Martins MTA,

10.1590/0103-8478cr20151218

Gao S, Li J, et al. Degenerative

journal.pone.0167069

[93] Langford DJ, Bailey AL,

[94] Miller AL, Kitson GL, Skalkoyannis B, Flecknell PA, Leach MC. Using the mouse grimace scale and behaviour to assess pain in CBA mice following vasectomy. Applied Animal Behaviour Science. 2016;**181**:160-165. DOI: 10.1016/j.

applanim.2016.05.020

pone.0092281

[95] Dalla Costa E, Minero M, Lebelt D, Stucke D, Canali E,

Leach MC. Development of the Horse Grimace Scale (HGS) as a pain assessment tool in horses undergoing routine castration. PLoS One.

2014;**9**(3):e92281. DOI: 10.1371/journal.

[96] Hockenhull J, Creighton E. The use of equipment and training practices and the prevalence of owner-reported ridden behaviour problems in UK leisure horses. Equine Veterinary Journal. 2013;**45**(1):15-19. DOI: 10.1111/j.2042-3306.2012.00567.x

[97] Dyson S. Proximal suspensory desmitis in the hindlimb. In: 9th

Echols S, et al. Coding of facial expressions of pain in the laboratory mouse. Nature Methods. 2010;**7**(6): 447-449. DOI: 10.1038/nmeth.1455

Possebon FS, Aguiar ACS. Relation between type and local of orthopaedic injuries with physical activity in horses. Ciência Rural. 2017;**47**(2):36-41. DOI:

[92] Luo W, Sandy J, Trella K, Gorski D,

suspensory ligament desmitis (DSLD) in Peruvian Paso horses is characterized by altered expression of TGFbeta signaling components in adiposederived stromal fibroblasts. PLoS One. 2016;**11**(11):e0167069. DOI: 10.1371/

Chanda ML, Clarke SE, Drummond TE,

*Investigation into Whether Proximal Suspensory Desmitis of the Hindlimb Could Predispose… DOI: http://dx.doi.org/10.5772/intechopen.92353*

[91] de Sousa NR, Luna SPL, Pizzigatti D, Martins MTA, Possebon FS, Aguiar ACS. Relation between type and local of orthopaedic injuries with physical activity in horses. Ciência Rural. 2017;**47**(2):36-41. DOI: 10.1590/0103-8478cr20151218

*Equine Science*

2005;**2**(1):37-41

ajvr.70.10.1220

10.1111/eve.12498

jevs.2010.06.001

and Comparative Equine Physiology.

[83] Jeffcott LB. Disorders of the thoracolumbar spine of the horse—A survey of 443 cases. Equine Veterinary Journal. 1980;**12**(4):197-210. DOI: 10.1111/j.2042-3306.1980.tb03427.x

mineralisation in degenerative suspensory ligament desmitis. Equine Veterinary Education. 2018;**30**(8):424-

426. DOI: 10.1111/eve.12732

[85] Alvarez CBG, Bobbert MF, Lamers L, Johnston C, Back W, van Weeren PR. The effect of induced hindlimb lameness on thoracolumbar

kinematics during treadmill locomotion. Equine Veterinary Journal. 2008;**40**(2):147-152. DOI: 10.2746/042516408x250184

asymmetry in working polo horses. Equine Veterinary Journal. 2016;**48**(4):517-522. DOI: 10.1111/

evj.12467

eve.12392

[86] Pfau T, Parkes RS, Burden ER, Bell N, Fairhurst H, Witte TH. Movement

[87] Pilsworth R, Dyson S. Where does it hurt? Problems with interpretation of regional and intra-synovial diagnostic analgesia. Equine Veterinary Education. 2015;**27**(11):595-603. DOI: 10.1111/

[88] Denoix JM, Jacquet S. Ultrasoundguided injections of the sacroiliac area in horses. Equine Veterinary Education. 2008;**20**(4):203-207. DOI:

10.2746/095777308x292128

Journal. 2013;**45**(44):9

[89] Burden ER, Pfau T, Witte TH. Objective assessment of gait asymmetry in polo ponies. Equine Veterinary

[90] Goff L, Jeffcott L, Riggs C, McGowan C. Sacroiliac joint morphology: Influence of age, body weight and previous back pain. In: International Conference on Equine Exercise Physiology. Chester, UK: Equine Veterinary Journal; 2014

[84] Halper J, Mueller POE. Dystrophic

[76] McMahon TA, Greene PR. The influence of track compliance on running. Journal of Biomechanics.

Campbell IP, Reese JL, Morgan JM, Kim SY, et al. Hoof accelerations and ground reaction forces of thoroughbred racehorses measured on dirt, synthetic, and turf track surfaces. American Journal of Veterinary Research. 2009;**70**(10):1220-1229. DOI: 10.2460/

[78] Dyson S. Evaluation of poor performance in competition horses: A musculoskeletal perspective. Part 2: Further investigation. Equine Veterinary Education. 2016;**28**(7):379-387. DOI:

[79] Dyson S, Murray R. Pain associated with the sacroiliac joint region: A clinical study of 74 horses. Equine Veterinary Journal. 2003;**35**(3):240-245. DOI: 10.2746/042516403776148255

[80] Dyson S, Murray R. Management of hindlimb proximal suspensory desmopathy by neurectomy of the deep branch of the lateral plantar nerve and plantar fasciotomy: 155 horses (2003-2008). Equine Veterinary Journal. 2012;**44**(3):361-367. DOI: 10.1111/j.2042-3306.2011.00445.x

[81] Orsini JA. Equine biomechanics. Journal of Equine Veterinary Science. 2010;**30**(7):344. DOI: 10.1016/j.

[82] van Weeren PR, McGowan C, Haussler KK. Science overview: Development of a structural and functional understanding of the equine back. Equine Veterinary Journal. 2010;**42**:393-400. DOI: 10.1111/j.2042-3306.2010.00207.x

1979;**12**(12):893-904. DOI: 10.1016/0021-9290(79)90057-5

[77] Setterbo JJ, Garcia TC,

**88**

[92] Luo W, Sandy J, Trella K, Gorski D, Gao S, Li J, et al. Degenerative suspensory ligament desmitis (DSLD) in Peruvian Paso horses is characterized by altered expression of TGFbeta signaling components in adiposederived stromal fibroblasts. PLoS One. 2016;**11**(11):e0167069. DOI: 10.1371/ journal.pone.0167069

[93] Langford DJ, Bailey AL, Chanda ML, Clarke SE, Drummond TE, Echols S, et al. Coding of facial expressions of pain in the laboratory mouse. Nature Methods. 2010;**7**(6): 447-449. DOI: 10.1038/nmeth.1455

[94] Miller AL, Kitson GL, Skalkoyannis B, Flecknell PA, Leach MC. Using the mouse grimace scale and behaviour to assess pain in CBA mice following vasectomy. Applied Animal Behaviour Science. 2016;**181**:160-165. DOI: 10.1016/j. applanim.2016.05.020

[95] Dalla Costa E, Minero M, Lebelt D, Stucke D, Canali E, Leach MC. Development of the Horse Grimace Scale (HGS) as a pain assessment tool in horses undergoing routine castration. PLoS One. 2014;**9**(3):e92281. DOI: 10.1371/journal. pone.0092281

[96] Hockenhull J, Creighton E. The use of equipment and training practices and the prevalence of owner-reported ridden behaviour problems in UK leisure horses. Equine Veterinary Journal. 2013;**45**(1):15-19. DOI: 10.1111/j.2042-3306.2012.00567.x

[97] Dyson S. Proximal suspensory desmitis in the hindlimb. In: 9th

International Congress of World Equine Veterinary Association (WEVA). Marrakech, Morocco: World Equine Veterinary Association; 2009

[98] Greve L, Dyson SJ. An investigation of the relationship between hindlimb lameness and saddle slip. Equine Veterinary Journal. 2013;**45**(5):570-577. DOI: 10.1111/evj.12029

**91**

damage [1–8].

**Chapter 6**

*Sinan Kandir*

**Abstract**

animals.

**1. Introduction**

molecular purification and cloning.

ADAMTS Proteases: Potential

Targets for Cartilage Health

Biomarkers and Novel Therapeutic

The equine locomotor system's health plays a key role on athletic performance. Bone and joint diseases are the major causes of lameness. Poor performance and diseases lead to great economic loss to equestrian sports and horse breeders. Therefore, prevention, early diagnosis, and therapy of joint diseases are important. A disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) proteinase family plays an important role in many physiological processes such as tissue reorganization, coagulation, and angiogenesis. Aggrecan proteinases ADAMTS-4 and ADAMTS-5 are physiologically responsible for the restructuring with enzymatic cleavage of the cartilage, specific biomarkers in the synovium or body fluids for early diagnosis, and potential specific therapeutic targets in order to their role on degenerative joint diseases physiopathology in humans and various

**Keywords:** ADAMTS, aggrecan, equine, lameness, metalloproteinase, proteoglycan

A disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) protease family plays an important role in many physiological and physiopathological processes. The ADAMTS family is an important potential biomarker for the evaluation of early diagnosis due to its roles in the physiopathological mechanisms of many diseases such as cancer, arthritis, and atherosclerosis. ADAMTS-4 and ADAMTS-5 have been reported to play an important role in the pathogenesis of osteoarthritis in humans and various animals, following their first

Articular cartilage is structurally composed of partially chondrocyte cells and a large number of extracellular matrix components. Many macromolecules have been identified in cartilage tissue, including collagen fibrils, aggregate proteoglycans, and glycoproteins. Although joint damage is caused by oxidative metabolisminduced free radicals and hypoxic conditions, the main reason is the increase in proteolytic enzymes. Matrix metalloproteinases (MMPs), pro- and anti-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α), and retinoic acid are the main biomarkers recommended for the diagnosis of joint

#### **Chapter 6**

## ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health

*Sinan Kandir*

#### **Abstract**

The equine locomotor system's health plays a key role on athletic performance. Bone and joint diseases are the major causes of lameness. Poor performance and diseases lead to great economic loss to equestrian sports and horse breeders. Therefore, prevention, early diagnosis, and therapy of joint diseases are important. A disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) proteinase family plays an important role in many physiological processes such as tissue reorganization, coagulation, and angiogenesis. Aggrecan proteinases ADAMTS-4 and ADAMTS-5 are physiologically responsible for the restructuring with enzymatic cleavage of the cartilage, specific biomarkers in the synovium or body fluids for early diagnosis, and potential specific therapeutic targets in order to their role on degenerative joint diseases physiopathology in humans and various animals.

**Keywords:** ADAMTS, aggrecan, equine, lameness, metalloproteinase, proteoglycan

#### **1. Introduction**

A disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) protease family plays an important role in many physiological and physiopathological processes. The ADAMTS family is an important potential biomarker for the evaluation of early diagnosis due to its roles in the physiopathological mechanisms of many diseases such as cancer, arthritis, and atherosclerosis. ADAMTS-4 and ADAMTS-5 have been reported to play an important role in the pathogenesis of osteoarthritis in humans and various animals, following their first molecular purification and cloning.

Articular cartilage is structurally composed of partially chondrocyte cells and a large number of extracellular matrix components. Many macromolecules have been identified in cartilage tissue, including collagen fibrils, aggregate proteoglycans, and glycoproteins. Although joint damage is caused by oxidative metabolisminduced free radicals and hypoxic conditions, the main reason is the increase in proteolytic enzymes. Matrix metalloproteinases (MMPs), pro- and anti-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α), and retinoic acid are the main biomarkers recommended for the diagnosis of joint damage [1–8].

In this chapter, we focus on ADAMTS proteases and their role on cartilage health for joints' stability, their possible usage as damage biomarkers for early diagnosis, and novel therapeutic properties.

#### **2. ADAMTS proteases**

Metzincins are a superfamily of zinc-dependent metalloproteases, metalloproteinases, or metalloendopeptidases, which are responsible for many physiological functions that also include cartilage turnover due to their regulatory activities on the extracellular matrix (ECM). The metzincins constitute by the zinc-binding catalytic motif consensus sequence HEXXHXXGXX (H/D) exactly; the binding of a zinc molecule is modulated by the three histidines (or an aspartic acid in the third position), the acid-base catalysis is facilitated by the glutamic acid residue in general, and the steric flexibility is acquired by the small glycine residue in the catalytic motif [9].

The first referenced equine gene mapping project was initiated in October 1995 by the "First International Equine Gene Mapping Workshop," in order to search answers of the main questions "speed gene" and "evidence of heritable trait", and the first construction of a low density, male linkage map in 1999 was reported [10, 11]. Thereafter, the first domestic horse gene map (EquCab2.0), a thoroughbred mare Twilight's gene sequence, was released in 2007 and published in November 2009 [12–16]. The latest version of high-quality equine gene map EquCab3.0 is available and enables to detailed data about genes and encoding proteins [16]. The Vertebrate Gene Nomenclature Committee (VGNC) [17] has standardized names to genes in vertebrate species including horse [18, 19]. According to these accessible latest data versions, the equine ADAMTS and ADAMTS-like family members with chromosomal locations are listed in **Table 1**.

#### **2.1 ADAMTS family**

Towards the end of 1990s, Kuno et al. [20] described a new family of metalloproteinase, which consists of sequence similarity with snake venom disintegrin that was upregulated in colon adenocarcinoma cell line as a specific gene for cachexigenic tumor and was named a disintegrin-like and metalloproteinase with thrombospondin type-1 motif (ADAMTS). The equine ADAMTS and ADAMTS-like proteins are a superfamily comprised of 19 and 7 members, respectively (**Table 1**). The ADAMTSs are secreted proteinases and multidomain enzymes constituted of zinc-binding active site motif similar to adamalysin (ADAMs) and ensued by a metalloproteinase domain with that of reprolysins (snake venom metalloproteinases) and disintegrin-like domain (**Figures 1** and **3**). ADAMTS-like (ADAMTSL) family and papilin are newly identified and secreted ECM-related proteins which are relatives to ADAMTS proteases. Additionally, they lack catalytic activity due to the absence of prometalloprotease and the disintegrin-like domain which are found in the ADAMTSs [21–24]. ADAMTSs differ from ADAMs with the lack of a transmembrane domain and the inclusion of well-conserved thrombospondin 1-like repeats, a cysteine-rich domain, and the CUB (complement subcomponents C1s/ C1r, Uegf, BMP1) domain, thus being soluble extracellular proteases [25–31].

The main physiological functions of equine ADAMTSLs and papilin are extensively unknown; thus they have some troubles and need to further detailed investigations. However, recent studies on genome-wide association analysis and transgenic animals have indicated that ADAMTS, ADAMTSL, and papilin gene mutations cause lethal embryonic defects and autosomal recessive Mendelian disorders such as human Ehlers-Danlos syndrome [32], bovine dermatosparaxis

**93**

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health*

ADAMTS1 26 15061 ENSECAG00000016339 F6YLN3 ADAMTS2 14 55397 ENSECAG00000016328 F6X633 ADAMTS3 3 — ENSECAG00000019061 F6ZC90/F7A3A4 ADAMTS4 5 15070 ENSECAG00000024172 F6YRD3/

ADAMTS5 26 59233 ENSECAG00000006500 F7ACI3/

ADAMTS6 21 15071 ENSECAG00000029347 F6X9L7 ADAMTS7 1 15072 ENSECAG00000007527 F6W0M1 ADAMTS8 7 15073 ENSECAG00000014164 F6ZXN7 ADAMTS9 16 15074 ENSECAG00000019880 F6VTC7 ADAMTS10 7 55772 ENSECAG00000016210 F6QIB9 ADAMTS12 21 15062 ENSECAG00000016121 F6TW13 ADAMTS13 25 — (NCBI Gene ID: 100069281) — ADAMTS14 1 15063 ENSECAG00000014713 F7D1G5 ADAMTS15 7 15064 ENSECAG00000015715 F6V0J9 ADAMTS16 21 15065 ENSECAG00000000787 F6W504 ADAMTS17 1 15066 ENSECAG00000000579 F7DNJ9 ADAMTS18 3 15067 ENSECAG00000019006 F7A7V7 ADAMTS19 14 15068 ENSECAG00000023694 F6YNK0 ADAMTS20 6 15069 ENSECAG00000020835 F6PZV0

23 15075 ENSECAG00000015972 F6W1K7

1 22941 ENSECAG00000012008 F6T6C4

1 51434 ENSECAG00000022944 F6UWV1

24 21150 ENSECAT00000008176 F6VT48

ADAMTSL2 25 15076 ENSECAG00000011887 F6TEW7

ADAMTSL4 5 15077 ENSECAG00000019154 F7A7L3 ADAMTSL5 7 50328 ENSECAG00000009642 F6X928

**ENSEMBL UniProt**

A0A3Q2H7G6

A0A5F5PZN1

**VGNC\_ ID**

[33, 34], human Weill-Marchesani syndrome [35], canine ectopia lentis [36], human Geleophysic dysplasia [37], canine Musladin-Lueke syndrome [38], and thrombotic thrombocytopenic purpura [39]. In consideration of this knowledge, ADAMTSs, ADAMTSLs, and papilin could be responsible as most commonly screened genetic disorders among horses as early embryonic death and abnormalities, junctional epidermolysis bullosa [40], hereditary equine regional dermal

*The equine ADAMTS and ADAMTS-like family members with chromosomal locations and accession numbers.*

asthenia [41], thrombocytopenia and, von Willebrand disease [42].

*DOI: http://dx.doi.org/10.5772/intechopen.93046*

**Chromosomal Location**

**Gene/protein name**

ADAMTSL1 (Punctin-1)

ADAMTSL3 (Punctin-2/ SH3GL3)

ADAMTSL6 (THSD4)

PAPLN (Papilin)

**Table 1.**


*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health DOI: http://dx.doi.org/10.5772/intechopen.93046*

**Table 1.**

*Equine Science*

and novel therapeutic properties.

somal locations are listed in **Table 1**.

**2.1 ADAMTS family**

**2. ADAMTS proteases**

In this chapter, we focus on ADAMTS proteases and their role on cartilage health for joints' stability, their possible usage as damage biomarkers for early diagnosis,

Metzincins are a superfamily of zinc-dependent metalloproteases, metalloproteinases, or metalloendopeptidases, which are responsible for many physiological functions that also include cartilage turnover due to their regulatory activities on the extracellular matrix (ECM). The metzincins constitute by the zinc-binding catalytic motif consensus sequence HEXXHXXGXX (H/D) exactly; the binding of a zinc molecule is modulated by the three histidines (or an aspartic acid in the third position), the acid-base catalysis is facilitated by the glutamic acid residue in general, and the steric flexibility is acquired by the small glycine residue in the catalytic motif [9]. The first referenced equine gene mapping project was initiated in October 1995 by the "First International Equine Gene Mapping Workshop," in order to search answers of the main questions "speed gene" and "evidence of heritable trait", and the first construction of a low density, male linkage map in 1999 was reported [10, 11]. Thereafter, the first domestic horse gene map (EquCab2.0), a thoroughbred mare Twilight's gene sequence, was released in 2007 and published in November 2009 [12–16]. The latest version of high-quality equine gene map EquCab3.0 is available and enables to detailed data about genes and encoding proteins [16]. The Vertebrate Gene Nomenclature Committee (VGNC) [17] has standardized names to genes in vertebrate species including horse [18, 19]. According to these accessible latest data versions, the equine ADAMTS and ADAMTS-like family members with chromo-

Towards the end of 1990s, Kuno et al. [20] described a new family of metalloproteinase, which consists of sequence similarity with snake venom disintegrin that was upregulated in colon adenocarcinoma cell line as a specific gene for cachexigenic tumor and was named a disintegrin-like and metalloproteinase with thrombospondin type-1 motif (ADAMTS). The equine ADAMTS and ADAMTS-like proteins are a superfamily comprised of 19 and 7 members, respectively (**Table 1**). The ADAMTSs are secreted proteinases and multidomain enzymes constituted of zinc-binding active site motif similar to adamalysin (ADAMs) and ensued by a metalloproteinase domain with that of reprolysins (snake venom metalloproteinases) and disintegrin-like domain (**Figures 1** and **3**). ADAMTS-like (ADAMTSL) family and papilin are newly identified and secreted ECM-related proteins which are relatives to ADAMTS proteases. Additionally, they lack catalytic activity due to the absence of prometalloprotease and the disintegrin-like domain which are found in the ADAMTSs [21–24]. ADAMTSs differ from ADAMs with the lack of a transmembrane domain and the inclusion of well-conserved thrombospondin 1-like repeats, a cysteine-rich domain, and the CUB (complement subcomponents C1s/ C1r, Uegf, BMP1) domain, thus being soluble extracellular proteases [25–31]. The main physiological functions of equine ADAMTSLs and papilin are extensively unknown; thus they have some troubles and need to further detailed investigations. However, recent studies on genome-wide association analysis and transgenic animals have indicated that ADAMTS, ADAMTSL, and papilin gene mutations cause lethal embryonic defects and autosomal recessive Mendelian disorders such as human Ehlers-Danlos syndrome [32], bovine dermatosparaxis

**92**

*The equine ADAMTS and ADAMTS-like family members with chromosomal locations and accession numbers.*

[33, 34], human Weill-Marchesani syndrome [35], canine ectopia lentis [36], human Geleophysic dysplasia [37], canine Musladin-Lueke syndrome [38], and thrombotic thrombocytopenic purpura [39]. In consideration of this knowledge, ADAMTSs, ADAMTSLs, and papilin could be responsible as most commonly screened genetic disorders among horses as early embryonic death and abnormalities, junctional epidermolysis bullosa [40], hereditary equine regional dermal asthenia [41], thrombocytopenia and, von Willebrand disease [42].

#### **Figure 1.**

*Phylogenetic analysis of equine ADAMTS protein family. The evolutionary history was inferred using the maximum parsimony method. The most parsimonious tree with length = 9505 is shown. The consistency index is (0.751174), the retention index is (0.551189), and the composite index is 0.440603 (0.414039) for all sites and parsimony-informative sites. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches [43]. The MP tree was obtained using the subtree-pruning-regrafting (SPR) algorithm (p. 126 in Ref. [44]) with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). Branch lengths were calculated using the average pathway method (see p. 132 in Ref. [2]) and are in the units of the number of changes over the whole sequence. They are shown next to the branches. This analysis involved 19 amino acid sequences. There were a total of 2768 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [45].*

#### **3. Role of ADAMTS family on equine cartilage health**

Musculoskeletal system health is crucial for equine locomotion. This system is responsible to deploy the mechanical energy to the joints for efficient movement and specific biomechanical functions. There are many types of joints presented, where a majority of the free movements are managed by diarthrodial or synovial joints in the body. Cartilage tissue, which is the most important part of the diarthrodial joints, is absorbed and the loading energy throughout locomotion is distributed. Alterations due to inaccurately loading or metabolic disruptions could lead to acute or chronic damage, namely as arthritis, osteoarthrosis, or osteoarthritis to the joint and its critical component cartilage tissue, and restrict the locomotor functions. It is important to understand the molecular mechanisms of healthy and damaged cartilage tissues by the novel candidate molecular biomarkers in order to

**95**

**Figure 2.**

*Schematic view of aggrecan proteoglycan [50, 55, 56].*

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health*

early detection, easily clinical application, and therapy. Hence, we will focus on the

Cartilages are divided into three major types as histological and biochemical properties in the body as hyaline, elastic and fibrocartilage. The distinctive features among these cartilage types are water content/dry matter balance and fiber types. In the diarthrodial joints, hyaline cartilage is existed on the articular surfaces and is well resist to pressure stress during various locomotion by its unique structure [46, 47]. Avascularized, unnerved, alymphatic hyaline cartilage (Latin words "hyălĭnus"

meaning "glassy; made of glass; transparent") tissue's matrix is fundamentally constituted by water (%63–70), collagens (the majority type II in normal hyaline cartilage), non-collagenous proteins, and proteoglycans (the majority of aggrecans), while the most compounds are glycosaminoglycans (GAG) [48, 49]. Proteoglycans are classified into four subgroups related to their function: intracellular, cell-surface, pericellular, and extracellular. In the cartilage tissue, hyaluronan- and lectin-binding proteoglycans (hyalectans; aggrecan, versican, neurocan, and brevican) and small leucine-rich proteoglycans exist. Hyalectans are compromised with a similar structure in their tridomain structure; the N-terminal domain binds to hyaluronan, a central domain with the core protein for attachment

of GAG chains, and the C-terminal region that binds lectins [50, 51].

could directly affect the aggrecanase activity by ADAMTSs [58].

The major proteoglycans in the diarthrodial joints aggrecans are the crucial elements for the biomechanical function with well-balanced load distribution and transmissions in order to provide the viscoelastic properties, the tight junctions, and the bridges of the extracellular matrix (ECM) [50, 52–55]. Aggrecans have a large molecular mass that contains GAG side chains comprising of the mostly chondroitin sulfate and keratan sulfate. They have three globular domains (G1, G2, and G3) to maintain the stabilization of protein complexes and to ensure mechanical features of cartilage. These highly conserved globular domains among the vertebrates have specific cleavage sites for proteases such as ADAMTSs (**Figure 2**) [50, 55–57]. The GAG's chondroitin sulfate and keratan sulfate contents of aggrecan

Although, the hyaline cartilage consists of the chondrocytes which are the only cell type; this cell population has shown different morphological properties under the microscope and has been identified as dark, light, and adipocyte-like

cartilage health and importance of the ADAMTS family in this section.

*DOI: http://dx.doi.org/10.5772/intechopen.93046*

**3.1 Healthy cartilage tissue**

early detection, easily clinical application, and therapy. Hence, we will focus on the cartilage health and importance of the ADAMTS family in this section.

#### **3.1 Healthy cartilage tissue**

*Equine Science*

**94**

**Figure 1.**

**3. Role of ADAMTS family on equine cartilage health**

Musculoskeletal system health is crucial for equine locomotion. This system is responsible to deploy the mechanical energy to the joints for efficient movement and specific biomechanical functions. There are many types of joints presented, where a majority of the free movements are managed by diarthrodial or synovial joints in the body. Cartilage tissue, which is the most important part of the diarthrodial joints, is absorbed and the loading energy throughout locomotion is distributed. Alterations due to inaccurately loading or metabolic disruptions could lead to acute or chronic damage, namely as arthritis, osteoarthrosis, or osteoarthritis to the joint and its critical component cartilage tissue, and restrict the locomotor functions. It is important to understand the molecular mechanisms of healthy and damaged cartilage tissues by the novel candidate molecular biomarkers in order to

*Phylogenetic analysis of equine ADAMTS protein family. The evolutionary history was inferred using the maximum parsimony method. The most parsimonious tree with length = 9505 is shown. The consistency index is (0.751174), the retention index is (0.551189), and the composite index is 0.440603 (0.414039) for all sites and parsimony-informative sites. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches [43]. The MP tree was obtained using the subtree-pruning-regrafting (SPR) algorithm (p. 126 in Ref. [44]) with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). Branch lengths were calculated using the average pathway method (see p. 132 in Ref. [2]) and are in the units of the number of changes over the whole sequence. They are shown next to the branches. This analysis involved 19 amino acid sequences. There were a total of 2768 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [45].*

Cartilages are divided into three major types as histological and biochemical properties in the body as hyaline, elastic and fibrocartilage. The distinctive features among these cartilage types are water content/dry matter balance and fiber types. In the diarthrodial joints, hyaline cartilage is existed on the articular surfaces and is well resist to pressure stress during various locomotion by its unique structure [46, 47].

Avascularized, unnerved, alymphatic hyaline cartilage (Latin words "hyălĭnus" meaning "glassy; made of glass; transparent") tissue's matrix is fundamentally constituted by water (%63–70), collagens (the majority type II in normal hyaline cartilage), non-collagenous proteins, and proteoglycans (the majority of aggrecans), while the most compounds are glycosaminoglycans (GAG) [48, 49].

Proteoglycans are classified into four subgroups related to their function: intracellular, cell-surface, pericellular, and extracellular. In the cartilage tissue, hyaluronan- and lectin-binding proteoglycans (hyalectans; aggrecan, versican, neurocan, and brevican) and small leucine-rich proteoglycans exist. Hyalectans are compromised with a similar structure in their tridomain structure; the N-terminal domain binds to hyaluronan, a central domain with the core protein for attachment of GAG chains, and the C-terminal region that binds lectins [50, 51].

The major proteoglycans in the diarthrodial joints aggrecans are the crucial elements for the biomechanical function with well-balanced load distribution and transmissions in order to provide the viscoelastic properties, the tight junctions, and the bridges of the extracellular matrix (ECM) [50, 52–55]. Aggrecans have a large molecular mass that contains GAG side chains comprising of the mostly chondroitin sulfate and keratan sulfate. They have three globular domains (G1, G2, and G3) to maintain the stabilization of protein complexes and to ensure mechanical features of cartilage. These highly conserved globular domains among the vertebrates have specific cleavage sites for proteases such as ADAMTSs (**Figure 2**) [50, 55–57]. The GAG's chondroitin sulfate and keratan sulfate contents of aggrecan could directly affect the aggrecanase activity by ADAMTSs [58].

Although, the hyaline cartilage consists of the chondrocytes which are the only cell type; this cell population has shown different morphological properties under the microscope and has been identified as dark, light, and adipocyte-like

**Figure 2.** *Schematic view of aggrecan proteoglycan [50, 55, 56].*

(adipochondrocytes) [59]. Chondrocytes manage the functional regulation of joint. These cell groups provide the synthesis and degradation of the ECM compnents, to support growth and regeneration, through the maintenance the gene expression and metabolism responded by mechanical stimuli. Extracellular matrix and proteoglycans are expressed by chondrocytes in articular joints [60–64].

The interleukins (ILs) and tumor necrosis factor α (TNF-α) cytokines are synthesized by chondrocytes, synovium, or inflammatory cells [7, 65]. Proteoglycan depletion has been stimulating physiopathologic processes, which is the main cause of degenerative joint diseases, e.g., osteoarthritis [66, 67]. Interleukins and TNF-α exert their effects on many diseases nonselectively from dental to cancer [68, 69], despite that a biomarker must be tissue-specific [70, 71]. Thus, in the last decade, the equine orthopedic researches have been deeply focused on proteoglycans, especially aggrecanases.

#### **3.2 Aggrecanases on cartilage physiopathology**

As it is well known, the proteases are responsible for proteolysis processes, which are catalytic enzymes to breakdown of the proteins into small polypeptides and amino acids by cleaved peptide bonds. The metalloproteinase superfamily members show their proteinase activity on osteoarthritis formation throughout the physiopathological processes. The matrix metalloproteinases (MMPs) and their endogenous inhibitors tissue inhibitors of metalloproteinases (TIMPs) are extensively studied; besides this, recent advances have indicated that the role of ADAMTS proteinase family is more considerable due to its abundant and specific aggrecanase activity by ADAMTS-4 and -5.

The aggrecan residues which cleaved at the glutamate 373-alanine 374 bond between the G1 and G2 interglobular domains were found at the synovial fluid analysis from various joint diseases (inflammatory or noninflammatory) in humans [72]. The first aggrecanase was purified and cloned by Tortorella et.al and named as aggrecanase-1 (currently termed as ADAMTS-4). They showed that ADAMTS-4 cleaves the aggrecan at the glutamic acid-373-alanine-374 bond [73]. After a while, Abbaszade et al. described aggrecanase-2 and named ADAMTS-11 (presently known as ADAMTS-5) [74]. ADAMTS-4 and ADAMTS-5 cleave the aggrecan at five common aggrecanase specific sites (Glu373-Ala374, Glu1480-Gly1481, Glu1667- Gly1668, Glu1771-Ala1772, and Glu1871-Leu1872,); nonetheless, ADAMTS-5 cleaves an additional site (Glu1480-Gly1481). Moreover, ADAMTS-5 is approximately twice slower than ADAMTS-4 [75, 76].

ADAMTS-4 and -5 are distributed in equine hoof lamina [78] and joints [79] and are expressed more in cartilage tissue than other tissues [80]. In our study, we observed concour horses after 50 minutes of a regular exercise program. As a result the serum ADAMTS-5 levels significantly increased but ADAMTS-4 did not. We concluded that ADAMTS-4 and ADAMTS-5 are using different pathways to physiologic and physiopathologic response [81]. Additionally and interestingly, the owners, whose horses had higher individual ADAMTS-5 serum levels, called the local veterinarians to complain about an orthopedic problem two or three weeks after our observations (unpublished data).

ADAMTS-4 needs to interact with sulfated GAGs that are attached to aggrecan core protein in order to effectively aggrecan degradation [58, 82]. ADAMTS-4 lacks the thrombospondin repeat domain on C-terminal region (**Figure 3**). This unique configuration allows bind to the adhesive glycoprotein fibronectin [82, 83]. Fibronectin is a glycoprotein that is found in low levels under physiologic conditions at the articular surface of cartilage and increases on pathologic conditions by activating innate immune response with toll-like receptors that are responded to

**97**

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health*

regulate the innate immune system in case of pathogen-related inflammation [84, 85]. Hashimoto et al. reported that fibronectin is a novel inhibitor of ADAMTS4 activity in addition to its original endogenous inhibitor TIMP-3. Hence, fibronectin could be a potent preventive therapeutic against aggrecan degradation related to degenerative joint diseases [82]. While ADAMTS-4 is mediated by TNF-α, IL-1β, and nuclear factor-kappa B (NFκB) released from synovial macrophages, the regulation of ADAMTS-5 is not totally but predominantly independent of the

*Domain organization of ADAMTS aggrecanases. SP: signal peptide; T: thrombospondin type 1 motif; and* 

The differences between synthesis pathways of ADAMTS-4 and ADAMTS-5 have to be taken into consideration on the TNF-α and IL-1 neutralization-targeted cytokine inhibitor therapies throughout degenerative joint diseases. In addition to classical therapy strategies, novel gene therapies are arising nowadays. An exciting work on this subject is a knockout murine model by the correction of ADAMTS-13 gene, which causes von Willebrand disease and leads to thrombotic thrombocytopenic purpura [87]. Transgenic animal studies with ADAMTS-4 and -5 double knockout mice [88, 89] revealed that aggrecan deletion protects from progressive osteoarthritis. These results have indicated that ADAMTS-4 and -5 may be potent therapeutic agents against laminitis and osteoarthritis, tendon, and ligament

Special thanks to Fulya Kandır for her patience and support during the writing process, Cemil Erdem (Cearts Creative Agency) for drawing the **Figure 3** and Mac

*DOI: http://dx.doi.org/10.5772/intechopen.93046*

these cytokine response [86].

injuries for equine gene therapy.

Hamidou Camara for his support.

**Acknowledgements**

**4. Conclusion**

**Figure 3.**

*CYS: cysteine [77].*

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health DOI: http://dx.doi.org/10.5772/intechopen.93046*


**Figure 3.**

*Equine Science*

cans, especially aggrecanases.

**3.2 Aggrecanases on cartilage physiopathology**

aggrecanase activity by ADAMTS-4 and -5.

mately twice slower than ADAMTS-4 [75, 76].

after our observations (unpublished data).

(adipochondrocytes) [59]. Chondrocytes manage the functional regulation of joint. These cell groups provide the synthesis and degradation of the ECM compnents, to support growth and regeneration, through the maintenance the gene expression and metabolism responded by mechanical stimuli. Extracellular matrix and proteo-

The interleukins (ILs) and tumor necrosis factor α (TNF-α) cytokines are synthesized by chondrocytes, synovium, or inflammatory cells [7, 65]. Proteoglycan depletion has been stimulating physiopathologic processes, which is the main cause of degenerative joint diseases, e.g., osteoarthritis [66, 67]. Interleukins and TNF-α exert their effects on many diseases nonselectively from dental to cancer [68, 69], despite that a biomarker must be tissue-specific [70, 71]. Thus, in the last decade, the equine orthopedic researches have been deeply focused on proteogly-

As it is well known, the proteases are responsible for proteolysis processes, which are catalytic enzymes to breakdown of the proteins into small polypeptides and amino acids by cleaved peptide bonds. The metalloproteinase superfamily members show their proteinase activity on osteoarthritis formation throughout the physiopathological processes. The matrix metalloproteinases (MMPs) and their endogenous inhibitors tissue inhibitors of metalloproteinases (TIMPs) are extensively studied; besides this, recent advances have indicated that the role of ADAMTS proteinase family is more considerable due to its abundant and specific

The aggrecan residues which cleaved at the glutamate 373-alanine 374 bond between the G1 and G2 interglobular domains were found at the synovial fluid analysis from various joint diseases (inflammatory or noninflammatory) in humans [72]. The first aggrecanase was purified and cloned by Tortorella et.al and named as aggrecanase-1 (currently termed as ADAMTS-4). They showed that ADAMTS-4 cleaves the aggrecan at the glutamic acid-373-alanine-374 bond [73]. After a while, Abbaszade et al. described aggrecanase-2 and named ADAMTS-11 (presently known as ADAMTS-5) [74]. ADAMTS-4 and ADAMTS-5 cleave the aggrecan at five common aggrecanase specific sites (Glu373-Ala374, Glu1480-Gly1481, Glu1667- Gly1668, Glu1771-Ala1772, and Glu1871-Leu1872,); nonetheless, ADAMTS-5 cleaves an additional site (Glu1480-Gly1481). Moreover, ADAMTS-5 is approxi-

ADAMTS-4 and -5 are distributed in equine hoof lamina [78] and joints [79] and are expressed more in cartilage tissue than other tissues [80]. In our study, we observed concour horses after 50 minutes of a regular exercise program. As a result the serum ADAMTS-5 levels significantly increased but ADAMTS-4 did not. We concluded that ADAMTS-4 and ADAMTS-5 are using different pathways to physiologic and physiopathologic response [81]. Additionally and interestingly, the owners, whose horses had higher individual ADAMTS-5 serum levels, called the local veterinarians to complain about an orthopedic problem two or three weeks

ADAMTS-4 needs to interact with sulfated GAGs that are attached to aggrecan

core protein in order to effectively aggrecan degradation [58, 82]. ADAMTS-4 lacks the thrombospondin repeat domain on C-terminal region (**Figure 3**). This unique configuration allows bind to the adhesive glycoprotein fibronectin [82, 83]. Fibronectin is a glycoprotein that is found in low levels under physiologic conditions at the articular surface of cartilage and increases on pathologic conditions by activating innate immune response with toll-like receptors that are responded to

glycans are expressed by chondrocytes in articular joints [60–64].

**96**

*Domain organization of ADAMTS aggrecanases. SP: signal peptide; T: thrombospondin type 1 motif; and CYS: cysteine [77].*

regulate the innate immune system in case of pathogen-related inflammation [84, 85]. Hashimoto et al. reported that fibronectin is a novel inhibitor of ADAMTS4 activity in addition to its original endogenous inhibitor TIMP-3. Hence, fibronectin could be a potent preventive therapeutic against aggrecan degradation related to degenerative joint diseases [82]. While ADAMTS-4 is mediated by TNF-α, IL-1β, and nuclear factor-kappa B (NFκB) released from synovial macrophages, the regulation of ADAMTS-5 is not totally but predominantly independent of the these cytokine response [86].

#### **4. Conclusion**

The differences between synthesis pathways of ADAMTS-4 and ADAMTS-5 have to be taken into consideration on the TNF-α and IL-1 neutralization-targeted cytokine inhibitor therapies throughout degenerative joint diseases. In addition to classical therapy strategies, novel gene therapies are arising nowadays. An exciting work on this subject is a knockout murine model by the correction of ADAMTS-13 gene, which causes von Willebrand disease and leads to thrombotic thrombocytopenic purpura [87]. Transgenic animal studies with ADAMTS-4 and -5 double knockout mice [88, 89] revealed that aggrecan deletion protects from progressive osteoarthritis. These results have indicated that ADAMTS-4 and -5 may be potent therapeutic agents against laminitis and osteoarthritis, tendon, and ligament injuries for equine gene therapy.

#### **Acknowledgements**

Special thanks to Fulya Kandır for her patience and support during the writing process, Cemil Erdem (Cearts Creative Agency) for drawing the **Figure 3** and Mac Hamidou Camara for his support.

*Equine Science*

### **Author details**

Sinan Kandir Department of Physiology, Faculty of Veterinary Medicine, Cukurova University Ceyhan, Adana, Turkey

\*Address all correspondence to: sinankandir@cu.edu.tr; skandir@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**99**

May 2009]

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health*

models. Cartilage. 2017;**8**(3):211-233. PubMed PMID: 28618869. PMCID: PMC5625856. [Epub: 18 June 2017]

[7] Wojdasiewicz P, Poniatowski LA, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis. Mediators of Inflammation. 2014;**2014**:561459. PubMed PMID: 24876674. PMCID: PMC4021678.

[8] Von den Hoff HW, van Kampen GP, van der Korst JK. Proteoglycan depletion of intact articular cartilage by retinoic acid is irreversible and involves loss of hyaluronate. Osteoarthritis and Cartilage. 1993;**1**(3):157-166. PubMed PMID: 15449421. [Epub: 01 July 1993]

[9] Brunet FG, Fraser FW, Binder MJ, Smith AD, Kintakas C, Dancevic CM, et al. The evolutionary conservation

[10] Modern Horse Breeding, Inc. First International Equine Gene Mapping Workshop [Internet]. Lexington, Ky: Modern Horse Breeding, Inc; 1995. Available from: https://www.uky.edu/ Ag/Horsemap/Workshop/first.html

[11] Guerin G, Bailey E, Bernoco D, Anderson I, Antczak DF, Bell K, et al. Report of the international equine gene mapping workshop: Male linkage map.

[12] Raudsepp T, Finno CJ, Bellone RR, Petersen JL. Ten years of the horse

Animal Genetics. 1999;**30**(5): 341-354. PubMed PMID: 10582279.

[Epub: 03 December 1999]

[cited 20 February 2020]

of the A disintegrin-like and metalloproteinase domain with Thrombospondin-1 motif metzincins across vertebrate species and their expression in teleost zebrafish. BMC Evolutionary Biology. 2015;**15**:22. PubMed PMID: 25879701. PMCID: PMC4349717. [Epub: 17 April 2015]

[Epub: 31 May 2014]

*DOI: http://dx.doi.org/10.5772/intechopen.93046*

[1] McIlwraith CW. Use of synovial fluid and serum biomarkers in equine bone and joint disease: A review. Equine

Veterinary Journal. 2005;**37**(5): 473-482. PubMed PMID: 16163952.

[2] Brama PA, van den Boom R, DeGroott J, Kiers GH, van

Weeren PR. Collagenase-1 (MMP-1) activity in equine synovial fluid: Influence of age, joint pathology, exercise and repeated arthrocentesis.

2004;**36**(1):34-40. PubMed PMID: 14756369. [Epub: 06 February 2004]

[3] Donabedian M, van Weeren PR, Perona G, Fleurance G, Robert C, Leger S, et al. Early changes in biomarkers of skeletal metabolism and their association to the occurrence

of osteochondrosis (OC) in the horse. Equine Veterinary Journal. 2008;**40**(3):253-259. PubMed PMID: 18267892. [Epub: 13 February 2008]

[4] Frisbie DD, Al-Sobayil F, Billinghurst RC, Kawcak CE,

McIlwraith CW. Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses. Osteoarthritis and Cartilage. 2008;**16**(10):1196-1204. PubMed PMID:

18442931. [Epub: 30 April 2008]

[5] Davies MR, Ribeiro LR, Downey-Jones M, Needham MR, Oakley C, Wardale J. Ligands for retinoic acid receptors are elevated in osteoarthritis and may contribute to pathologic processes in the osteoarthritic joint. Arthritis and Rheumatism. 2009;**60**(6):1722-1732. PubMed PMID: 19479829. [Epub: 30

[6] Legrand CB, Lambert CJ,

Comblain FV, Sanchez C, Henrotin YE. Review of soluble biomarkers of osteoarthritis: Lessons from animal

[Epub: 17 September 2005]

Equine Veterinary Journal.

**References**

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health DOI: http://dx.doi.org/10.5772/intechopen.93046*

#### **References**

*Equine Science*

**98**

**Author details**

Ceyhan, Adana, Turkey

provided the original work is properly cited.

Department of Physiology, Faculty of Veterinary Medicine, Cukurova University

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: sinankandir@cu.edu.tr; skandir@gmail.com

Sinan Kandir

[1] McIlwraith CW. Use of synovial fluid and serum biomarkers in equine bone and joint disease: A review. Equine Veterinary Journal. 2005;**37**(5): 473-482. PubMed PMID: 16163952. [Epub: 17 September 2005]

[2] Brama PA, van den Boom R, DeGroott J, Kiers GH, van Weeren PR. Collagenase-1 (MMP-1) activity in equine synovial fluid: Influence of age, joint pathology, exercise and repeated arthrocentesis. Equine Veterinary Journal. 2004;**36**(1):34-40. PubMed PMID: 14756369. [Epub: 06 February 2004]

[3] Donabedian M, van Weeren PR, Perona G, Fleurance G, Robert C, Leger S, et al. Early changes in biomarkers of skeletal metabolism and their association to the occurrence of osteochondrosis (OC) in the horse. Equine Veterinary Journal. 2008;**40**(3):253-259. PubMed PMID: 18267892. [Epub: 13 February 2008]

[4] Frisbie DD, Al-Sobayil F, Billinghurst RC, Kawcak CE, McIlwraith CW. Changes in synovial fluid and serum biomarkers with exercise and early osteoarthritis in horses. Osteoarthritis and Cartilage. 2008;**16**(10):1196-1204. PubMed PMID: 18442931. [Epub: 30 April 2008]

[5] Davies MR, Ribeiro LR, Downey-Jones M, Needham MR, Oakley C, Wardale J. Ligands for retinoic acid receptors are elevated in osteoarthritis and may contribute to pathologic processes in the osteoarthritic joint. Arthritis and Rheumatism. 2009;**60**(6):1722-1732. PubMed PMID: 19479829. [Epub: 30 May 2009]

[6] Legrand CB, Lambert CJ, Comblain FV, Sanchez C, Henrotin YE. Review of soluble biomarkers of osteoarthritis: Lessons from animal

models. Cartilage. 2017;**8**(3):211-233. PubMed PMID: 28618869. PMCID: PMC5625856. [Epub: 18 June 2017]

[7] Wojdasiewicz P, Poniatowski LA, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis. Mediators of Inflammation. 2014;**2014**:561459. PubMed PMID: 24876674. PMCID: PMC4021678. [Epub: 31 May 2014]

[8] Von den Hoff HW, van Kampen GP, van der Korst JK. Proteoglycan depletion of intact articular cartilage by retinoic acid is irreversible and involves loss of hyaluronate. Osteoarthritis and Cartilage. 1993;**1**(3):157-166. PubMed PMID: 15449421. [Epub: 01 July 1993]

[9] Brunet FG, Fraser FW, Binder MJ, Smith AD, Kintakas C, Dancevic CM, et al. The evolutionary conservation of the A disintegrin-like and metalloproteinase domain with Thrombospondin-1 motif metzincins across vertebrate species and their expression in teleost zebrafish. BMC Evolutionary Biology. 2015;**15**:22. PubMed PMID: 25879701. PMCID: PMC4349717. [Epub: 17 April 2015]

[10] Modern Horse Breeding, Inc. First International Equine Gene Mapping Workshop [Internet]. Lexington, Ky: Modern Horse Breeding, Inc; 1995. Available from: https://www.uky.edu/ Ag/Horsemap/Workshop/first.html [cited 20 February 2020]

[11] Guerin G, Bailey E, Bernoco D, Anderson I, Antczak DF, Bell K, et al. Report of the international equine gene mapping workshop: Male linkage map. Animal Genetics. 1999;**30**(5): 341-354. PubMed PMID: 10582279. [Epub: 03 December 1999]

[12] Raudsepp T, Finno CJ, Bellone RR, Petersen JL. Ten years of the horse

reference genome: Insights into equine biology, domestication and population dynamics in the post-genome era. Animal Genetics. 2019;**50**(6):569-597. PubMed PMID: 31568563. PMCID: PMC6825885. [Epub: 01 October 2019]

[13] Finno CJ, Bannasch DL. Applied equine genetics. Equine Veterinary Journal. 2014;**46**(5):538-544. PubMed PMID: 24802051. PMCID: PMC4327934. [Epub: 08 May 2014]

[14] Brosnahan MM, Brooks SA, Antczak DF. Equine clinical genomics: A clinician's primer. Equine Veterinary Journal. 2010;**42**(7):658-670. PubMed PMID: 20840582. PMCID: PMC3297474. [Epub: 16 September 2010]

[15] Wade CM, Giulotto E, Sigurdsson S, Zoli M, Gnerre S, Imsland F, et al. Genome sequence, comparative analysis, and population genetics of the domestic horse. Science. 2009;**326**(5954):865-867. PubMed PMID: 19892987. PMCID: PMC3785132. [Epub: 07 November 2009]

[16] Kalbfleisch TS, Rice ES, DePriest MS Jr, Walenz BP, Hestand MS, Vermeesch JR, et al. Improved reference genome for the domestic horse increases assembly contiguity and composition. Communications Biology. 2018;**1**:197. PubMed PMID: 30456315. PMCID: PMC6240028 adviser of Dovetail Genomics, LLC. The other authors declare no competing interests. [Epub: 21 November 2018]

[17] Braschi B, Denny P, Gray K, Jones T, Seal R, Tweedie S, et al. Genenames. org: The HGNC and VGNC resources in 2019. Nucleic Acids Research. 2019;**47**(D1):D786-DD92. PubMed PMID: 30304474. PMCID: PMC6324057. [Epub: 12 October 2018]

[18] Cunningham F, Achuthan P, Akanni W, Allen J, Amode MR, Armean IM, et al. Ensembl 2019. Nucleic Acids Research.

2019;**47**(D1):D745-DD51. PubMed PMID: 30407521. PMCID: PMC6323964. [Epub: 09 November 2018]

[19] Hestand MS, Kalbfleisch TS, Coleman SJ, Zeng Z, Liu J, Orlando L, et al. Annotation of the protein coding regions of the equine genome. PLoS One. 2015;**10**(6):e0124375. PubMed PMID: 26107351. PMCID: PMC4481266. [Epub: 25 June 2015]

[20] Kuno K, Kanada N, Nakashima E, Fujiki F, Ichimura F, Matsushima K. Molecular cloning of a gene encoding a new type of metalloproteinasedisintegrin family protein with thrombospondin motifs as an inflammation associated gene. The Journal of Biological Chemistry. 1997;**272**(1):556-562. PubMed PMID: 8995297. [Epub: 03 January 1997]

[21] Fessler JH, Kramerova I, Kramerov A, Chen Y, Fessler LI. Papilin, a novel component of basement membranes, in relation to ADAMTS metalloproteases and ECM development. The International Journal of Biochemistry & Cell Biology. 2004;**36**(6):1079-1084. PubMed PMID: 15094122. [Epub: 20 April 2004]

[22] Dubail J, Apte SS. Insights on ADAMTS proteases and ADAMTS-like proteins from mammalian genetics. Matrix Biology. 2015;**44-46**:24-37. PubMed PMID: 25770910. [Epub: 17 March 2015]

[23] Hirohata S, Wang LW, Miyagi M, Yan L, Seldin MF, Keene DR, et al. Punctin, a novel ADAMTS-like molecule, ADAMTSL-1, in extracellular matrix. The Journal of Biological Chemistry. 2002;**277**(14):12182-12189. PubMed PMID: 11805097. [Epub: 24 January 2002]

[24] Kramerova IA, Kawaguchi N, Fessler LI, Nelson RE, Chen Y, Kramerov AA, et al. Papilin in development; a pericellular protein

**101**

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health*

[31] Tang BL, Hong W. ADAMTS: A novel family of proteases with an ADAM protease domain and thrombospondin 1

[32] Colige A, Nuytinck L, Hausser I, van Essen AJ, Thiry M, Herens C, et al. Novel types of mutation responsible for the dermatosparactic type of Ehlers-Danlos syndrome (type VIIC) and common polymorphisms in the ADAMTS2 gene. The Journal of Investigative Dermatology.

2004;**123**(4):656-663. PubMed PMID: 15373769. [Epub: 18 September 2004]

[33] Carty CI, Lee AM, Wienandt NA, Stevens EL, Alves DA, Browne JA, et al. Dermatosparaxis in two limousin calves. Irish Veterinary Journal. 2016;**69**:15. PubMed PMID: 27777746. PMCID: PMC5070005. [Epub: 26 November 2016]

[34] Colige A, Sieron AL, Li SW, Schwarze U, Petty E, Wertelecki W, et al. Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis

are caused by mutations in the procollagen I N-proteinase gene. American Journal of Human Genetics. 1999;**65**(2):308-317. PubMed PMID: 10417273. PMCID: PMC1377929. [Epub:

[35] Dagoneau N, Benoist-Lasselin C, Huber C, Faivre L, Megarbane A, Alswaid A, et al. ADAMTS10 mutations in autosomal recessive Weill-Marchesani syndrome. American Journal of Human Genetics. 2004;**75**(5):801-806. PubMed PMID: 15368195. PMCID: PMC1182109.

[36] Farias FH, Johnson GS, Taylor JF, Giuliano E, Katz ML, Sanders DN, et al. An ADAMTS17 splice donor site mutation in dogs with primary lens luxation. Investigative Ophthalmology & Visual Science. 2010;**51**(9):4716-4721. PubMed PMID: 20375329. [Epub: 09

[Epub: 16 September 2004]

27 July 1999]

April 2010]

repeats. FEBS Letters. 1999;**445** (2-3):223-225. PubMed PMID: 10094461. [Epub: 27 March 1999]

*DOI: http://dx.doi.org/10.5772/intechopen.93046*

with a homology to the ADAMTS metalloproteinases. Development. 2000;**127**(24):5475-5485. PubMed PMID: 11076767. [Epub: 15 November

[25] Cerda-Costa N, Gomis-Ruth FX.

[26] Petri A, Kim HJ, Xu Y, de Groot R, Li C, Vandenbulcke A, et al. Crystal structure and substrate-induced activation of ADAMTS13. Nature Communications. 2019;**10**(1):3781. PubMed PMID: 31439947. PMCID: PMC6706451. [Epub: 24 August 2019]

[27] Bork P, Beckmann G. The CUB domain. A widespread module in developmentally regulated proteins. Journal of Molecular Biology.

1993;**231**(2):539-545. PubMed PMID:

8510165. [Epub: 20 May 1993]

01 July 2005]

August 2009]

[28] Jones GC, Riley GP. ADAMTS proteinases: A multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis. Arthritis Research & Therapy. 2005;**7**(4):160-169. PubMed PMID: 15987500. PMCID: PMC1175049. [Epub:

[29] van Goor H, Melenhorst WB, Turner AJ, Holgate ST. Adamalysins in biology and disease. The Journal of Pathology. 2009;**219**(3):277-286. PubMed PMID: 19662664. [Epub: 08

[30] Cal S, Obaya AJ, Llamazares M, Garabaya C, Quesada V, Lopez-Otin C. Cloning, expression analysis, and structural characterization of seven novel human ADAMTSs, a family of metalloproteinases with disintegrin and thrombospondin-1 domains. Gene. 2002;**283**(1-2):49-62. PubMed PMID: 11867212. [Epub: 28 February 2002]

Architecture and function of metallopeptidase catalytic domains. Protein Science. 2014;**23**(2):123-144. PubMed PMID: 24596965. PMCID: PMC3926739. [Epub: 07 March 2014]

2000]

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health DOI: http://dx.doi.org/10.5772/intechopen.93046*

with a homology to the ADAMTS metalloproteinases. Development. 2000;**127**(24):5475-5485. PubMed PMID: 11076767. [Epub: 15 November 2000]

*Equine Science*

reference genome: Insights into equine biology, domestication and population dynamics in the post-genome era. Animal Genetics. 2019;**50**(6):569-597. PubMed PMID: 31568563. PMCID: PMC6825885. [Epub: 01 October 2019] 2019;**47**(D1):D745-DD51. PubMed PMID: 30407521. PMCID: PMC6323964.

[19] Hestand MS, Kalbfleisch TS, Coleman SJ, Zeng Z, Liu J, Orlando L, et al. Annotation of the protein coding regions of the equine genome. PLoS One. 2015;**10**(6):e0124375. PubMed PMID: 26107351. PMCID: PMC4481266.

[20] Kuno K, Kanada N, Nakashima E, Fujiki F, Ichimura F, Matsushima K. Molecular cloning of a gene encoding a new type of metalloproteinasedisintegrin family protein with thrombospondin motifs as an inflammation associated gene. The Journal of Biological Chemistry. 1997;**272**(1):556-562. PubMed PMID: 8995297. [Epub: 03 January 1997]

[21] Fessler JH, Kramerova I,

metalloproteases and ECM development. The International

a novel component of basement membranes, in relation to ADAMTS

[22] Dubail J, Apte SS. Insights on ADAMTS proteases and ADAMTS-like proteins from mammalian genetics. Matrix Biology. 2015;**44-46**:24-37. PubMed PMID: 25770910. [Epub: 17

[23] Hirohata S, Wang LW, Miyagi M, Yan L, Seldin MF, Keene DR, et al. Punctin, a novel ADAMTS-like

molecule, ADAMTSL-1, in extracellular matrix. The Journal of Biological Chemistry. 2002;**277**(14):12182-12189. PubMed PMID: 11805097. [Epub: 24

[24] Kramerova IA, Kawaguchi N, Fessler LI, Nelson RE, Chen Y, Kramerov AA, et al. Papilin in development; a pericellular protein

March 2015]

January 2002]

Kramerov A, Chen Y, Fessler LI. Papilin,

Journal of Biochemistry & Cell Biology. 2004;**36**(6):1079-1084. PubMed PMID: 15094122. [Epub: 20 April 2004]

[Epub: 09 November 2018]

[Epub: 25 June 2015]

[13] Finno CJ, Bannasch DL. Applied equine genetics. Equine Veterinary Journal. 2014;**46**(5):538-544. PubMed PMID: 24802051. PMCID: PMC4327934.

[14] Brosnahan MM, Brooks SA, Antczak DF. Equine clinical genomics: A clinician's primer. Equine Veterinary Journal. 2010;**42**(7):658-670. PubMed PMID: 20840582. PMCID: PMC3297474.

[Epub: 16 September 2010]

[15] Wade CM, Giulotto E, Sigurdsson S, Zoli M, Gnerre S, Imsland F, et al. Genome sequence, comparative analysis, and population genetics of the domestic horse. Science. 2009;**326**(5954):865-867. PubMed PMID: 19892987. PMCID: PMC3785132.

[Epub: 07 November 2009]

21 November 2018]

[Epub: 12 October 2018]

Nucleic Acids Research.

[18] Cunningham F, Achuthan P, Akanni W, Allen J, Amode MR, Armean IM, et al. Ensembl 2019.

[16] Kalbfleisch TS, Rice ES, DePriest MS Jr, Walenz BP, Hestand MS,

Vermeesch JR, et al. Improved reference genome for the domestic horse increases assembly contiguity and composition. Communications Biology. 2018;**1**:197. PubMed PMID: 30456315. PMCID: PMC6240028 adviser of Dovetail Genomics, LLC. The other authors declare no competing interests. [Epub:

[17] Braschi B, Denny P, Gray K, Jones T, Seal R, Tweedie S, et al. Genenames. org: The HGNC and VGNC resources in 2019. Nucleic Acids Research. 2019;**47**(D1):D786-DD92. PubMed PMID: 30304474. PMCID: PMC6324057.

[Epub: 08 May 2014]

**100**

[25] Cerda-Costa N, Gomis-Ruth FX. Architecture and function of metallopeptidase catalytic domains. Protein Science. 2014;**23**(2):123-144. PubMed PMID: 24596965. PMCID: PMC3926739. [Epub: 07 March 2014]

[26] Petri A, Kim HJ, Xu Y, de Groot R, Li C, Vandenbulcke A, et al. Crystal structure and substrate-induced activation of ADAMTS13. Nature Communications. 2019;**10**(1):3781. PubMed PMID: 31439947. PMCID: PMC6706451. [Epub: 24 August 2019]

[27] Bork P, Beckmann G. The CUB domain. A widespread module in developmentally regulated proteins. Journal of Molecular Biology. 1993;**231**(2):539-545. PubMed PMID: 8510165. [Epub: 20 May 1993]

[28] Jones GC, Riley GP. ADAMTS proteinases: A multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis. Arthritis Research & Therapy. 2005;**7**(4):160-169. PubMed PMID: 15987500. PMCID: PMC1175049. [Epub: 01 July 2005]

[29] van Goor H, Melenhorst WB, Turner AJ, Holgate ST. Adamalysins in biology and disease. The Journal of Pathology. 2009;**219**(3):277-286. PubMed PMID: 19662664. [Epub: 08 August 2009]

[30] Cal S, Obaya AJ, Llamazares M, Garabaya C, Quesada V, Lopez-Otin C. Cloning, expression analysis, and structural characterization of seven novel human ADAMTSs, a family of metalloproteinases with disintegrin and thrombospondin-1 domains. Gene. 2002;**283**(1-2):49-62. PubMed PMID: 11867212. [Epub: 28 February 2002]

[31] Tang BL, Hong W. ADAMTS: A novel family of proteases with an ADAM protease domain and thrombospondin 1 repeats. FEBS Letters. 1999;**445** (2-3):223-225. PubMed PMID: 10094461. [Epub: 27 March 1999]

[32] Colige A, Nuytinck L, Hausser I, van Essen AJ, Thiry M, Herens C, et al. Novel types of mutation responsible for the dermatosparactic type of Ehlers-Danlos syndrome (type VIIC) and common polymorphisms in the ADAMTS2 gene. The Journal of Investigative Dermatology. 2004;**123**(4):656-663. PubMed PMID: 15373769. [Epub: 18 September 2004]

[33] Carty CI, Lee AM, Wienandt NA, Stevens EL, Alves DA, Browne JA, et al. Dermatosparaxis in two limousin calves. Irish Veterinary Journal. 2016;**69**:15. PubMed PMID: 27777746. PMCID: PMC5070005. [Epub: 26 November 2016]

[34] Colige A, Sieron AL, Li SW, Schwarze U, Petty E, Wertelecki W, et al. Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations in the procollagen I N-proteinase gene. American Journal of Human Genetics. 1999;**65**(2):308-317. PubMed PMID: 10417273. PMCID: PMC1377929. [Epub: 27 July 1999]

[35] Dagoneau N, Benoist-Lasselin C, Huber C, Faivre L, Megarbane A, Alswaid A, et al. ADAMTS10 mutations in autosomal recessive Weill-Marchesani syndrome. American Journal of Human Genetics. 2004;**75**(5):801-806. PubMed PMID: 15368195. PMCID: PMC1182109. [Epub: 16 September 2004]

[36] Farias FH, Johnson GS, Taylor JF, Giuliano E, Katz ML, Sanders DN, et al. An ADAMTS17 splice donor site mutation in dogs with primary lens luxation. Investigative Ophthalmology & Visual Science. 2010;**51**(9):4716-4721. PubMed PMID: 20375329. [Epub: 09 April 2010]

[37] Allali S, Le Goff C, Pressac-Diebold I, Pfennig G, Mahaut C, Dagoneau N, et al. Molecular screening of ADAMTSL2 gene in 33 patients reveals the genetic heterogeneity of geleophysic dysplasia. Journal of Medical Genetics. 2011;**48**(6):417-421. PubMed PMID: 21415077. PMCID: PMC4413937. [Epub: 19 March 2011]

[38] Bader HL, Ruhe AL, Wang LW, Wong AK, Walsh KF, Packer RA, et al. An ADAMTSL2 founder mutation causes Musladin-Lueke syndrome, a heritable disorder of beagle dogs, featuring stiff skin and joint contractures. PLoS One. 2010;**5**(9):e12817. PubMed PMID: 20862248. PMCID: PMC2941456. [Epub: 24 September 2010]

[39] Zheng XL. ADAMTS13 and von Willebrand factor in thrombotic thrombocytopenic purpura. Annual Review of Medicine. 2015;**66**:211-225. PubMed PMID: 25587650. PMCID: PMC4599565. [Epub: 15 January 2015]

[40] Graves KT, Henney PJ, Ennis RB. Partial deletion of the LAMA3 gene is responsible for hereditary junctional epidermolysis bullosa in the American Saddlebred horse. Animal Genetics. 2009;**40**(1):35-41. PubMed PMID: 19016681. [Epub: 20 November 2008]

[41] White SD, Affolter VK, Bannasch DL, Schultheiss PC, Hamar DW, Chapman PL, et al. Hereditary equine regional dermal asthenia ("hyperelastosis cutis") in 50 horses: Clinical, histological, immunohistological and ultrastructural findings. Veterinary Dermatology. 2004;**15**(4):207-217. PubMed PMID: 15305927. [Epub: 13 August 2004]

[42] Rathgeber RA, Brooks MB, Bain FT, Byars TD. Clinical vignette. Von Willebrand disease in a thoroughbred mare and foal. Journal of Veterinary Internal Medicine. 2001;**15**(1):63-66. PubMed PMID: 11215915. [Epub: 24 February 2001]

[43] Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985;**39**(4):783- 791. PubMed PMID: 28561359. [Epub: 01 July 1985]

[44] Nei M, Kumar S. Molecular Evolution and Phylogenetics. Oxford, New York: Oxford University Press; 2000. xiv, p. 333

[45] Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 2018;**35**(6):1547-1549. PubMed PMID: 29722887. PMCID: PMC5967553. [Epub: 04 July 2018]

[46] Sophia Fox AJ, Bedi A, Rodeo SA. The basic science of articular cartilage: Structure, composition, and function. Sports Health. 2009;**1**(6):461-468. PubMed PMID: 23015907. PMCID: PMC3445147. [Epub: 01 November 2009]

[47] Hall BK. Bones and Cartilage : Developmental and Evolutionary Skeletal Biology. Amsterdam/London: Elsevier Academic; 2005

[48] Seibel MJ, Robins SP, Bilezikian JP. Dynamics of Bone and Cartilage Metabolism. 2nd ed. United States of America: Academic Press; 2006

[49] Smith DW, Gardiner BS, Zhang L, Grodzinsky AJ. Articular Cartilage Dynamics. Berlin, Heidelberg, New York, NY: Springer; 2018

[50] Iozzo RV, Schaefer L. Proteoglycan form and function: A comprehensive nomenclature of proteoglycans. Matrix Biology. 2015;**42**:11-55. PubMed PMID: 25701227. PMCID: PMC4859157. [Epub: 24 February 2015]

[51] Kjellen L, Lindahl U. Proteoglycans: Structures and interactions. Annual Review of Biochemistry. 1991;**60**:

**103**

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health*

2000]

aggrecan glycosylation affect cleavage

2000;**275**(50):39096-39102. PubMed PMID: 10991945. [Epub: 19 September

heterogeneity; it is the time to update the understanding of cartilage histology. SVU-International Journal of Veterinary

[60] Hall AC. The role of chondrocyte

controlling phenotype-implications for osteoarthritis, cartilage repair, and cartilage engineering. Current Rheumatology Reports. 2019;**21**(8):38. PubMed PMID: 31203465. PMCID: PMC6571082. [Epub: 17 June 2019]

[61] Hall AC, Bush PG, Davidson ME, Kempson SA. Equine articular cartilage chondrocytes: Opening the black box. Equine Veterinary Journal. 2003;**35**(5):425-428. PubMed PMID: 12875317. [Epub: 24 July 2003]

[62] Wilkins RJ, Browning JA, Ellory JC. Surviving in a matrix: Membrane transport in articular chondrocytes. The Journal of Membrane Biology. 2000;**177**(2):95-108. PubMed PMID: 11003684. [Epub: 26 September 2000]

[63] Mobasheri A, Matta C, Uzieliene I, Budd E, Martin-Vasallo P, Bernotiene E. The chondrocyte channelome: A narrative review. Joint, Bone, Spine. 2019;**86**(1):29-35. PubMed PMID: 29452304. [Epub: 17 February 2018]

[64] O'Conor CJ, Leddy HA,

Benefield HC, Liedtke WB, Guilak F. TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**(4):1316-1321. PubMed PMID: 24474754. PMCID: PMC3910592. [Epub: 30 January 2014]

by aggrecanase. The Journal of Biological Chemistry.

[59] Ahmed Y. Chondrocyte

Sciences. 2018;**1**(2):1-3

morphology and volume in

*DOI: http://dx.doi.org/10.5772/intechopen.93046*

443-475. PubMed PMID: 1883201.

[53] Viitanen M, Bird J, Smith R, Tulamo RM, May SA. Biochemical characterisation of navicular hyaline cartilage, navicular fibrocartilage and the deep digital flexor tendon in horses with navicular disease. Research in Veterinary Science. 2003;**75**(2):113-120. PubMed PMID: 12893159. [Epub: 02

[54] Nugent GE, Law AW, Wong EG, Temple MM, Bae WC, Chen AC, et al. Site- and exercise-related variation in structure and function of cartilage from equine distal metacarpal

condyle. Osteoarthritis and Cartilage. 2004;**12**(10):826-833. PubMed PMID: 15450533. [Epub: 29 September 2004]

[55] Kiani C, Chen L, Wu YJ, Yee AJ, Yang BB. Structure and function of aggrecan. Cell Research. 2002;**12**(1): 19-32. PubMed PMID: 11942407. [Epub:

[56] Caporali EH, Kuykendall T, Stewart MC. Complete sequencing and characterization of equine

aggrecan. Veterinary and Comparative Orthopaedics and Traumatology. 2015;**28**(2):79-87. PubMed PMID: 25632964. [Epub: 31 January 2015]

[57] Aspberg A. The different roles of aggrecan interaction domains. The Journal of Histochemistry and Cytochemistry. 2012;**60**(12):987-996. PubMed PMID: 23019016. PMCID: PMC3527881. [Epub: 29 September 2012]

[58] Pratta MA, Tortorella MD, Arner EC. Age-related changes in

[52] Murray RC, Birch HL, Lakhani K, Goodship AE. Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specific manner. Osteoarthritis and Cartilage. 2001;**9**(7):625-632. PubMed PMID: 11597175. [Epub: 13 October

[Epub: 01 January 1991]

2001]

August 2003]

11 April 2002]

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health DOI: http://dx.doi.org/10.5772/intechopen.93046*

443-475. PubMed PMID: 1883201. [Epub: 01 January 1991]

*Equine Science*

[37] Allali S, Le Goff C, Pressac-Diebold I, Pfennig G, Mahaut C, Dagoneau N, et al. Molecular screening of ADAMTSL2 gene in 33 patients reveals the genetic heterogeneity of geleophysic dysplasia. Journal of Medical Genetics. 2011;**48**(6):417-421. PubMed PMID: 21415077. PMCID: PMC4413937. [Epub: 19 March 2011]

[43] Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985;**39**(4):783- 791. PubMed PMID: 28561359. [Epub:

[44] Nei M, Kumar S. Molecular Evolution and Phylogenetics. Oxford, New York: Oxford University Press;

[45] Kumar S, Stecher G, Li M,

Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 2018;**35**(6):1547-1549. PubMed PMID: 29722887. PMCID: PMC5967553. [Epub: 04 July 2018]

[46] Sophia Fox AJ, Bedi A, Rodeo SA. The basic science of articular cartilage: Structure, composition, and function. Sports Health. 2009;**1**(6):461-468. PubMed PMID: 23015907. PMCID: PMC3445147. [Epub: 01 November

[47] Hall BK. Bones and Cartilage : Developmental and Evolutionary Skeletal Biology. Amsterdam/London:

[48] Seibel MJ, Robins SP, Bilezikian JP. Dynamics of Bone and Cartilage Metabolism. 2nd ed. United States of America: Academic Press; 2006

[49] Smith DW, Gardiner BS, Zhang L, Grodzinsky AJ. Articular Cartilage Dynamics. Berlin, Heidelberg, New

[50] Iozzo RV, Schaefer L. Proteoglycan form and function: A comprehensive nomenclature of proteoglycans. Matrix Biology. 2015;**42**:11-55. PubMed PMID: 25701227. PMCID: PMC4859157. [Epub:

[51] Kjellen L, Lindahl U. Proteoglycans: Structures and interactions. Annual Review of Biochemistry. 1991;**60**:

Elsevier Academic; 2005

York, NY: Springer; 2018

24 February 2015]

01 July 1985]

2000. xiv, p. 333

2009]

[38] Bader HL, Ruhe AL, Wang LW, Wong AK, Walsh KF, Packer RA, et al. An ADAMTSL2 founder mutation causes Musladin-Lueke syndrome, a heritable disorder of beagle dogs, featuring stiff skin and joint contractures. PLoS One. 2010;**5**(9):e12817. PubMed PMID: 20862248. PMCID: PMC2941456. [Epub: 24 September 2010]

[39] Zheng XL. ADAMTS13 and von Willebrand factor in thrombotic thrombocytopenic purpura. Annual Review of Medicine. 2015;**66**:211-225. PubMed PMID: 25587650. PMCID: PMC4599565. [Epub: 15 January 2015]

[40] Graves KT, Henney PJ, Ennis RB. Partial deletion of the LAMA3 gene is responsible for hereditary junctional epidermolysis bullosa in the American Saddlebred horse. Animal Genetics. 2009;**40**(1):35-41. PubMed PMID: 19016681. [Epub: 20 November 2008]

[41] White SD, Affolter VK, Bannasch DL, Schultheiss PC, Hamar DW, Chapman PL, et al. Hereditary equine regional dermal asthenia ("hyperelastosis cutis") in 50 horses: Clinical, histological, immunohistological and ultrastructural findings. Veterinary Dermatology. 2004;**15**(4):207-217. PubMed PMID: 15305927. [Epub: 13 August 2004]

[42] Rathgeber RA, Brooks MB,

Bain FT, Byars TD. Clinical vignette. Von Willebrand disease in a thoroughbred mare and foal. Journal of Veterinary Internal Medicine. 2001;**15**(1):63-66. PubMed PMID: 11215915. [Epub: 24

**102**

February 2001]

[52] Murray RC, Birch HL, Lakhani K, Goodship AE. Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specific manner. Osteoarthritis and Cartilage. 2001;**9**(7):625-632. PubMed PMID: 11597175. [Epub: 13 October 2001]

[53] Viitanen M, Bird J, Smith R, Tulamo RM, May SA. Biochemical characterisation of navicular hyaline cartilage, navicular fibrocartilage and the deep digital flexor tendon in horses with navicular disease. Research in Veterinary Science. 2003;**75**(2):113-120. PubMed PMID: 12893159. [Epub: 02 August 2003]

[54] Nugent GE, Law AW, Wong EG, Temple MM, Bae WC, Chen AC, et al. Site- and exercise-related variation in structure and function of cartilage from equine distal metacarpal condyle. Osteoarthritis and Cartilage. 2004;**12**(10):826-833. PubMed PMID: 15450533. [Epub: 29 September 2004]

[55] Kiani C, Chen L, Wu YJ, Yee AJ, Yang BB. Structure and function of aggrecan. Cell Research. 2002;**12**(1): 19-32. PubMed PMID: 11942407. [Epub: 11 April 2002]

[56] Caporali EH, Kuykendall T, Stewart MC. Complete sequencing and characterization of equine aggrecan. Veterinary and Comparative Orthopaedics and Traumatology. 2015;**28**(2):79-87. PubMed PMID: 25632964. [Epub: 31 January 2015]

[57] Aspberg A. The different roles of aggrecan interaction domains. The Journal of Histochemistry and Cytochemistry. 2012;**60**(12):987-996. PubMed PMID: 23019016. PMCID: PMC3527881. [Epub: 29 September 2012]

[58] Pratta MA, Tortorella MD, Arner EC. Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase. The Journal of Biological Chemistry. 2000;**275**(50):39096-39102. PubMed PMID: 10991945. [Epub: 19 September 2000]

[59] Ahmed Y. Chondrocyte heterogeneity; it is the time to update the understanding of cartilage histology. SVU-International Journal of Veterinary Sciences. 2018;**1**(2):1-3

[60] Hall AC. The role of chondrocyte morphology and volume in controlling phenotype-implications for osteoarthritis, cartilage repair, and cartilage engineering. Current Rheumatology Reports. 2019;**21**(8):38. PubMed PMID: 31203465. PMCID: PMC6571082. [Epub: 17 June 2019]

[61] Hall AC, Bush PG, Davidson ME, Kempson SA. Equine articular cartilage chondrocytes: Opening the black box. Equine Veterinary Journal. 2003;**35**(5):425-428. PubMed PMID: 12875317. [Epub: 24 July 2003]

[62] Wilkins RJ, Browning JA, Ellory JC. Surviving in a matrix: Membrane transport in articular chondrocytes. The Journal of Membrane Biology. 2000;**177**(2):95-108. PubMed PMID: 11003684. [Epub: 26 September 2000]

[63] Mobasheri A, Matta C, Uzieliene I, Budd E, Martin-Vasallo P, Bernotiene E. The chondrocyte channelome: A narrative review. Joint, Bone, Spine. 2019;**86**(1):29-35. PubMed PMID: 29452304. [Epub: 17 February 2018]

[64] O'Conor CJ, Leddy HA, Benefield HC, Liedtke WB, Guilak F. TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**(4):1316-1321. PubMed PMID: 24474754. PMCID: PMC3910592. [Epub: 30 January 2014]

[65] Beutler BA. The role of tumor necrosis factor in health and disease. The Journal of Rheumatology. Supplement. 1999;**57**:16-21. PubMed PMID: 10328138. [Epub: 18 May 1999]

[66] Loeser RF, Goldring SR, Scanzello CR, Goldring MB. OSteoarthritis: A disease of the joint as an organ. Arthritis and Rheumatism. 2012;64(6):1697-1707. PubMed PMID: 22392533. PMCID: PMC3366018. [Epub: 07 March 2012]

[67] Malemud CJ. Changes in proteoglycans in osteoarthritis: Biochemistry, ultrastructure and biosynthetic processing. The Journal of Rheumatology. Supplement. 1991;**27**:60-62. PubMed PMID: 2027133. [Epub: 01 February 1991]

[68] Idris A, Ghazali NB, Koh D. Interleukin 1beta-A potential salivary biomarker for cancer progression? Biomark Cancer. 2015;**7**:25- 29. PubMed PMID: 26244033. PMCID: PMC4498652. [Epub: 06 August 2015]

[69] Gomes FI, Aragao MG, Barbosa FC, Bezerra MM, de Paulo Teixeira Pinto V, Chaves HV. Inflammatory cytokines interleukin-1beta and tumour necrosis factor-alpha—Novel biomarkers for the detection of periodontal diseases: A literature review. Journal of Oral & Maxillofacial Research. 2016;**7**(2):e2. PubMed PMID: 27489606. PMCID: PMC4970502. [Epub: 05 August 2016]

[70] Strimbu K, Tavel JA. What are biomarkers? Current Opinion in HIV and AIDS. 2010;**5**(6):463-466. PubMed PMID: 20978388. PMCID: PMC3078627. [Epub: 28 October 2010]

[71] McIlwraith CW, Kawcak CE, Frisbie DD, Little CB, Clegg PD, Peffers MJ, et al. Biomarkers for equine joint injury and osteoarthritis. Journal of Orthopaedic Research. 2018;**36**(3):823-831. PubMed PMID: 28921609. [Epub: 19 September 2017]

[72] Lohmander LS, Neame PJ, Sandy JD. The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis and Rheumatism. 1993;**36**(9):1214-1222. PubMed PMID: 8216415. [Epub: 01 September 1993]

[73] Tortorella MD, Burn TC, Pratta MA, Abbaszade I, Hollis JM, Liu R, et al. Purification and cloning of aggrecanase-1: A member of the ADAMTS family of proteins. Science. 1999;**284**(5420):1664-1666. PubMed PMID: 10356395. [Epub: 05 June 1999]

[74] Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross OH, Link JR, et al. Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. The Journal of Biological Chemistry. 1999;**274**(33):23443-23450. PubMed PMID: 10438522. [Epub: 07 August 1999]

[75] Fosang AJ, Rogerson FM, East CJ, Stanton H. ADAMTS-5: The story so far. European Cells & Materials. 2008;**15**:11- 26. PubMed PMID: 18247274. [Epub: 06 February 2008]

[76] Tortorella MD, Liu RQ, Burn T, Newton RC, Arner E. Characterization of human aggrecanase 2 (ADAM-TS5): Substrate specificity studies and comparison with aggrecanase 1 (ADAM-TS4). Matrix Biology. 2002;**21**(6):499-511. PubMed PMID: 12392761. [Epub: 24 October 2002]

[77] Nagase H, Kashiwagi M. Aggrecanases and cartilage matrix degradation. Arthritis Research & Therapy. 2003;**5**(2):94-103. PubMed PMID: 12718749. PMCID: PMC165039. [Epub: 30 April 2003]

[78] Pawlak E, Wang L, Johnson PJ, Nuovo G, Taye A, Belknap JK, et al. Distribution and processing of a

**105**

May 2004]

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health*

Journal of Cell Science. 2003;**116** (Pt 22):4663-4674. PubMed PMID: 14576359. [Epub: 25 October 2003]

[85] Kelsh R, You R, Horzempa C, Zheng M, McKeown-Longo PJ. Regulation of the innate immune response by fibronectin: Synergism between the III-1 and EDA domains. PLoS One. 2014;**9**(7):e102974. PubMed PMID: 25051083. PMCID: PMC4106844.

[86] Bondeson J, Wainwright S, Hughes C, Caterson B. The regulation of the ADAMTS4 and ADAMTS5 aggrecanases in osteoarthritis: A review. Clinical and Experimental Rheumatology. 2008;**26**(1):139-145. PubMed PMID: 18328163. eng

[87] Laje P, Shang D, Cao W, Niiya M, Endo M, Radu A, et al. Correction of murine ADAMTS13 deficiency by hematopoietic progenitor cell-mediated gene therapy. Blood. 2009;**113**(10):2172- 2180. PubMed PMID: 19141866. PMCID: PMC2652365. [Epub: 15 January 2009]

[88] Majumdar MK, Askew R, Schelling S, Stedman N, Blanchet T, Hopkins B, et al. Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis. Arthritis and Rheumatism. 2007;**56**(11):3670-3674. PubMed PMID: 17968948. [Epub: 31

[89] Ilic MZ, East CJ, Rogerson FM, Fosang AJ, Handley CJ. Distinguishing aggrecan loss from aggrecan proteolysis

in ADAMTS-4 and ADAMTS-5 single and double deficient mice. The Journal of Biological Chemistry. 2007;**282**(52):37420-37428. PubMed PMID: 17938173. [Epub: 17 October 2007]

October 2007]

[Epub: 23 July 2014]

*DOI: http://dx.doi.org/10.5772/intechopen.93046*

disintegrin and metalloproteinase with thrombospondin motifs-4, aggrecan, versican, and hyaluronan in equine digital laminae. American Journal of Veterinary Research. 2012;**73**(7):1035- 1046. PubMed PMID: 22738056. PMCID: PMC3535468. [Epub: 29 June 2012]

[79] Peffers MJ, Thornton DJ, Clegg PD. Characterization of neopeptides in equine articular cartilage degradation. Journal of Orthopaedic Research. 2016;**34**(1):106-120. PubMed PMID: 26124002. PMCID: PMC4737130.

[80] Moon J-W, Ahn K, Bae J-H, Nam G-H, Cho B-W, Park K-D, et al. mRNA sequence analysis and quantitative expression of the ADAMTS4 gene in the thoroughbred horse. Genes & Genomics. 2012;**34**(4):441-445

[81] Kandir S, Tekin G, Er C, Karakurt S. Effects of exercise on ADAMTS-4 and ADAMTS-5 levels in sport horses. Acta Physiologica. 2017;**221**:194-194. PubMed PMID: WOS:000408842000350. English

[82] Hashimoto G, Shimoda M, Okada Y. ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan. The Journal of Biological Chemistry. 2004;**279**(31):32483-32491. PubMed PMID: 15161923. [Epub: 27

[83] Tortorella M, Pratta M, Liu RQ, Abbaszade I, Ross H, Burn T, et al. The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage. The Journal of Biological Chemistry. 2000;**275**(33):25791-25797. PubMed PMID: 10827174. [Epub: 29 May 2000]

McKeown-Longo PJ. Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin.

[84] Klein RM, Zheng M, Ambesi A, Van De Water L,

[Epub: 01 July 2015]

*ADAMTS Proteases: Potential Biomarkers and Novel Therapeutic Targets for Cartilage Health DOI: http://dx.doi.org/10.5772/intechopen.93046*

disintegrin and metalloproteinase with thrombospondin motifs-4, aggrecan, versican, and hyaluronan in equine digital laminae. American Journal of Veterinary Research. 2012;**73**(7):1035- 1046. PubMed PMID: 22738056. PMCID: PMC3535468. [Epub: 29 June 2012]

*Equine Science*

[65] Beutler BA. The role of tumor necrosis factor in health and disease. The Journal of Rheumatology. Supplement. 1999;**57**:16-21. PubMed PMID: 10328138. [Epub: 18 May 1999] [72] Lohmander LS, Neame PJ, Sandy JD. The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis and Rheumatism. 1993;**36**(9):1214-1222. PubMed PMID: 8216415. [Epub: 01

[73] Tortorella MD, Burn TC, Pratta MA, Abbaszade I, Hollis JM, Liu R, et al. Purification and cloning of aggrecanase-1: A member of the ADAMTS family of proteins. Science. 1999;**284**(5420):1664-1666. PubMed PMID: 10356395. [Epub: 05 June 1999]

[74] Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross OH, Link JR, et al.

[75] Fosang AJ, Rogerson FM, East CJ, Stanton H. ADAMTS-5: The story so far. European Cells & Materials. 2008;**15**:11- 26. PubMed PMID: 18247274. [Epub: 06

[76] Tortorella MD, Liu RQ, Burn T, Newton RC, Arner E. Characterization of human aggrecanase 2 (ADAM-TS5): Substrate specificity studies and comparison with aggrecanase 1 (ADAM-TS4). Matrix Biology. 2002;**21**(6):499-511. PubMed PMID: 12392761. [Epub: 24 October 2002]

[77] Nagase H, Kashiwagi M. Aggrecanases and cartilage matrix degradation. Arthritis Research & Therapy. 2003;**5**(2):94-103. PubMed PMID: 12718749. PMCID: PMC165039.

[78] Pawlak E, Wang L, Johnson PJ, Nuovo G, Taye A, Belknap JK, et al. Distribution and processing of a

[Epub: 30 April 2003]

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. The Journal of Biological Chemistry. 1999;**274**(33):23443-23450. PubMed PMID: 10438522. [Epub: 07 August 1999]

February 2008]

September 1993]

[66] Loeser RF, Goldring SR, Scanzello CR, Goldring MB. OSteoarthritis: A disease of the joint as an organ. Arthritis and Rheumatism. 2012;64(6):1697-1707. PubMed PMID: 22392533. PMCID: PMC3366018. [Epub: 07 March 2012]

1991;**27**:60-62. PubMed PMID: 2027133.

Koh D. Interleukin 1beta-A potential salivary biomarker for cancer

progression? Biomark Cancer. 2015;**7**:25- 29. PubMed PMID: 26244033. PMCID: PMC4498652. [Epub: 06 August 2015]

[69] Gomes FI, Aragao MG, Barbosa FC, Bezerra MM, de Paulo Teixeira Pinto V, Chaves HV. Inflammatory cytokines interleukin-1beta and tumour necrosis factor-alpha—Novel biomarkers for the detection of periodontal diseases: A literature review. Journal of Oral & Maxillofacial Research. 2016;**7**(2):e2. PubMed PMID: 27489606. PMCID: PMC4970502. [Epub: 05 August 2016]

[70] Strimbu K, Tavel JA. What are biomarkers? Current Opinion in HIV and AIDS. 2010;**5**(6):463-466. PubMed PMID: 20978388. PMCID: PMC3078627.

[71] McIlwraith CW, Kawcak CE, Frisbie DD, Little CB, Clegg PD, Peffers MJ, et al. Biomarkers for equine

joint injury and osteoarthritis. Journal of Orthopaedic Research. 2018;**36**(3):823-831. PubMed PMID: 28921609. [Epub: 19 September 2017]

[Epub: 28 October 2010]

[67] Malemud CJ. Changes in proteoglycans in osteoarthritis: Biochemistry, ultrastructure and biosynthetic processing. The Journal of Rheumatology. Supplement.

[Epub: 01 February 1991]

[68] Idris A, Ghazali NB,

**104**

[79] Peffers MJ, Thornton DJ, Clegg PD. Characterization of neopeptides in equine articular cartilage degradation. Journal of Orthopaedic Research. 2016;**34**(1):106-120. PubMed PMID: 26124002. PMCID: PMC4737130. [Epub: 01 July 2015]

[80] Moon J-W, Ahn K, Bae J-H, Nam G-H, Cho B-W, Park K-D, et al. mRNA sequence analysis and quantitative expression of the ADAMTS4 gene in the thoroughbred horse. Genes & Genomics. 2012;**34**(4):441-445

[81] Kandir S, Tekin G, Er C, Karakurt S. Effects of exercise on ADAMTS-4 and ADAMTS-5 levels in sport horses. Acta Physiologica. 2017;**221**:194-194. PubMed PMID: WOS:000408842000350. English

[82] Hashimoto G, Shimoda M, Okada Y. ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan. The Journal of Biological Chemistry. 2004;**279**(31):32483-32491. PubMed PMID: 15161923. [Epub: 27 May 2004]

[83] Tortorella M, Pratta M, Liu RQ, Abbaszade I, Ross H, Burn T, et al. The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage. The Journal of Biological Chemistry. 2000;**275**(33):25791-25797. PubMed PMID: 10827174. [Epub: 29 May 2000]

[84] Klein RM, Zheng M, Ambesi A, Van De Water L, McKeown-Longo PJ. Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin. Journal of Cell Science. 2003;**116** (Pt 22):4663-4674. PubMed PMID: 14576359. [Epub: 25 October 2003]

[85] Kelsh R, You R, Horzempa C, Zheng M, McKeown-Longo PJ. Regulation of the innate immune response by fibronectin: Synergism between the III-1 and EDA domains. PLoS One. 2014;**9**(7):e102974. PubMed PMID: 25051083. PMCID: PMC4106844. [Epub: 23 July 2014]

[86] Bondeson J, Wainwright S, Hughes C, Caterson B. The regulation of the ADAMTS4 and ADAMTS5 aggrecanases in osteoarthritis: A review. Clinical and Experimental Rheumatology. 2008;**26**(1):139-145. PubMed PMID: 18328163. eng

[87] Laje P, Shang D, Cao W, Niiya M, Endo M, Radu A, et al. Correction of murine ADAMTS13 deficiency by hematopoietic progenitor cell-mediated gene therapy. Blood. 2009;**113**(10):2172- 2180. PubMed PMID: 19141866. PMCID: PMC2652365. [Epub: 15 January 2009]

[88] Majumdar MK, Askew R, Schelling S, Stedman N, Blanchet T, Hopkins B, et al. Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis. Arthritis and Rheumatism. 2007;**56**(11):3670-3674. PubMed PMID: 17968948. [Epub: 31 October 2007]

[89] Ilic MZ, East CJ, Rogerson FM, Fosang AJ, Handley CJ. Distinguishing aggrecan loss from aggrecan proteolysis in ADAMTS-4 and ADAMTS-5 single and double deficient mice. The Journal of Biological Chemistry. 2007;**282**(52):37420-37428. PubMed PMID: 17938173. [Epub: 17 October 2007]

**107**

**Chapter 7**

**Abstract**

**1. Introduction**

the animal [2].

Equine Sarcoid

*Beatrice Funiciello and Paola Roccabianca*

vidual patient, facilities, owner, and financial issues.

**2. Etiopathogenesis of the equine sarcoid**

**2.1 Bovine papillomavirus infection**

The equine sarcoid is the most common skin neoplasia in the horse. It has a worldwide distribution and can also affect other equids such as donkeys, zebras, and mules. All breeds can develop the disease at any age, with no sex predilection, although geldings seem to be overrepresented. This fibroblastic neoplasm has several clinical presentations and microscopic features and has a nonmetastatic behavior but can be severely locally invasive. In many cases, multiple sarcoids may develop simultaneously or sequentially during their life and spontaneous remission is rarely reported. The etiology is multifactorial and involves bovine papillomaviruses, genetic, and environmental factors. Treatment options include different modalities depending on multiple factors: lesion type, location and extent, indi-

**Keywords:** sarcoid, neoplasia, tumor, skin, horse, equids, donkey, mule, zebra

The equine sarcoid is the most common skin neoplasia in the horse. This fibroblastic neoplasm has a multifactorial etiology and is nonmetastatic but can be severely locally invasive. First described in 1936, it has a worldwide distribution and can also affect other equids such as donkeys, zebras, and mules as well as other mammals [1–3]. Prevalence of sarcoid varies among published studies; however, many reports include cases from referral clinics that may not exactly reflect the entire equine population. Reported percentages of sarcoid among skin diseases and skin neoplasms vary from 13% to 90% and 8% to 38% when considering ocular neoplasms. There are also some geographical variations that may correlate with variations of risk factors, including the presence of cattle and vectors near horses [1, 4–6]. Horses of all breeds and colors can develop the disease at any age, most presenting a first lesion between 2 and 9 years of age. There is no demonstrated sex predilection, although geldings seem to be overrepresented [3, 5, 7]. Data about the incidence of sarcoids in the population are available only for donkeys and not for horses [8].

Affected animals can never be considered free of the disease even after successful treatment and presence or history of sarcoid can lower the likely sale value of

To date, it is widely recognized that sarcoids are associated with the presence of bovine papillomaviruses (BPV), typically BPV-1 and/or BPV-2. In two Brazilian

## **Chapter 7** Equine Sarcoid

*Beatrice Funiciello and Paola Roccabianca*

### **Abstract**

The equine sarcoid is the most common skin neoplasia in the horse. It has a worldwide distribution and can also affect other equids such as donkeys, zebras, and mules. All breeds can develop the disease at any age, with no sex predilection, although geldings seem to be overrepresented. This fibroblastic neoplasm has several clinical presentations and microscopic features and has a nonmetastatic behavior but can be severely locally invasive. In many cases, multiple sarcoids may develop simultaneously or sequentially during their life and spontaneous remission is rarely reported. The etiology is multifactorial and involves bovine papillomaviruses, genetic, and environmental factors. Treatment options include different modalities depending on multiple factors: lesion type, location and extent, individual patient, facilities, owner, and financial issues.

**Keywords:** sarcoid, neoplasia, tumor, skin, horse, equids, donkey, mule, zebra

#### **1. Introduction**

The equine sarcoid is the most common skin neoplasia in the horse. This fibroblastic neoplasm has a multifactorial etiology and is nonmetastatic but can be severely locally invasive. First described in 1936, it has a worldwide distribution and can also affect other equids such as donkeys, zebras, and mules as well as other mammals [1–3]. Prevalence of sarcoid varies among published studies; however, many reports include cases from referral clinics that may not exactly reflect the entire equine population. Reported percentages of sarcoid among skin diseases and skin neoplasms vary from 13% to 90% and 8% to 38% when considering ocular neoplasms. There are also some geographical variations that may correlate with variations of risk factors, including the presence of cattle and vectors near horses [1, 4–6]. Horses of all breeds and colors can develop the disease at any age, most presenting a first lesion between 2 and 9 years of age. There is no demonstrated sex predilection, although geldings seem to be overrepresented [3, 5, 7]. Data about the incidence of sarcoids in the population are available only for donkeys and not for horses [8].

Affected animals can never be considered free of the disease even after successful treatment and presence or history of sarcoid can lower the likely sale value of the animal [2].

#### **2. Etiopathogenesis of the equine sarcoid**

#### **2.1 Bovine papillomavirus infection**

To date, it is widely recognized that sarcoids are associated with the presence of bovine papillomaviruses (BPV), typically BPV-1 and/or BPV-2. In two Brazilian studies, newly proposed BPV 'BsR-UEL-4' and BPV-13 were found in some equine sarcoids, suggesting the need for further research regarding BPV serotype involvement in the development of these tumors [9–12].

The prevalence of BPV-1 and BPV-2 types seem to vary among geographical areas. In Europe and Australia, BPV-1 is most detected. In eastern USA, an almost equal proportion of both virus types was found, whereas in Canada and Western USA, BPV-2 was demonstrated in most of the samples [12–17].

The bovine papillomavirus genome comprises early and late coding regions. The early (E) genes encode nonstructural proteins involved in viral replication, maintenance of the episomal state, and activation of cell proliferation. The late (L) genes encode structural proteins (viral capsid) produced only in the life cycle of keratinocytes in natural hosts. A non-coding long control region is also present playing a role in viral replication and transcription [18, 19]. The main factors identified in sarcoid oncogenesis are the E2, E5, E6, E7, and p53 proteins. The E2 protein has regulatory effects on viral transcription and on the expression of matrix metalloproteinases (MMPs) that may be implicated in neoplastic cell invasiveness. The E5 protein exerts its function by binding to platelet-derived growth factor-β receptor (PDGFβ-R) thus activating p38 mitogen-activated protein kinase (MAPK) to induce fibroblastic transformation in sarcoids and down-regulate the major histocompatibility complex (MHC) I to facilitate the evasion of the immune system. The E6 protein can interfere in the activity of the p53 protein and has anti-apoptotic activity. The E7 protein cooperates in evading innate immunity [19–27]. The oncogenesis of equine sarcoids also involves loss of expression of the Fragile Histidine Triad (FHIT) and of the O6 -methylguanine-DNA methyltransferase (MGMT) tumor suppressor proteins [28, 29]. Recent studies have evaluated the role of small non-coding RNAs that regulate gene expression (microRNAs) in the development of sarcoids, and the role of aberrant methylation (S100A14 gene) is under research [30–34].

It seems that BPV infection in horses starts in the epidermis, where it can remain latent, with a subsequent presence of viral material within sub-epidermal fibroblasts where full transformation takes place [35–37]. Latency seems to take place also in peripheral blood mononuclear cells (PBMC) [38]. The infection in horses is abortive, the virus is present episomally but intact virions have never been detected. Furthermore, intralesional viral load seems to be correlated to disease severity [39, 40].

#### **2.2 BPV transmission**

Viral transmission between animals has not been completely elucidated yet. Direct contact with cattle, contaminated surfaces, and flies are presumably the most common routes of transmission [35, 36, 41, 42]. Infected equids may possibly spread BPV infection to horses and donkeys through contact. Appropriate fly protection and hygiene should be basic control measures in the presence of cattle and sarcoid-affected animals [35, 36].

#### **2.3 Genetic risk factors**

Bovine papillomavirus infection alone is not sufficient to promote normal cells transformation into sarcoid tumors, the presence of genetic factors and trauma are associated with the disease [43]. All breeds can be affected but Quarter Horses, Appaloosas, and Arabian horses are reported to be at greater risk than Thoroughbreds. Standardbreds have an even lower risk of developing sarcoids [5, 44, 45]. Certain equine families have an increased prevalence of sarcoid lesions and an association between the disease and equine leukocyte antigen (ELA) alleles has been observed in several breeds. The ELA W13 allele associated with the MHC

**109**

**2.4 Trauma**

**Figure 1.**

*Equine Sarcoid*

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

II has been linked with sarcoid susceptibility in studies involving different breeds such as Swiss, Irish, French, and Swedish Warmbloods, and Thoroughbreds. The ELA W13 allele is not expressed in Standardbreds, a breed at lower risk of developing lesions [43, 46, 47]. Other MCH-encoded antigens are reported to play a role in sarcoid development: W3, B1, A3, A5, A16, A20, W5, W11, and W21 [1, 48–50]. A breed specific antigen, the Abe108, has been associated with sarcoids in Freiberger

*Sarcoid development on the jugular groove, possibly triggered by injection micro-trauma.*

Skin trauma is involved in sarcoid initiation, progression, and possibly recurrence. Micro-trauma due to injections (**Figure 1**) or even insect bites can be followed by sarcoid development, even long after apparent healing. Furthermore, sarcoids are a well-recognized possible complication and cause for delayed healing

An individual animal may present only with one sarcoid, but most commonly, affected horses develop multiple sarcoids during their lives. These neoplasms may remain static for months or years and then, slowly or suddenly become aggressive and progress in type and/or extension without apparent reason (**Figure 2A** and **B**). Sarcoids tend to be locally invasive, sometimes extending into subcutaneous and

horses that lack A3, A5, and W13 antigens [50].

in both traumatic and surgical wounds in horses [2, 5, 51].

**3. The biological behavior of the equine sarcoid**

*Equine Science*

studies, newly proposed BPV 'BsR-UEL-4' and BPV-13 were found in some equine sarcoids, suggesting the need for further research regarding BPV serotype involve-

The prevalence of BPV-1 and BPV-2 types seem to vary among geographical areas. In Europe and Australia, BPV-1 is most detected. In eastern USA, an almost equal proportion of both virus types was found, whereas in Canada and Western

The bovine papillomavirus genome comprises early and late coding regions. The early (E) genes encode nonstructural proteins involved in viral replication, maintenance of the episomal state, and activation of cell proliferation. The late (L) genes encode structural proteins (viral capsid) produced only in the life cycle of keratinocytes in natural hosts. A non-coding long control region is also present playing a role in viral replication and transcription [18, 19]. The main factors identified in sarcoid oncogenesis are the E2, E5, E6, E7, and p53 proteins. The E2 protein has regulatory effects on viral transcription and on the expression of matrix metalloproteinases (MMPs) that may be implicated in neoplastic cell invasiveness. The E5 protein exerts its function by binding to platelet-derived growth factor-β receptor (PDGFβ-R) thus activating p38 mitogen-activated protein kinase (MAPK) to induce fibroblastic transformation in sarcoids and down-regulate the major histocompatibility complex (MHC) I to facilitate the evasion of the immune system. The E6 protein can interfere in the activity of the p53 protein and has anti-apoptotic activity. The E7 protein cooperates in evading innate immunity [19–27]. The oncogenesis of equine sarcoids also involves loss of expression of the Fragile Histidine Triad (FHIT) and of the


It seems that BPV infection in horses starts in the epidermis, where it can remain latent, with a subsequent presence of viral material within sub-epidermal fibroblasts where full transformation takes place [35–37]. Latency seems to take place also in peripheral blood mononuclear cells (PBMC) [38]. The infection in horses is abortive, the virus is present episomally but intact virions have never been detected. Furthermore, intralesional viral load seems to be correlated to disease severity [39, 40].

Viral transmission between animals has not been completely elucidated yet. Direct contact with cattle, contaminated surfaces, and flies are presumably the most common routes of transmission [35, 36, 41, 42]. Infected equids may possibly spread BPV infection to horses and donkeys through contact. Appropriate fly protection and hygiene should be basic control measures in the presence of cattle

Bovine papillomavirus infection alone is not sufficient to promote normal cells transformation into sarcoid tumors, the presence of genetic factors and trauma are associated with the disease [43]. All breeds can be affected but Quarter Horses, Appaloosas, and Arabian horses are reported to be at greater risk than Thoroughbreds. Standardbreds have an even lower risk of developing sarcoids [5, 44, 45]. Certain equine families have an increased prevalence of sarcoid lesions and an association between the disease and equine leukocyte antigen (ELA) alleles has been observed in several breeds. The ELA W13 allele associated with the MHC

of aberrant methylation (S100A14 gene) is under research [30–34].

ment in the development of these tumors [9–12].

USA, BPV-2 was demonstrated in most of the samples [12–17].

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O6

**2.2 BPV transmission**

**2.3 Genetic risk factors**

and sarcoid-affected animals [35, 36].

II has been linked with sarcoid susceptibility in studies involving different breeds such as Swiss, Irish, French, and Swedish Warmbloods, and Thoroughbreds. The ELA W13 allele is not expressed in Standardbreds, a breed at lower risk of developing lesions [43, 46, 47]. Other MCH-encoded antigens are reported to play a role in sarcoid development: W3, B1, A3, A5, A16, A20, W5, W11, and W21 [1, 48–50]. A breed specific antigen, the Abe108, has been associated with sarcoids in Freiberger horses that lack A3, A5, and W13 antigens [50].

#### **2.4 Trauma**

Skin trauma is involved in sarcoid initiation, progression, and possibly recurrence. Micro-trauma due to injections (**Figure 1**) or even insect bites can be followed by sarcoid development, even long after apparent healing. Furthermore, sarcoids are a well-recognized possible complication and cause for delayed healing in both traumatic and surgical wounds in horses [2, 5, 51].

#### **3. The biological behavior of the equine sarcoid**

An individual animal may present only with one sarcoid, but most commonly, affected horses develop multiple sarcoids during their lives. These neoplasms may remain static for months or years and then, slowly or suddenly become aggressive and progress in type and/or extension without apparent reason (**Figure 2A** and **B**). Sarcoids tend to be locally invasive, sometimes extending into subcutaneous and

#### **Figure 2.**

*(A) Ear sarcoid slowly grown over years. (B) Same horse (hair clipped) few weeks after, the sarcoid underwent rapid growth at the beginning of the fly season. (C) Fibroblastic sarcoid development on a recently treated occult sarcoid. Note the "healthy" scar on the right where a similar occult sarcoid was successfully treated simultaneously.*

muscular planes, especially periocular lesions. They do not metastasize, however, with the exception of the malignant form that can spread to lymphatics and cause the formation of multiple masses along the lymphatic vessels and at remote sites such as lymph nodes [52].

Spontaneous regression is rarely reported and usually these horses do not develop new sarcoid tumors. Only in one recent study on a population of Franches-Montagnes horses in Switzerland has a high proportion of spontaneous remission been observed [53]. The mechanisms for spontaneous regression are not clear and antibodies have been detected only in donkeys [5].

The equine sarcoid has high frequency of recurrence after treatment (**Figure 2C**), especially following surgical excision. Recurrent tumors are usually more aggressive than the initial lesion and tend to grow rapidly and be more invasive. Recurring sarcoids can appear within a few days or weeks to months or years. The recurrence is often due to incomplete removal or spread of sarcoid cells during the procedure [1, 2, 52].

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**Table 1.**

*Equine Sarcoid*

**4.1 Occult sarcoid**

toward verrucose growth.

A—no cutaneous involvement

B—cutaneous involvement

and forearms. They rarely affect the limbs.

**Type Subtype Features**

conditions (pemphigus foliaceous, and vasculitis), and burns.

Nodular Subcutaneous spherical masses

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

**4. Clinical presentations of the equine sarcoid**

Sarcoids have been classified into six different types depending on their macroscopic appearance (**Table 1**). This clinical classification is important because different types require different therapeutic approaches and have differing prognoses. One subject may carry more than one type of sarcoid and commonly, though

The occult sarcoid presents as an area of hairless skin, generally roughly circular. The skin may be thinned and/or have variably hyperkeratotic or roughened areas and contain one or more nodules, usually about 2–5 mm in diameter (**Figure 3A** and **B**). Occult sarcoids may involve extensive surfaces and individual horses may carry several lesions (**Figure 3C**). In some cases, only partial alopecia with thin hair and mild changes in skin and/or hair pigmentation (darker or paler) can be detected. Pruritus and pain are not present. These sarcoids have a slow progression

Occult lesions can develop at any site but with predilection for the skin around mouth, eyes, the neck, and areas with less hair such as the medial thighs (**Figure 3D**)

Differential diagnoses for occult sarcoids are: idiopathic hypotrichosis/alopecia, dermatophytosis, alopecia areata, rub marks, chronic rubbing and scarring, bullous

Occult — Roughly circular, hairless thinned and/or hyperkeratotic skin, may contain nodules Verrucose — Warty, hyperkeratotic area, may have nodules and/or occult halo

margins

margins

1—pedunculated 1a Distinct pedicle without palpable or histological presence of tumor extensions

Mixed — Verrucose, nodular and fibroblastic features present in variable

Malignant — Multiple, locally invasive nodular and fibroblastic sarcoids with

Fibroblastic Fleshy, ulcerated appearance, fibrocelullar scab

2—sessile/broad-based Poorly defined margins, invasive 'bound-down'

*Summary of the clinical classification of sarcoid types and features.*

A1 Deeper tissues are not involved, loose capsule and defined

A2 Deep tissue involvement with poorly defined margins and invasive 'bound-down' nature

B1 Deeper tissues are not involved, loose capsule and defined

B2 Deep tissue involvement with poorly defined margins and invasive 'bound-down' nature

1b Distinct pedicle with palpable root, poorly defined margins,

subcutaneous connections, may spread to lymphatics

invasive 'bound-down' nature

proportions within the same lesion

unpredictably, milder forms can progress to more severe types [52, 54, 55].

### **4. Clinical presentations of the equine sarcoid**

Sarcoids have been classified into six different types depending on their macroscopic appearance (**Table 1**). This clinical classification is important because different types require different therapeutic approaches and have differing prognoses. One subject may carry more than one type of sarcoid and commonly, though unpredictably, milder forms can progress to more severe types [52, 54, 55].

#### **4.1 Occult sarcoid**

*Equine Science*

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such as lymph nodes [52].

**Figure 2.**

*simultaneously.*

procedure [1, 2, 52].

antibodies have been detected only in donkeys [5].

muscular planes, especially periocular lesions. They do not metastasize, however, with the exception of the malignant form that can spread to lymphatics and cause the formation of multiple masses along the lymphatic vessels and at remote sites

*(A) Ear sarcoid slowly grown over years. (B) Same horse (hair clipped) few weeks after, the sarcoid underwent rapid growth at the beginning of the fly season. (C) Fibroblastic sarcoid development on a recently treated occult sarcoid. Note the "healthy" scar on the right where a similar occult sarcoid was successfully treated* 

Spontaneous regression is rarely reported and usually these horses do not develop new sarcoid tumors. Only in one recent study on a population of Franches-Montagnes horses in Switzerland has a high proportion of spontaneous remission been observed [53]. The mechanisms for spontaneous regression are not clear and

The equine sarcoid has high frequency of recurrence after treatment (**Figure 2C**), especially following surgical excision. Recurrent tumors are usually more aggressive than the initial lesion and tend to grow rapidly and be more invasive. Recurring sarcoids can appear within a few days or weeks to months or years. The recurrence is often due to incomplete removal or spread of sarcoid cells during the

The occult sarcoid presents as an area of hairless skin, generally roughly circular. The skin may be thinned and/or have variably hyperkeratotic or roughened areas and contain one or more nodules, usually about 2–5 mm in diameter (**Figure 3A** and **B**). Occult sarcoids may involve extensive surfaces and individual horses may carry several lesions (**Figure 3C**). In some cases, only partial alopecia with thin hair and mild changes in skin and/or hair pigmentation (darker or paler) can be detected. Pruritus and pain are not present. These sarcoids have a slow progression toward verrucose growth.

Occult lesions can develop at any site but with predilection for the skin around mouth, eyes, the neck, and areas with less hair such as the medial thighs (**Figure 3D**) and forearms. They rarely affect the limbs.

Differential diagnoses for occult sarcoids are: idiopathic hypotrichosis/alopecia, dermatophytosis, alopecia areata, rub marks, chronic rubbing and scarring, bullous conditions (pemphigus foliaceous, and vasculitis), and burns.


#### **Table 1.**

*Summary of the clinical classification of sarcoid types and features.*

#### **Figure 3.**

*(A) Occult sarcoid in the pectoral region: circular roughened hairless area with one small nodule within. (B) Large occult sarcoid: note mild hair loss, alterations in skin pigmentation, and presence of nodules. (C) Multiple occult sarcoids, the central one has a verrucose central area with occult halo. (D) Early occult sarcoid (blue circle) on the medial thigh. Sparsely haired shining skin with mild pigmentary changes.*

#### **4.2 Verrucose sarcoid**

The verrucose form has a characteristic "wart-like" appearance, which is the main reason for calling sarcoids "warts". These lesions are alopecic and neither pruritic nor painful unless secondarily infected (**Figure 4A**). Some may ulcerate and bleed (**Figure 4B**). Thickness and size vary, small nodules may develop in the hyperkeratotic area and some lesions may present a pathognomonic occult margin/ halo (**Figure 4C**). They usually grow slowly but progression to a more aggressive form is possible, especially with trauma. As occult sarcoids, the verrucose ones can coalesce and cover large body areas (**Figure 5**).

Verrucose sarcoids can develop in any region with predilection sites being face (periorbital), axillae, groin, body, and sheath. Limbs are rarely affected.

Differential diagnoses for verrucose sarcoids are: papillomatosis (warts), linear keratosis/epidermal nevus, dermatophytosis, chronic blistering.

#### **4.3 Nodular sarcoid**

Nodular sarcoids are firm and well-defined subcutaneous masses, usually spherical with variable diameters from few mm to 7 cm. In many cases, the nodules may be multiple and coalescing. Pain and pruritus are not typical features.

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**Figure 5.**

*coverage by hair.*

**Figure 4.**

*(A) Verrucose sarcoid in the axillary region (hair has been clipped), note another one in the sternal region.* 

*(A) Large verrucose sarcoid with nodular formations on the side of the neck. (B) Same horse after hair clipping: note extended hyperkeratotic and occult areas that were not previously visible because of* 

*(B) Verrucose sarcoid with fissures and mild bleeding. (C) Verrucose sarcoid with occult halo.*

*Equine Sarcoid*

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

*Equine Science*

**112**

**4.2 Verrucose sarcoid**

**Figure 3.**

**4.3 Nodular sarcoid**

coalesce and cover large body areas (**Figure 5**).

The verrucose form has a characteristic "wart-like" appearance, which is the main reason for calling sarcoids "warts". These lesions are alopecic and neither pruritic nor painful unless secondarily infected (**Figure 4A**). Some may ulcerate and bleed (**Figure 4B**). Thickness and size vary, small nodules may develop in the hyperkeratotic area and some lesions may present a pathognomonic occult margin/ halo (**Figure 4C**). They usually grow slowly but progression to a more aggressive form is possible, especially with trauma. As occult sarcoids, the verrucose ones can

*(A) Occult sarcoid in the pectoral region: circular roughened hairless area with one small nodule within. (B) Large occult sarcoid: note mild hair loss, alterations in skin pigmentation, and presence of nodules. (C) Multiple occult sarcoids, the central one has a verrucose central area with occult halo. (D) Early occult sarcoid (blue circle) on the medial thigh. Sparsely haired shining skin with mild pigmentary changes.*

Verrucose sarcoids can develop in any region with predilection sites being face

Differential diagnoses for verrucose sarcoids are: papillomatosis (warts), linear

Nodular sarcoids are firm and well-defined subcutaneous masses, usually spherical with variable diameters from few mm to 7 cm. In many cases, the nodules may be multiple and coalescing. Pain and pruritus are not typical features.

(periorbital), axillae, groin, body, and sheath. Limbs are rarely affected.

keratosis/epidermal nevus, dermatophytosis, chronic blistering.

#### **Figure 4.**

*(A) Verrucose sarcoid in the axillary region (hair has been clipped), note another one in the sternal region. (B) Verrucose sarcoid with fissures and mild bleeding. (C) Verrucose sarcoid with occult halo.*

#### **Figure 5.**

*(A) Large verrucose sarcoid with nodular formations on the side of the neck. (B) Same horse after hair clipping: note extended hyperkeratotic and occult areas that were not previously visible because of coverage by hair.*

Similar to other forms, they very rarely develop on the limbs and the predilection sites are the groin, sheath, and eyelids.

A further classification has been suggested for these sarcoids based on skin and deep tissues involvement.

	- Type A1: the nodule can be moved from both the skin and the underlying tissues, usually has a fibrocelullar capsule. In some lesions, a skin pedicle is palpable.
	- Type A2: no skin involvement but the nodule cannot be moved independently from the underlying tissues, it has a 'bound-down' nature. Very common around the eye.
	- Type B1: no involvement of the deeper structures (**Figure 6A**).
	- Type B2: locally invasive with 'bound-down' nature and no separation from deeper layers (**Figure 6B**).

Differential diagnoses for nodular sarcoids are: fibroma/fibrosarcoma, neurofibroma, eosinophilic/collagenolytic granuloma, melanoma, equine cutaneous mastocytosis/malignant cutaneous mastocytosis/congenital mastocytoma, lymphosarcoma/lymphoma/cutaneous histiocytic lymphoma, dermoid cyst, and *Hypoderma* spp./foreign body cyst.

#### **4.4 Fibroblastic sarcoid**

Fibroblastic sarcoids are a more aggressive form with fleshy and ulcerated appearance, often covered by a fibrocellular scab and possibly secondary infection.

#### **Figure 6.**

*(A) Type B1 nodular sarcoid on the medial thigh. (B) Three type B2 nodular sarcoids around the eye, an occult area is also present.*

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**Figure 7.**

*the pedicle. (C) Type 2 sessile fibroblastic sarcoid.*

*Equine Sarcoid*

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

(**Figure 7B**).

tissues (**Figure 7C**).

Bleeding and serum exudation are common and can be heavy with trauma. These surface characteristics attract flies that may contribute to irritation and self-trauma.

• Type 1 pedunculated fibroblastic sarcoids: characterized by a narrow pedicle

○ Type 1a: pedunculated with no palpable tumor and thickening at the base,

○ Type 1b: this is pedunculated and rooted, where palpable alterations are detected beneath the pedicle, sometimes alteration are also visible

• Type 2 sessile fibroblastic sarcoid: the lesion is broad-based with invariably ill-defined margins and extensive invasion of the lateral and deeper

Differential diagnoses for fibroblastic sarcoids are: exuberant granulation tissue, habronemiasis, pythiosis, botryomycosis/pyogranuloma/pseudomycetoma,

*(A) Type 1a fibroblastic sarcoid on the penis. (B) Type 1b fibroblastic sarcoid with clear tumor involvement of* 

Fibroblastic sarcoids commonly develop at wound sites (both traumatic and surgical), on the site of other sarcoids, especially if treatment attempts have been unsuccessful (**Figure 2C**) and they are usually more difficult to manage. Excessive granulation tissue may develop especially at wound sites thus complicating the diagnosis of sarcoid. Pruritus and pain rarely characterize these lesions. Predilection sites for fibroblastic sarcoids are groin, eyelid, wounds, coronets, and distal limbs. At some of these sites they carry a very poor prognosis. The classification of this form includes:

with apparently normal skin and a fleshy crown. Subtypes are:

no extensions detected on histology (**Figure 7A**).

#### *Equine Sarcoid DOI: http://dx.doi.org/10.5772/intechopen.91013*

*Equine Science*

sites are the groin, sheath, and eyelids.

over the nodule. Two subtypes exist:

common around the eye.

deeper layers (**Figure 6B**).

*Hypoderma* spp./foreign body cyst.

**4.4 Fibroblastic sarcoid**

deep tissues involvement.

palpable.

Similar to other forms, they very rarely develop on the limbs and the predilection

A further classification has been suggested for these sarcoids based on skin and

• Type A nodules do not involve skin that is not altered and can be freely moved

○ Type A1: the nodule can be moved from both the skin and the underlying tissues, usually has a fibrocelullar capsule. In some lesions, a skin pedicle is

○ Type A2: no skin involvement but the nodule cannot be moved independently from the underlying tissues, it has a 'bound-down' nature. Very

• Type B nodules are characterized by visible and/or palpable alterations of the skin. They cannot be freely moved from the overlying skin that may look normal or be alopecic, thinned, hyperkeratotic, or ulcerated. Some may have

adjacent occult changes. Also, two subtypes are recognized:

○ Type B1: no involvement of the deeper structures (**Figure 6A**).

○ Type B2: locally invasive with 'bound-down' nature and no separation from

Differential diagnoses for nodular sarcoids are: fibroma/fibrosarcoma, neurofibroma, eosinophilic/collagenolytic granuloma, melanoma, equine cutaneous mastocytosis/malignant cutaneous mastocytosis/congenital mastocytoma, lymphosarcoma/lymphoma/cutaneous histiocytic lymphoma, dermoid cyst, and

Fibroblastic sarcoids are a more aggressive form with fleshy and ulcerated appearance, often covered by a fibrocellular scab and possibly secondary infection.

*(A) Type B1 nodular sarcoid on the medial thigh. (B) Three type B2 nodular sarcoids around the eye, an occult* 

**114**

**Figure 6.**

*area is also present.*

Bleeding and serum exudation are common and can be heavy with trauma. These surface characteristics attract flies that may contribute to irritation and self-trauma. Fibroblastic sarcoids commonly develop at wound sites (both traumatic and surgical), on the site of other sarcoids, especially if treatment attempts have been unsuccessful (**Figure 2C**) and they are usually more difficult to manage. Excessive granulation tissue may develop especially at wound sites thus complicating the diagnosis of sarcoid. Pruritus and pain rarely characterize these lesions. Predilection sites for fibroblastic sarcoids are groin, eyelid, wounds, coronets, and distal limbs. At some of these sites they carry a very poor prognosis. The classification of this form includes:

	- Type 1a: pedunculated with no palpable tumor and thickening at the base, no extensions detected on histology (**Figure 7A**).
	- Type 1b: this is pedunculated and rooted, where palpable alterations are detected beneath the pedicle, sometimes alteration are also visible (**Figure 7B**).

Differential diagnoses for fibroblastic sarcoids are: exuberant granulation tissue, habronemiasis, pythiosis, botryomycosis/pyogranuloma/pseudomycetoma,

#### **Figure 7.**

*(A) Type 1a fibroblastic sarcoid on the penis. (B) Type 1b fibroblastic sarcoid with clear tumor involvement of the pedicle. (C) Type 2 sessile fibroblastic sarcoid.*

**Figure 8.** *Mixed sarcoid: a type B2 nodule with a small fibroblastic sarcoid within an occult area.*

hemangioma/hemangiosarcoma, cavernous hemangioma/vascular hamartoma, neurofibroma/neurofibrosarcoma (ulcerated), fibrosarcoma, squamous cell carcinoma, sweat gland tumor, giant cell sarcoma, and mycosis fungoides.

#### **4.5 Mixed sarcoid**

Most sarcoids could be classified as mixed since different types (verrucose, nodular, and fibroblastic) are often present in variable proportions within the same lesion. Nevertheless, the definition of mixed sarcoid is usually reserved for those where a specific sarcoid type is not considered predominant. These cases may probably represent the transition/progression phase between one clinical type into the other. The combinations and extents of the various types are multiple, and they usually tend to become more aggressive, especially as the fibroblastic type grows (**Figure 8**).

Predilection sites for mixed sarcoids are the face, eyelids, groin, and medial thigh but mixed sarcoids can appear everywhere.

Differential diagnoses for mixed sarcoids are mixtures of granulation tissue within verrucose or fibroblastic lesions, habronemiasis, pemphigus complex.

#### **4.6 Malignant sarcoid**

The most recently described form of sarcoid tumor is the malignant type. It is usually, but not always, characterized by a history of repeated trauma or interference (also with inappropriate treatments) with another type of sarcoid. The particular behavior of the malignant type is the development of multiple, locally invasive nodular and fibroblastic sarcoids. Often cords of nodules and ulcerated lesions are visible and/or palpable, when these connections are subcutaneous the classification should be that of malignant sarcoid. They can be localized or spread through the lymphatic vessels invading local tissues with possible associated lymph node enlargement. No disseminated metastasis has been reported even for this form. A rare particular and dangerous form presents with a ring of nodules surrounding a verrucose or occult central area, especially on the neck/ jugular and buttock regions. Predilection sites include jaw, face (**Figure 9**), elbow, and medial thigh.

Differential diagnoses for malignant sarcoids are squamous cell carcinoma, lymphoma/lymphosarcoma, subcutaneous mycosis, lymphangitis, glanders, epizootic lymphangitis/histoplasmosis, and hypertrophic scarring/cheloid.

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**5.1 Equine sarcoid pathology**

*Equine Sarcoid*

**Figure 9.**

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

**5. Clinical examination and diagnostic procedures**

*changes, ulceration, and a fibroblastic component.*

opportunity of taking a biopsy should be carefully evaluated [2, 55].

Histopathology is deemed necessary to confirm the diagnosis of many equine sarcoids [57]. It is important to stress that due to the variable microscopic features of equine sarcoids, small biopsies may not provide enough tissue to differentiate sarcoids from other lesions such as granulation tissue, fibromas, or fibrosarcomas.

The clinical examination should include signalment and a full thorough history with details on lesion development, especially about the behavior and progression. The clinical presentation of sarcoid lesions and their features are usually clearly recognizable, especially if multiple tumors of different types are present on the same horse. The confirmation of the diagnosis is not always straightforward and possible differential diagnoses and concurrent conditions should be considered [55, 56]. Depending on the sarcoid type, the full list of differential diagnoses should be considered when choosing the diagnostic procedures. The diagnosis of sarcoid is confirmed by histopathology, thus a biopsy sample is needed. Partial or excisional biopsy should provide sufficient information but a risk of exacerbation due to the surgical trauma should always be taken into account. If possible, a total excisional biopsy is preferable, the owner should be carefully advised about the implicit risks and a proper therapeutic plan should be prepared when taking the biopsy to avoid any exacerbation triggered by the procedure. If benign neglect is the plan, the

*Malignant sarcoid on the face: 'bound-down' invasive nodules with a central area with occult to verrucose* 

#### **Figure 9.**

*Equine Science*

**4.5 Mixed sarcoid**

**Figure 8.**

grows (**Figure 8**).

**4.6 Malignant sarcoid**

elbow, and medial thigh.

thigh but mixed sarcoids can appear everywhere.

hemangioma/hemangiosarcoma, cavernous hemangioma/vascular hamartoma, neurofibroma/neurofibrosarcoma (ulcerated), fibrosarcoma, squamous cell carci-

Most sarcoids could be classified as mixed since different types (verrucose, nodular, and fibroblastic) are often present in variable proportions within the same lesion. Nevertheless, the definition of mixed sarcoid is usually reserved for those where a specific sarcoid type is not considered predominant. These cases may probably represent the transition/progression phase between one clinical type into the other. The combinations and extents of the various types are multiple, and they usually tend to become more aggressive, especially as the fibroblastic type

Predilection sites for mixed sarcoids are the face, eyelids, groin, and medial

Differential diagnoses for mixed sarcoids are mixtures of granulation tissue within verrucose or fibroblastic lesions, habronemiasis, pemphigus complex.

The most recently described form of sarcoid tumor is the malignant type. It is usually, but not always, characterized by a history of repeated trauma or interference (also with inappropriate treatments) with another type of sarcoid. The particular behavior of the malignant type is the development of multiple, locally invasive nodular and fibroblastic sarcoids. Often cords of nodules and ulcerated lesions are visible and/or palpable, when these connections are subcutaneous the classification should be that of malignant sarcoid. They can be localized or spread through the lymphatic vessels invading local tissues with possible associated lymph node enlargement. No disseminated metastasis has been reported even for this form. A rare particular and dangerous form presents with a ring of nodules surrounding a verrucose or occult central area, especially on the neck/ jugular and buttock regions. Predilection sites include jaw, face (**Figure 9**),

Differential diagnoses for malignant sarcoids are squamous cell carcinoma, lymphoma/lymphosarcoma, subcutaneous mycosis, lymphangitis, glanders, epizootic lymphangitis/histoplasmosis, and hypertrophic scarring/cheloid.

noma, sweat gland tumor, giant cell sarcoma, and mycosis fungoides.

*Mixed sarcoid: a type B2 nodule with a small fibroblastic sarcoid within an occult area.*

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*Malignant sarcoid on the face: 'bound-down' invasive nodules with a central area with occult to verrucose changes, ulceration, and a fibroblastic component.*

#### **5. Clinical examination and diagnostic procedures**

The clinical examination should include signalment and a full thorough history with details on lesion development, especially about the behavior and progression. The clinical presentation of sarcoid lesions and their features are usually clearly recognizable, especially if multiple tumors of different types are present on the same horse. The confirmation of the diagnosis is not always straightforward and possible differential diagnoses and concurrent conditions should be considered [55, 56].

Depending on the sarcoid type, the full list of differential diagnoses should be considered when choosing the diagnostic procedures. The diagnosis of sarcoid is confirmed by histopathology, thus a biopsy sample is needed. Partial or excisional biopsy should provide sufficient information but a risk of exacerbation due to the surgical trauma should always be taken into account. If possible, a total excisional biopsy is preferable, the owner should be carefully advised about the implicit risks and a proper therapeutic plan should be prepared when taking the biopsy to avoid any exacerbation triggered by the procedure. If benign neglect is the plan, the opportunity of taking a biopsy should be carefully evaluated [2, 55].

#### **5.1 Equine sarcoid pathology**

Histopathology is deemed necessary to confirm the diagnosis of many equine sarcoids [57]. It is important to stress that due to the variable microscopic features of equine sarcoids, small biopsies may not provide enough tissue to differentiate sarcoids from other lesions such as granulation tissue, fibromas, or fibrosarcomas. This is especially true if samples are obtained from ulcerated areas of the tumors [58]. Notably, trauma and reparative processes (wound healing) may activate cell growth and facilitate the development or heighten the progression of equine sarcoids [57–59], particularly for verrucose, occult and small nodular sarcoids [60]. Thus, excisional biopsies with wide margins should be favored for clinical reasons and because they provide with the most diagnostic material [46, 58]. If a non-excisional biopsy must be performed, sites within the mass must be carefully chosen to minimize the confounding factors of surrounding inflammation and granulation and to include intact epidermis [46].

Sarcoids derive from the proliferation of two components: the dermal fibroblasts and epidermal keratinocytes. They are regarded as biphasic tumors. Histopathology is heterogeneous and microscopic aspects and number of components varies according to the type of sarcoid [61].

Microscopic features of the epidermis may include orthokeratotic to compact hyperkeratosis, parakeratosis, irregular hyperplasia with epithelial proliferations producing long and pointed branches, termed rete pegs or rete ridges, extending deep into the dermal proliferation (**Figure 10A**) [46]. Epidermal ulceration is variable but frequent in nodular and fibroblastic sarcoids.

The amount of epithelial cell proliferation varies according with the type of sarcoid and ranges from severe hyperplasia to epidermal atrophy [1, 61]. Overall up to 46% of sarcoids lack epidermal hyperplasia and 54% lack rete peg formation [61]. Epidermal changes are maximal in verrucous sarcoids [58] and can be minimal to absent in nodular and occult sarcoids. Epidermal ulceration is common especially in nodular sarcoids [58].

All sarcoids are characterized by variable substitution of normal dermal components by neoplastic fibroblasts embedded in variable amounts of collagen. Histopathological findings consist of poorly demarcated, unencapsulated, variably infiltrative proliferation of large spindle to stellate, bland to highly atypical fibroblasts with plump, oval, nuclei with granular chromatin and variable hyperchromasia and with prominent nucleoli. Cellular atypia is low to absent and increases with time, number of excisions, ulceration, inflammation and type of sarcoid, being higher in malignant and mixed sarcoids. Number of mitoses is generally low (0–1 per HPF) if excluding malignant sarcoids. Density of neoplastic fibroblast is oftentimes higher in the superficial dermis [61]. At the dermal-epidermal junction, fibroblasts may be oriented perpendicularly to the basement membrane in the so-called "picket fence" arrangement (**Figure 10B** and **C**) [61–63]. This feature is considered highly diagnostic but is missing in up to 52% of sarcoids [61]. Additional patterns that can been seen at all levels of the dermis are whorling (**Figure 11A**), present in over 86% of tumors [64], parallel to interlacing short bundles (**Figure 11B**), storiform, herringbone, tangles or fibroblasts may be haphazardly arranged, this latter arrangement occurring more often in flat sarcoids (**Figure 11C**) [46, 62]. Amount of collagen matrix varies from minimal to abundant and can be dense, edematous, or myxoid (**Figure 11D**). Adnexal structures are variably reduced in density or obscured by the neoplasm [46].

Of all types of equine sarcoids, flat/occult sarcoids at initial stages can be easily overlooked at histopathology [63]. For this type of sarcoid, the only histopathologic finding may be an increased density of subepidermal neoplastic fibroblasts infiltrating between a reduced number of hair follicles and sweat glands [61]. The density of dermal fibroblasts is lower compared with the other types of sarcoids [58].

Immunohistochemistry can assist in the diagnosis of sarcoids although protein expression patterns are not considered highly specific. Fibroblasts in sarcoid express vimentin, the intermediate filament identifying mesodermal origin, and may be variably positive for laminin, smooth muscle actin, and type IV

**119**

negative [62].

*of equine sarcoids. Hematoxylin and eosin, 100×.*

**Figure 10.**

collagen [59, 65, 66]. Sarcoids are generally S100 negative [65], however, S100 focal expression has been observed [66]. Bovine papillomavirus is involved in the pathogenesis of equine sarcoids, however, BPV infection of fibroblasts is mainly nonproductive [10]. Therefore immunohistochemistry against BPV is mostly

*(A) Moderate hyperkeratosis and severe epidermal irregular hyperplasia with rete peg formation. In the superficial dermis higher density of neoplastic fibroblasts compared to mid dermis is evident. Hematoxylin and eosin, 200×. (B) Moderate compact hyperkeratosis with mild epidermal hyperplasia and mild rete peg formation. In the superficial and mid dermis, typical picket fence arrangement of fibroblasts is present. The picket fence pattern is considered a highly diagnostic pattern but is observed in less than 50% of equine sarcoids. Hematoxylin and eosin, 20×. (C) Moderate compact hyperkeratosis with mild epidermal hyperplasia and rete peg formation. In the superficial dermis, high cellularity and typical picket fence arrangement of fibroblasts are present. The picket fence pattern is considered a highly diagnostic pattern but is observed in less than 50%* 

BPV DNA can be detected by in situ hybridization and PCR on formalin fixed and paraffin embedded tissue sections of biopsy samples [36, 65, 67] or by PCR fresh cytological specimens obtained by swabbing or scraping of equine sarcoid tissue in non-healing wounds and recurrent cases and following recurrence after surgery [9].

*Equine Sarcoid*

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

*Equine Science*

This is especially true if samples are obtained from ulcerated areas of the tumors [58]. Notably, trauma and reparative processes (wound healing) may activate cell growth and facilitate the development or heighten the progression of equine sarcoids [57–59], particularly for verrucose, occult and small nodular sarcoids [60]. Thus, excisional biopsies with wide margins should be favored for clinical reasons and because they provide with the most diagnostic material [46, 58]. If a non-excisional biopsy must be performed, sites within the mass must be carefully chosen to minimize the confounding factors of surrounding inflammation and

Sarcoids derive from the proliferation of two components: the dermal fibroblasts and epidermal keratinocytes. They are regarded as biphasic tumors. Histopathology is heterogeneous and microscopic aspects and number of components varies

Microscopic features of the epidermis may include orthokeratotic to compact hyperkeratosis, parakeratosis, irregular hyperplasia with epithelial proliferations producing long and pointed branches, termed rete pegs or rete ridges, extending deep into the dermal proliferation (**Figure 10A**) [46]. Epidermal ulceration is vari-

The amount of epithelial cell proliferation varies according with the type of sarcoid and ranges from severe hyperplasia to epidermal atrophy [1, 61]. Overall up to 46% of sarcoids lack epidermal hyperplasia and 54% lack rete peg formation [61]. Epidermal changes are maximal in verrucous sarcoids [58] and can be minimal to absent in nodular and occult sarcoids. Epidermal ulceration is common especially

All sarcoids are characterized by variable substitution of normal dermal components by neoplastic fibroblasts embedded in variable amounts of collagen. Histopathological findings consist of poorly demarcated, unencapsulated, variably infiltrative proliferation of large spindle to stellate, bland to highly atypical fibroblasts with plump, oval, nuclei with granular chromatin and variable hyperchromasia and with prominent nucleoli. Cellular atypia is low to absent and increases with time, number of excisions, ulceration, inflammation and type of sarcoid, being higher in malignant and mixed sarcoids. Number of mitoses is generally low (0–1 per HPF) if excluding malignant sarcoids. Density of neoplastic fibroblast is oftentimes higher in the superficial dermis [61]. At the dermal-epidermal junction, fibroblasts may be oriented perpendicularly to the basement membrane in the so-called "picket fence" arrangement (**Figure 10B** and **C**) [61–63]. This feature is considered highly diagnostic but is missing in up to 52% of sarcoids [61]. Additional patterns that can been seen at all levels of the dermis are whorling (**Figure 11A**), present in over 86% of tumors [64], parallel to interlacing short bundles (**Figure 11B**), storiform, herringbone, tangles or fibroblasts may be haphazardly arranged, this latter arrangement occurring more often in flat sarcoids (**Figure 11C**) [46, 62]. Amount of collagen matrix varies from minimal to abundant and can be dense, edematous, or myxoid (**Figure 11D**). Adnexal structures are variably reduced in density or

Of all types of equine sarcoids, flat/occult sarcoids at initial stages can be easily overlooked at histopathology [63]. For this type of sarcoid, the only histopathologic finding may be an increased density of subepidermal neoplastic fibroblasts infiltrating between a reduced number of hair follicles and sweat glands [61]. The density of

Immunohistochemistry can assist in the diagnosis of sarcoids although protein

dermal fibroblasts is lower compared with the other types of sarcoids [58].

expression patterns are not considered highly specific. Fibroblasts in sarcoid express vimentin, the intermediate filament identifying mesodermal origin, and may be variably positive for laminin, smooth muscle actin, and type IV

granulation and to include intact epidermis [46].

able but frequent in nodular and fibroblastic sarcoids.

according to the type of sarcoid [61].

in nodular sarcoids [58].

obscured by the neoplasm [46].

**118**

#### **Figure 10.**

*(A) Moderate hyperkeratosis and severe epidermal irregular hyperplasia with rete peg formation. In the superficial dermis higher density of neoplastic fibroblasts compared to mid dermis is evident. Hematoxylin and eosin, 200×. (B) Moderate compact hyperkeratosis with mild epidermal hyperplasia and mild rete peg formation. In the superficial and mid dermis, typical picket fence arrangement of fibroblasts is present. The picket fence pattern is considered a highly diagnostic pattern but is observed in less than 50% of equine sarcoids. Hematoxylin and eosin, 20×. (C) Moderate compact hyperkeratosis with mild epidermal hyperplasia and rete peg formation. In the superficial dermis, high cellularity and typical picket fence arrangement of fibroblasts are present. The picket fence pattern is considered a highly diagnostic pattern but is observed in less than 50% of equine sarcoids. Hematoxylin and eosin, 100×.*

collagen [59, 65, 66]. Sarcoids are generally S100 negative [65], however, S100 focal expression has been observed [66]. Bovine papillomavirus is involved in the pathogenesis of equine sarcoids, however, BPV infection of fibroblasts is mainly nonproductive [10]. Therefore immunohistochemistry against BPV is mostly negative [62].

BPV DNA can be detected by in situ hybridization and PCR on formalin fixed and paraffin embedded tissue sections of biopsy samples [36, 65, 67] or by PCR fresh cytological specimens obtained by swabbing or scraping of equine sarcoid tissue in non-healing wounds and recurrent cases and following recurrence after surgery [9].

#### **Figure 11.**

*(A) Bland neoplastic fibroblasts whorling around a thick collagen bundle. Whorling is considered a highly diagnostic pattern described in over 85% of equine sarcoids. Hematoxylin and eosin, 200×. (B) Area of high cellularity with plump neoplastic fibroblasts embedded in finely fibrillar to dense collagen and organized in parallel and perpendicular rows. Moderate anisocytosis and anisokaryosis are evident. Hematoxylin and eosin, 400×. (C) Area of moderate cellularity with parallel to haphazardly arranged plump fibroblasts embedded in abundant finely fibrillar to dense collagen. Moderate anisokaryosis is evident. Hematoxylin and eosin, 400×. (D) Area of high cellularity with haphazardly arranged highly atypical fibroblasts with spindle to stellate morphology embedded in finely fibrillar to myxoid stroma observed in a recurrent sarcoid. Hematoxylin and eosin, 200×.*

DNA from BPV 1, 2 [14, 67] and 13 [12] is detected in up to 90% of equine sarcoids by in situ hybridization in the nuclei of fibroblasts and keratinocytes [37, 67]. Disadvantages of DNA detection are the unsuitability for diagnosing occult sarcoids, the lower sensitivity compared to clinical diagnosis, and the low specificity due to high prevalence of BPV DNA positivity in normal equine skin samples [35], cutaneous inflammation [68], and in other skin-associated spindle cell soft tissue tumors such as peripheral nerve sheath tumors (PNSTs), fibrosarcomas, myxosarcomas, and fibromas [66].

In summary, the most diagnostic histopathologic features, when present, are the epidermal changes of hyperkeratosis, hyperplasia with elongated rete pegs and "picket fence" aspect in conjunction by proliferation of fibroblasts [9, 35, 62]. However, common to most sarcoids are the fibroblastic dermal proliferation and presence of BPV DNA [10, 35, 67].

Microscopic features of sarcoids can overlap with other lesions. Differential diagnosis may be challenging because of the variable histological configuration of the dermal proliferation especially in cases with extensive ulceration or lack of distinctive epidermal lesions. Major histopathological differentials include granulation tissue (proud flesh), fibroma, fibrosarcoma, and peripheral nerve sheath tumors (e.g. schwannoma and neurofibroma) [57, 63]. Granulation tissue is characterized by fibrous tissue oriented at right angles to newly formed capillaries and is often associated with edema

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S100 positivity has been reported in sarcoids [62, 66].

**6. Management of the equine sarcoid**

in the literature [3].

made [3, 55]:

**6.1 General considerations**

successful treatment.

the malignant form.

and a prominent inflammatory component. When fibroblastic sarcoids are ulcerated, it may not be possible to differentiate them from granulation tissue and clinical follow-up becomes necessary. Fibromas can be differentiated morphologically as well-circumscribed, expansile, sparsely cellular tumors composed of a monomorphic population of mature fibroblasts with no epidermal proliferation. Fibrosarcoma is more pleomorphic with higher cytological atypia but multiple patterns are rarely observed, and the epidermal component is absent. Peripheral nerve sheath tumors (PNST)/Schwannoma are characterized by variable presence of highly cellular often palisading areas (Antoni A pattern) and low cellular myxoid areas (Antoni B pattern). These areas are associated with the presence of typical Verocay bodies composed of acellular areas between areas of nuclear palisading. Immunohistochemical staining for S-100 protein may be useful in differentiating PNSTs from sarcoids; however, focal

A treatment should be prompted as soon as possible following diagnosis, and in

Before choosing a proper therapeutic plan, some general considerations must be

• Sarcoid-affected animals can never be considered free of the disease, even after

• Each lesion can require a specific treatment and can react in a different way

• The extent and location of the tumor greatly affect the decisional process. Periorbital sarcoids (**Figure 12**) tend to penetrate the underlying musculature. Function of the upper eyelid must be preserved and any possible deformation in the healing process must be avoided. Sarcoids over tendons, joints or the facial nerve can have severe complications. The worst sites are the elbow and the face, where sarcoids much more tend to local invasion and progression to

• The duration of the lesion is important as early intervention usually requires less aggressive treatments. It is also easier to treat small lesions that extensive ones that may also be under transformation from one type to the other.

• Previous therapies and/or interferences influence the response to a new treatment course and possibly a different approach may be indicated.

compared to other sarcoids even on the same horse.

• The prognosis is usually very guarded and owners must be thoroughly informed about possible complications associated with the condition.

some cases, suspicious lesions could be treated immediately after biopsy [5]. Several treatment modalities for the management of equine sarcoids are historically 'known' and anecdotal reports and retrospective studies on more or less effective therapies exist, but valuable prospective double-blinded trials are lacking

#### *Equine Sarcoid DOI: http://dx.doi.org/10.5772/intechopen.91013*

*Equine Science*

**120**

and fibromas [66].

**Figure 11.**

*eosin, 200×.*

presence of BPV DNA [10, 35, 67].

DNA from BPV 1, 2 [14, 67] and 13 [12] is detected in up to 90% of equine sarcoids by in situ hybridization in the nuclei of fibroblasts and keratinocytes [37, 67]. Disadvantages of DNA detection are the unsuitability for diagnosing occult sarcoids, the lower sensitivity compared to clinical diagnosis, and the low specificity due to high prevalence of BPV DNA positivity in normal equine skin samples [35], cutaneous inflammation [68], and in other skin-associated spindle cell soft tissue tumors such as peripheral nerve sheath tumors (PNSTs), fibrosarcomas, myxosarcomas,

*(A) Bland neoplastic fibroblasts whorling around a thick collagen bundle. Whorling is considered a highly diagnostic pattern described in over 85% of equine sarcoids. Hematoxylin and eosin, 200×. (B) Area of high cellularity with plump neoplastic fibroblasts embedded in finely fibrillar to dense collagen and organized in parallel and perpendicular rows. Moderate anisocytosis and anisokaryosis are evident. Hematoxylin and eosin, 400×. (C) Area of moderate cellularity with parallel to haphazardly arranged plump fibroblasts embedded in abundant finely fibrillar to dense collagen. Moderate anisokaryosis is evident. Hematoxylin and eosin, 400×. (D) Area of high cellularity with haphazardly arranged highly atypical fibroblasts with spindle to stellate morphology embedded in finely fibrillar to myxoid stroma observed in a recurrent sarcoid. Hematoxylin and* 

In summary, the most diagnostic histopathologic features, when present, are the epidermal changes of hyperkeratosis, hyperplasia with elongated rete pegs and "picket fence" aspect in conjunction by proliferation of fibroblasts [9, 35, 62]. However, common to most sarcoids are the fibroblastic dermal proliferation and

Microscopic features of sarcoids can overlap with other lesions. Differential diagnosis may be challenging because of the variable histological configuration of the dermal proliferation especially in cases with extensive ulceration or lack of distinctive epidermal lesions. Major histopathological differentials include granulation tissue (proud flesh), fibroma, fibrosarcoma, and peripheral nerve sheath tumors (e.g. schwannoma and neurofibroma) [57, 63]. Granulation tissue is characterized by fibrous tissue oriented at right angles to newly formed capillaries and is often associated with edema

and a prominent inflammatory component. When fibroblastic sarcoids are ulcerated, it may not be possible to differentiate them from granulation tissue and clinical follow-up becomes necessary. Fibromas can be differentiated morphologically as well-circumscribed, expansile, sparsely cellular tumors composed of a monomorphic population of mature fibroblasts with no epidermal proliferation. Fibrosarcoma is more pleomorphic with higher cytological atypia but multiple patterns are rarely observed, and the epidermal component is absent. Peripheral nerve sheath tumors (PNST)/Schwannoma are characterized by variable presence of highly cellular often palisading areas (Antoni A pattern) and low cellular myxoid areas (Antoni B pattern). These areas are associated with the presence of typical Verocay bodies composed of acellular areas between areas of nuclear palisading. Immunohistochemical staining for S-100 protein may be useful in differentiating PNSTs from sarcoids; however, focal S100 positivity has been reported in sarcoids [62, 66].

### **6. Management of the equine sarcoid**

A treatment should be prompted as soon as possible following diagnosis, and in some cases, suspicious lesions could be treated immediately after biopsy [5].

Several treatment modalities for the management of equine sarcoids are historically 'known' and anecdotal reports and retrospective studies on more or less effective therapies exist, but valuable prospective double-blinded trials are lacking in the literature [3].

#### **6.1 General considerations**

Before choosing a proper therapeutic plan, some general considerations must be made [3, 55]:


**Figure 12.** *Periorbital sarcoid, eyelid function must be preserved when treating these lesions.*

Wrong interference is a major cause of exacerbation and the prognosis significantly reduces with each treatment failure.


#### **6.2 Benign neglect**

As previously discussed, a proper treatment should follow the diagnosis of equine sarcoid, but in some cases benign neglect may be an option. Horses may present with such extensive lesions that any treatment method would be impractical. In other patients, the sarcoids may be small enough to render the procedure too expensive. Clinicians should opt for benign neglect with caution, both patient welfare and the lesions should be strictly monitored as sarcoids can progress. Furthermore, their presence may contribute to spread to other sites and horses [3, 55].

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**6.3 Surgical methods**

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using surgery alone [55].

recognized in equine practice [55].

exacerbation or recurrence [3, 55].

• *Sharp surgical excision*: this technique is often appealing to practitioners and, in some cases, easy and successful but carries rates of recurrence as high as 70%, with recurrences occurring mostly within few months or even during the healing process and being much more aggressive (commonly fibroblastic) than the original sarcoid [2, 3, 46]. Wide excision is necessary to reduce the risk of recurrence, but it is not always practical or feasible and a safe margin is impossible to define. The principle of smart surgery should be applied to minimize cell contamination during surgery. Protecting the tumor with adhesive dressings before surgery reduces contamination and in case of recurrence another therapeutic method or combined treatments are indicated [3, 55]. Occult and verrucose sarcoids can be effectively removed with wide margins, nodules in the eyelids are invasive thus very dangerous, whereas other nodular lesions may respond better. However, the prognosis is usually very guarded when

• *Cryosurgery*: this method causes tumor necrosis and is commonly used but has the same limitations as surgical excision. It can be used successfully on superficial lesions but restriction of blood flow, a defined safety margin and adjuvant chemotherapy (intralesional or topical) during the procedure can improve outcome. It can be repeated if necessary until the tumor is completely removed but the ability of the patient to resist the cold can be a limitation [3, 55, 69].

• *Hyperthermia/radiofrequency hyperthermia*: the tumor, being more sensitive to temperature than normal tissue, is heated for 30 s to 50°C weekly for up to 5 weeks. Very few cases are reported using this technique that is not generally

• *Surgical electrocautery*: this method was recently reported with a high rate of success, its advantages are the minimal bleeding into the wound site with a reduced risk of tumor cell contamination and usually limited scarring. Electrocautery is one of the few options for sarcoids on the ear pinna [3, 55, 70].

• *Laser surgery*: surgical ablation with CO2-YAG laser or diode laser devices is reported with success rates as high as more than 80%. When accurately used, this method is associated with the ability to sterilize the wound, no bleeding and avoids seeding tumor cells during the procedure. CO2 lasers cause less thermal injury than diode ones. Primary closure may be possible, but a high rate of wound dehiscence and slow healing are disadvantages. Careful selection of the lesion is important: recurrence is most likely in verrucose sarcoids with poorly defined margins, whereas localized pinnal sarcoids and fibroblastic

• *Ligation*: this method can be used only on pedunculated sarcoids where no tumor extensions are present in the pedicle below the ligature. This means that it is suitable for nodular type A1 and B1 or fibroblastic type 1a sarcoids, or any sarcoid where an artificial tumor-free pedicle can be created. The pedicle is ligated with castration/elastration bands, it works better if several bands can be placed and if adjunctive intralesional or chemotherapy are combined. The use of plastic ties or suture material that cut the lesion and partial ligation should be avoided as it carries a poorer prognosis and is associated with

type 1a tumors around the eye may respond well [3, 55, 71, 72].

#### **6.3 Surgical methods**

*Equine Science*

Wrong interference is a major cause of exacerbation and the prognosis signifi-

• Planning combined, prolonged or repeated treatments can be necessary for

• Costs and logistics can have a great influence on the choice of the therapeutic

• Professional skills and experience of the veterinarian can also affect the rate of success and the same treatment used by different clinicians can result in

• Animal and owner compliance for the best treatment: some are very painful, some sites (e.g. ear) are more sensitive, general anesthesia may be necessary in

• Careful fly protection, wound management and regular checks must be part of

• Spontaneous remission is reported but rare, the decision to delay treatment

As previously discussed, a proper treatment should follow the diagnosis of equine sarcoid, but in some cases benign neglect may be an option. Horses may present with such extensive lesions that any treatment method would be impractical. In other patients, the sarcoids may be small enough to render the procedure too expensive. Clinicians should opt for benign neglect with caution, both patient welfare and the lesions should be strictly monitored as sarcoids can progress. Furthermore, their

the long-term management of any sarcoid-affected horse.

based on a possible spontaneous remission is discouraged.

presence may contribute to spread to other sites and horses [3, 55].

cantly reduces with each treatment failure.

*Periorbital sarcoid, eyelid function must be preserved when treating these lesions.*

many sarcoids.

different outcomes.

certain cases.

**6.2 Benign neglect**

modality.

**Figure 12.**

**122**


#### **6.4 Chemotherapy**

Different chemotherapeutic agents and compounds can be used to treat sarcoids, usually they are topically or intralesionally administered with little or no systemic effects [55]. Systemic doxorubicin was used only in one study, but limitations and constraints to its use reserve this treatment only to very extensive or wide-spread lesions referred to specialist centers [3, 73].


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margins [3, 55, 70, 80].

[3, 79, 82, 83].

**6.6 Immunotherapy**

remission [3].

repeated injections are performed [55, 90].

but further studies are needed [3, 91].

**6.5 Photodynamic therapy**

concentration and a slower release. Due to the high toxicity, self-protection measures must be strictly respected when handling cisplatin [3, 55, 79]. The use of biodegradable beads containing cisplatin is also reported with or without surgical debulking, the latter is usually necessary in tumors larger than 1.5 cm in diameter. Beads are placed at 1–1.5 cm intervals along the wound or tumor

• *Bleomycin*: bleomycin is a glycopeptide antibiotic with antineoplastic activity, it has been used to treat sarcoids intralesionally and with the use of electrochemotherapy. Recently the topical use on occult and verrucose sarcoids of an ultradeformable liposomal preparation of bleomycin, alone or following 5-fluorouracil or tazarotene application, has shown good efficacy with the

• *Electrochemotherapy*: electrochemotherapy is based on the use of electrically induced increases in cell membrane permeability to increase the effects of cytotoxic agents such as cisplatin, carboplatin, and bleomycin. It requires repeated general anesthesia, up to 8 treatments, and specialist equipment

This method is based on photosensitization of tumor cells with a topical or intralesional photosensitizer (e.g. hypericin or 5-aminolevulinic acid and derivatives) followed by the application of a specific light wavelength emitted by a proper light source, for a defined time (minutes). The mechanism is complex and takes advantage of the production of reactive oxygen species that kill sensitized cells, so it is very localized. The literature on its use on sarcoids is limited but significant

Since the involvement of BPV infection, much research is being focused on immunologic methods but without practical results so far. Moreover, horses do not seroconvert for BPV and vaccination does not prevent sarcoid development [3].

• *Spontaneous remission*: it is generally reported as rare, however, a recent study just reported a high proportion of spontaneous remission in a population of Franches-Montagnes horses in Switzerland [53]. The mechanism is not clear yet and antibodies have been detected only in donkeys [5]. Long-term immunity appear to occur in horses with sarcoids that undergo spontaneous

• *Immunomodulation*: the use of intralesional injection of the bacillus Calmette-Guérin (BCG) is reported in different studies with high success rates, especially around the eye. Sarcoids on the distal limbs respond less or may even exacerbate [3, 69, 89, 90]. This method gives best results on nodular and fibroblastic sarcoids but may be associated with anaphylaxis, especially when

• *Vaccines*: attempts to stimulate sarcoid regression or potential preventive effects through autogenous and BPV-1 L1virus-like particles vaccines have been made

absence of pain and inflammation as an advantage [3, 81].

benefits are reported, with or without surgical debulking [3, 84–88].

#### *Equine Sarcoid DOI: http://dx.doi.org/10.5772/intechopen.91013*

*Equine Science*

**6.4 Chemotherapy**

lesions referred to specialist centers [3, 73].

of 5-fluorouracil at the dose of 50 mg/cm3

ment history [3, 52, 55].

sion at the dose of 1 mg/cm3

veterinary advice with deleterious effects [3].

be applied to very localized small lesions [55].

poorer prognosis compared with smaller lesions [75].

helpful in some cases to control the discomfort [55, 70, 76].

Different chemotherapeutic agents and compounds can be used to treat sarcoids, usually they are topically or intralesionally administered with little or no systemic effects [55]. Systemic doxorubicin was used only in one study, but limitations and constraints to its use reserve this treatment only to very extensive or wide-spread

• *Topical and intralesional 5-fluorouracil*: this cytotoxic and antimitotic agent can be topically applied as 5% ointment with a twice daily protocol over a few weeks. It is usually successful on small occult and verrucose lesions, or to control large areas that cannot be treated with other modalities. During treatment an inflammatory reaction can be marked but usually minimal scarring follows. It can also be combined with surgery [3, 55, 74]. The intralesional injection

reported with a successful rate of 61.5%, sarcoids larger than 13.5 cm3

• *Topical imiquimod*: this agent is an immune response modifier with potent antiviral and antitumor activity and is used to treat human genital warts. The reported protocol for equine sarcoid is to apply the cream three times a week for 16–32 weeks. The treatment is usually associated with inflammation, alopecia and depigmentation. Administration of oral phenylbutazone can be

• *Topical AW5*: it is a cream containing heavy metal salts, fluorouracil, thiouracil and steroid. Its use is restricted to veterinarians only, protocols include repeated applications but it can be contraindicated in some cases such as around the eye or other structures (facial nerve) that can be damaged. The reported success rate is around 74% depending on lesion and previous treat-

• *Topical acyclovir*: topical 5% acyclovir cream has been used to treat sarcoids with some benefits reported in one study [77]. A subsequent retrospective case-series and a double-blinded placebo-controlled trial resulted in no advantages from this agent compared to other treatments or placebo [70, 78]. The cream is used without prescription for human herpes virus infection and this may be attractive for owners that desire to treat horses without looking for

• *Silver nitrate*: silver nitrate caustic pencil is an old-fashioned treatment that can

every 2 weeks for four times but intervals may

• *Intralesional cisplatin*: this chemotherapeutic agent has been used in several studies in the form of injectable solution, emulsion and of biodegradable beads. Resolution rates are up to 93% in sarcoids less than 5 cm in diameter, larger lesions can be cured combining surgical debulking and intralesional cisplatin [3, 55]. A general protocol includes repeated injections of cisplatin oily emul-

change upon patient needs. The material does not diffuse more than 5 mm in tissues so several injections every 6 mm–1 cm of tumor and margin of normal tissue are necessary. The aqueous solution has a clearance of minutes whereas the medical-grade sesame seed oil emulsion has the advantage of a lower

every 2 weeks for up to 7 weeks is

had a

**124**

concentration and a slower release. Due to the high toxicity, self-protection measures must be strictly respected when handling cisplatin [3, 55, 79]. The use of biodegradable beads containing cisplatin is also reported with or without surgical debulking, the latter is usually necessary in tumors larger than 1.5 cm in diameter. Beads are placed at 1–1.5 cm intervals along the wound or tumor margins [3, 55, 70, 80].


#### **6.5 Photodynamic therapy**

This method is based on photosensitization of tumor cells with a topical or intralesional photosensitizer (e.g. hypericin or 5-aminolevulinic acid and derivatives) followed by the application of a specific light wavelength emitted by a proper light source, for a defined time (minutes). The mechanism is complex and takes advantage of the production of reactive oxygen species that kill sensitized cells, so it is very localized. The literature on its use on sarcoids is limited but significant benefits are reported, with or without surgical debulking [3, 84–88].

#### **6.6 Immunotherapy**

Since the involvement of BPV infection, much research is being focused on immunologic methods but without practical results so far. Moreover, horses do not seroconvert for BPV and vaccination does not prevent sarcoid development [3].


#### **6.7 Gene therapy**

Mediator-governed therapy and genetic manipulation are under research but no practical treatment for the equine sarcoid has been reported yet [3, 27].

#### **6.8 Radiotherapy**

Facilities and special equipment are required for radiation therapy, which contribute to its high costs and limited availability. Different techniques exist:


#### **6.9 Adjunctive therapy**

To remove secondary epidermal changes in sarcoid tumors, tazarotene can be used as adjunctive treatment. It is a retinoid 0.1% gel commonly used in human medicine for the management of keratinization disorders [3].

#### **6.10 Phytotherapy**


#### **6.11 Other remedies**

Several 'natural' or herbal or homeopathic remedies are often used to treat sarcoids, usually with a delay in proper treatment and a risk of interference causing exacerbation of the tumor. Caution should be used considering the use of any material suggested to treat every condition in every species [3].

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*Equine Sarcoid*

**8. Conclusions**

**Author details**

Beatrice Funiciello1

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

and zebras is sometimes reported as outbreaks [105–109].

Sarcoid tumors are reported also in animals other than horses. Donkeys, mules, and zebras can be affected, but reports of sarcoid tumors associated to BPV infection exist also in cats, giraffes, sable antelopes, and captive tapirs [98–104].

As far as equids are concerned, the reported prevalence of sarcoids in zebras is 25–53%, whereas incidence in UK donkeys is 0.6 per 100 animal years with apparent increased risk for young males [5, 105, 106]. The equine sarcoid is reported as the most common tumor in donkeys and presence of sarcoids among these equids

Diagnostic and treatment methods are the same as for horses, one study reports

The equine sarcoid is a locally invasive skin neoplasm commonly encountered in practice. It has different clinical presentations, and early diagnosis with prompt treatment can improve the prognosis, but their importance is often underestimated. Several treatment options are available with variability in lesion and patient response. Spontaneous regression is rare, recurrence is common, and exacerbation is a possible complication, especially when a wrong therapy is attempted. Sarcoidaffected animals can never be considered free of the disease and horse owners must

be correctly informed about the features and behavior of this tumor.

\* and Paola Roccabianca<sup>2</sup>

2 DIMEVET Università degli Studi di Milano—La Statale, Lodi, Italy

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: beatrice.funiciello@bemavet.com

1 Private Practitioner, Torre Boldone (BG), Italy

provided the original work is properly cited.

the use of surgical excision, intralesional 5-fluorouracil, allogenous vaccine or 5-fluorouracil in combination with autogenous vaccine in zebras [98, 110].

**7. Sarcoids in other equids**

### **7. Sarcoids in other equids**

*Equine Science*

**6.7 Gene therapy**

**6.8 Radiotherapy**

**6.9 Adjunctive therapy**

**6.10 Phytotherapy**

**6.11 Other remedies**

compared to placebo [3, 97].

• *Autoinoculation/autografting*: the inoculation of sarcoid tissue is reported in two studies, but doubts are raised about this method due to the risk of complications and the fact that it is not described for other cancers in any species [3, 92].

• *Hemotherapy*: no literature is available describing the effectiveness of this method. However, it is widely used in Central America and consists of withdrawal of venous blood and its intramuscular injection with anecdotal success [3].

Mediator-governed therapy and genetic manipulation are under research but no

Facilities and special equipment are required for radiation therapy, which contribute to its high costs and limited availability. Different techniques exist:

• *Teletherapy*: it is expected to be effective, but few reports exist [3, 93].

• *Brachytherapy*: using radioactive radon, iridium and gold isotopes, it has

• *Plesiotherapy*: this surface brachytherapy method uses beta radiation from

To remove secondary epidermal changes in sarcoid tumors, tazarotene can be used as adjunctive treatment. It is a retinoid 0.1% gel commonly used in human

• *Viscus album austriacus*: the use of the injectable extract of the white mistletoe plant is reported to have immunomodulating effects in humans and was used in one double-blinded placebo-controlled trial in horses with sarcoids. Repeated subcutaneous injections for 15 weeks provided a positive outcome

• *Sanguinaria canadensis/zinc chloride*: commercially available compounds

Several 'natural' or herbal or homeopathic remedies are often used to treat sarcoids, usually with a delay in proper treatment and a risk of interference causing exacerbation of the tumor. Caution should be used considering the use of any mate-

literature and dangerous toxicity in humans is reported [3].

rial suggested to treat every condition in every species [3].

containing bloodroot (*S. canadensis*) and zinc chloride are anecdotally used for the treatment of equine sarcoids. Although high rates of success are reported on the internet, the use of this material by owners without veterinary advice carries risks. The use of this product on horses is not supported by scientific

strontium90 and is reported on small superficial sarcoids [3, 93].

medicine for the management of keratinization disorders [3].

become the gold standard for sarcoids, especially periorbital lesions [3, 93–96].

practical treatment for the equine sarcoid has been reported yet [3, 27].

**126**

Sarcoid tumors are reported also in animals other than horses. Donkeys, mules, and zebras can be affected, but reports of sarcoid tumors associated to BPV infection exist also in cats, giraffes, sable antelopes, and captive tapirs [98–104].

As far as equids are concerned, the reported prevalence of sarcoids in zebras is 25–53%, whereas incidence in UK donkeys is 0.6 per 100 animal years with apparent increased risk for young males [5, 105, 106]. The equine sarcoid is reported as the most common tumor in donkeys and presence of sarcoids among these equids and zebras is sometimes reported as outbreaks [105–109].

Diagnostic and treatment methods are the same as for horses, one study reports the use of surgical excision, intralesional 5-fluorouracil, allogenous vaccine or 5-fluorouracil in combination with autogenous vaccine in zebras [98, 110].

#### **8. Conclusions**

The equine sarcoid is a locally invasive skin neoplasm commonly encountered in practice. It has different clinical presentations, and early diagnosis with prompt treatment can improve the prognosis, but their importance is often underestimated. Several treatment options are available with variability in lesion and patient response. Spontaneous regression is rare, recurrence is common, and exacerbation is a possible complication, especially when a wrong therapy is attempted. Sarcoidaffected animals can never be considered free of the disease and horse owners must be correctly informed about the features and behavior of this tumor.

#### **Author details**

Beatrice Funiciello1 \* and Paola Roccabianca2

1 Private Practitioner, Torre Boldone (BG), Italy

2 DIMEVET Università degli Studi di Milano—La Statale, Lodi, Italy

\*Address all correspondence to: beatrice.funiciello@bemavet.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Marti E, Lazary S, Antczak DF, Gerber H. Report of the first international workshop on equine sarcoid. Equine Veterinary Journal. 1993;**25**(5):397-407. DOI: 10.1111/ j.2042-3306.1993.tb02981.x

[2] Bergvall KE. Sarcoids. The Veterinary Clinics of North America. Equine Practice. 2013;**29**:657-671. DOI: 10.1016/j.cveq.2013.09.002

[3] Knottenbelt DC. The equine sarcoid: Why Are There So Many Treatment Options? Veterinary Clinics of North America: Equine Practice. 2019;**35**:243- 262. DOI: 10.1016/j.cveq.2019.03.006

[4] Valentine BA. Survey of equine cutaneous neoplasia in the Pacific Northwest. Journal of Veterinary Diagnostic Investigation. 2006;**18**:123-126. DOI: 10.1177/104063870601800121

[5] Knottenbelt DC, Patterson-Kane JC, Snalune KL, editors. Sarcoids. In: Clinical Equine Oncology. Elsevier; 2015. pp. 203-219. DOI: 10.1016/ C2009-0-61955-3

[6] Knowles EJ, Tremaine WH, Pearson GR, Mair TS. A database survey of equine tumours in the United Kingdom. Equine Veterinary Journal. 2016;**48**:280-284. DOI: 10.1111/ evj.12421

[7] Wobeser BK, Davies JL, Hill JE, Jackson ML, Kidney BA, Mayer MN, et al. Epidemiology of equine sarcoids in horses in western Canada. The Canadian Veterinary Journal. 2010;**51**:1103-1108

[8] Reid SW, Gettinby G, Fowler JN, Ikin P. Epidemiological observations on sarcoids in a population of donkeys (*Equus asinus*). The Veterinary Record. 1994;**134**:207-211. DOI: 10.1136/ vr.134.9.207

[9] Martens A, De Moor A, Ducatelle R. PCR detection of bovine papilloma virus DNA in superficial swabs and scrapings from equine sarcoids. Veterinary Journal. 2001;**161**:280-286. DOI: 10.1053/tvjl.2000.0524

[10] Chambers G, Ellsmore VA, O'Brien PM, Reid SWJ, Love S, Campo MS, et al. Association of bovine papillomavirus with the equine sarcoid. The Journal of General Virology. 2003;**84**:1055-1062. DOI: 10.1099/ vir.0.18947-0

[11] Silva MS, Weiss M, Brum MC, Dos Anjos BL, Torres FD, Weiblen R, et al. Molecular identification of bovine papillomaviruses associated with cutaneous warts in southern Brazil. Journal of Veterinary Diagnostic Investigation. 2010;**22**(4):603-606. DOI: 10.1177/104063871002200417

[12] Lunardi M, de Alcântara BK, Otonel RA, Rodrigues WB, Alfieri AF, Alfieri AA. Bovine papillomavirus type 13 DNA in equine sarcoids. Journal of Clinical Microbiology. 2013;**51**(7):2167- 2171. DOI: 10.1128/JCM.00371-13

[13] Angelos JA, Marti E, Lazary S, Carmichael LE. Characterization of BPV-like DNA in equine sarcoids. Archives of Virology. 1991;**119**:95-109. DOI: 10.1007/bf01314326

[14] Otten N, von Tscharner C, Lazary S, Antczak DF, Gerber H. DNA of bovine papillomavirus type 1 and 2 in equine sarcoids: PCr detection and direct sequencing. Archives of Virology. 1993;**132**:121-131. DOI: 10.1007/ bf01309847

[15] Bloch N, Breen M, Spradbrow PB. Genomic sequences of bovine papillomaviruses in formalin-fixed sarcoids from Australian horses revealed by polymerase chain reaction.

**129**

*Equine Sarcoid*

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

[23] Chambers G, Ellsmore VA, O'Brien PM, Reid SW, Love S, Campo MS, et al. Sequence variants of bovine papillomavirus E5 detected in equine sarcoids. Virus Research. 2003;**96**:141-145. DOI: 10.1016/

[24] Nixon C, Chambers G, Ellsmore V, Campo MS, Burr P, Argyle DJ, et al. Cancer Letters. 2005;**221**:237-245. DOI:

[25] Bogaert L, Van Poucke M, De Baere C, Dewulf J, Peelman L, Ducatelle R, et al. Bovine papillomavirus load and mRNA expression, cell proliferation and p53 expression in four clinical types of equine sarcoid. The Journal of General Virology.

s0168-1702(03)00175-8

10.1016/j.canlet.2004.08.039

2007;**88**:2155-2161. DOI: 10.1099/

[26] Yuan ZQ, Bennett L, Campo MS, Nasir L. Bovine papillomavirus type 1 e2 and E7 proteins down-regulate Toll Like Receptor 4 (*TLR4*) expression in equine fibroblasts. Virus Research. 2010;**149**:124-127. DOI: 10.1016/j.

[27] Yuan ZQ, Gault EA, Campo MS, Nasir L. p38 mitogen-activated protein

papillomavirus type-1 transformation of equine fibroblasts. The Journal of General Virology. 2011;**92**:1778-1786.

vir.0.82876-0

virusres.2010.01.008

kinase is crucial for bovine

DOI: 10.1099/vir.0.031516-0

10.1186/1746-6148-8-30

[29] Altamura G, Strazzullo M, Corteggio A, Francioso R, Roperto F, D'Esposito M, et al. (6)-methylguanine-

DNA methyltransferase in equine sarcoids: Molecular and

[28] Strazzullo M, Corteggio A, Altamura G, Francioso R, Roperto F, D'Esposito M, et al. Molecular and epigenetic analysis of the fragile histidine triad tumour suppressor gene in equine sarcoids. BMC

Veterinary Research. 2012;**8**:30. DOI:

[16] Carr EA, Théon AP, Madewell BR, Griffey SM, Hitchcock ME. Bovine papillomavirus DNA in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western United States. American Journal of Veterinary Research. 2001;**62**:741- 744. DOI: 10.2460/ajvr.2001.62.741

[17] Szczerba-Turek A, Siemionek J, Ras A, Bancerz-Kisiel A, Platt\_ Samoraj A, Lipczynska-Ilczuk K, et al. Genetic evaluation of bovine papillomavirus types detected in equine sarcoids in Poland. Polish Journal of Veterinary Sciences. 2019;**22**(1):25-29. DOI: 10.24425/pjvs.2018.125602

[18] Campo MS. Viral and cellular oncogenes in papillomavirus-associated cancers. British Journal of Cancer.

[19] Carr EA, Théon AP, Madewell BR, Hitchcock ME, Schlegel R, Schiller JT. Expression of a transforming gene (E5) of bovine papillomavirus in sarcoids obtained from horses. American Journal of Veterinary Research. 2001;**62**(8):1212-1217. DOI: 10.2460/

1988;**58**(Suppl. IX):80-84

ajvr.2001.62.1212

[20] Nasir L, Reid SW. Bovine papillomaviral gene expression in equine sarcoid tumours. Virus Research. 1999;**61**(2):171-175. DOI: 10.1016/

[21] Yuan Z, Gobeil PA, Campo MS, Nasir L. Equine sarcoid fibroblasts overexpress matrix metalloproteinases and are invasive. Virology. 2010;**396**(1):143-151.

DOI: 10.1016/j.virol.2009.10.010

[22] Campo MS. Animal models of papillomavirus pathogenesis. Virus Research. 2002;**89**:249-261. DOI: 10.1016/s0168-1702(02)00193-4

s0168-1702(99)00022-2

Veterinary Microbiology. 1994; **41**:163-172. DOI: 10.1016/ 0378-1135(94)90145-7

*Equine Sarcoid DOI: http://dx.doi.org/10.5772/intechopen.91013*

Veterinary Microbiology. 1994; **41**:163-172. DOI: 10.1016/ 0378-1135(94)90145-7

[16] Carr EA, Théon AP, Madewell BR, Griffey SM, Hitchcock ME. Bovine papillomavirus DNA in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western United States. American Journal of Veterinary Research. 2001;**62**:741- 744. DOI: 10.2460/ajvr.2001.62.741

[17] Szczerba-Turek A, Siemionek J, Ras A, Bancerz-Kisiel A, Platt\_ Samoraj A, Lipczynska-Ilczuk K, et al. Genetic evaluation of bovine papillomavirus types detected in equine sarcoids in Poland. Polish Journal of Veterinary Sciences. 2019;**22**(1):25-29. DOI: 10.24425/pjvs.2018.125602

[18] Campo MS. Viral and cellular oncogenes in papillomavirus-associated cancers. British Journal of Cancer. 1988;**58**(Suppl. IX):80-84

[19] Carr EA, Théon AP, Madewell BR, Hitchcock ME, Schlegel R, Schiller JT. Expression of a transforming gene (E5) of bovine papillomavirus in sarcoids obtained from horses. American Journal of Veterinary Research. 2001;**62**(8):1212-1217. DOI: 10.2460/ ajvr.2001.62.1212

[20] Nasir L, Reid SW. Bovine papillomaviral gene expression in equine sarcoid tumours. Virus Research. 1999;**61**(2):171-175. DOI: 10.1016/ s0168-1702(99)00022-2

[21] Yuan Z, Gobeil PA, Campo MS, Nasir L. Equine sarcoid fibroblasts overexpress matrix metalloproteinases and are invasive. Virology. 2010;**396**(1):143-151. DOI: 10.1016/j.virol.2009.10.010

[22] Campo MS. Animal models of papillomavirus pathogenesis. Virus Research. 2002;**89**:249-261. DOI: 10.1016/s0168-1702(02)00193-4

[23] Chambers G, Ellsmore VA, O'Brien PM, Reid SW, Love S, Campo MS, et al. Sequence variants of bovine papillomavirus E5 detected in equine sarcoids. Virus Research. 2003;**96**:141-145. DOI: 10.1016/ s0168-1702(03)00175-8

[24] Nixon C, Chambers G, Ellsmore V, Campo MS, Burr P, Argyle DJ, et al. Cancer Letters. 2005;**221**:237-245. DOI: 10.1016/j.canlet.2004.08.039

[25] Bogaert L, Van Poucke M, De Baere C, Dewulf J, Peelman L, Ducatelle R, et al. Bovine papillomavirus load and mRNA expression, cell proliferation and p53 expression in four clinical types of equine sarcoid. The Journal of General Virology. 2007;**88**:2155-2161. DOI: 10.1099/ vir.0.82876-0

[26] Yuan ZQ, Bennett L, Campo MS, Nasir L. Bovine papillomavirus type 1 e2 and E7 proteins down-regulate Toll Like Receptor 4 (*TLR4*) expression in equine fibroblasts. Virus Research. 2010;**149**:124-127. DOI: 10.1016/j. virusres.2010.01.008

[27] Yuan ZQ, Gault EA, Campo MS, Nasir L. p38 mitogen-activated protein kinase is crucial for bovine papillomavirus type-1 transformation of equine fibroblasts. The Journal of General Virology. 2011;**92**:1778-1786. DOI: 10.1099/vir.0.031516-0

[28] Strazzullo M, Corteggio A, Altamura G, Francioso R, Roperto F, D'Esposito M, et al. Molecular and epigenetic analysis of the fragile histidine triad tumour suppressor gene in equine sarcoids. BMC Veterinary Research. 2012;**8**:30. DOI: 10.1186/1746-6148-8-30

[29] Altamura G, Strazzullo M, Corteggio A, Francioso R, Roperto F, D'Esposito M, et al. (6)-methylguanine-DNA methyltransferase in equine sarcoids: Molecular and

**128**

vr.134.9.207

*Equine Science*

**References**

[1] Marti E, Lazary S, Antczak DF, Gerber H. Report of the first international workshop on equine sarcoid. Equine Veterinary Journal. 1993;**25**(5):397-407. DOI: 10.1111/ j.2042-3306.1993.tb02981.x

[9] Martens A, De Moor A, Ducatelle R. PCR detection of bovine papilloma virus DNA in superficial swabs and scrapings from equine sarcoids. Veterinary Journal. 2001;**161**:280-286.

Campo MS, et al. Association of bovine papillomavirus with the equine sarcoid. The Journal of General Virology. 2003;**84**:1055-1062. DOI: 10.1099/

[11] Silva MS, Weiss M, Brum MC, Dos Anjos BL, Torres FD, Weiblen R, et al. Molecular identification of bovine papillomaviruses associated with cutaneous warts in southern Brazil. Journal of Veterinary Diagnostic

Investigation. 2010;**22**(4):603-606. DOI:

10.1177/104063871002200417

[12] Lunardi M, de Alcântara BK, Otonel RA, Rodrigues WB, Alfieri AF, Alfieri AA. Bovine papillomavirus type 13 DNA in equine sarcoids. Journal of Clinical Microbiology. 2013;**51**(7):2167- 2171. DOI: 10.1128/JCM.00371-13

[13] Angelos JA, Marti E, Lazary S, Carmichael LE. Characterization of BPV-like DNA in equine sarcoids. Archives of Virology. 1991;**119**:95-109.

[14] Otten N, von Tscharner C, Lazary S, Antczak DF, Gerber H. DNA of bovine papillomavirus type 1 and 2 in equine sarcoids: PCr detection and direct sequencing. Archives of Virology. 1993;**132**:121-131. DOI: 10.1007/

[15] Bloch N, Breen M, Spradbrow PB.

Genomic sequences of bovine papillomaviruses in formalin-fixed sarcoids from Australian horses revealed by polymerase chain reaction.

DOI: 10.1007/bf01314326

bf01309847

DOI: 10.1053/tvjl.2000.0524

vir.0.18947-0

[10] Chambers G, Ellsmore VA, O'Brien PM, Reid SWJ, Love S,

[2] Bergvall KE. Sarcoids. The Veterinary Clinics of North America. Equine Practice. 2013;**29**:657-671. DOI: 10.1016/j.cveq.2013.09.002

[3] Knottenbelt DC. The equine sarcoid: Why Are There So Many Treatment Options? Veterinary Clinics of North America: Equine Practice. 2019;**35**:243- 262. DOI: 10.1016/j.cveq.2019.03.006

Investigation. 2006;**18**:123-126. DOI:

[5] Knottenbelt DC, Patterson-Kane JC, Snalune KL, editors. Sarcoids. In: Clinical Equine Oncology. Elsevier; 2015. pp. 203-219. DOI: 10.1016/

[4] Valentine BA. Survey of equine cutaneous neoplasia in the Pacific Northwest. Journal of Veterinary Diagnostic

10.1177/104063870601800121

[6] Knowles EJ, Tremaine WH, Pearson GR, Mair TS. A database survey of equine tumours in the United Kingdom. Equine Veterinary Journal. 2016;**48**:280-284. DOI: 10.1111/

[7] Wobeser BK, Davies JL, Hill JE, Jackson ML, Kidney BA, Mayer MN, et al. Epidemiology of equine sarcoids in horses in western Canada. The Canadian Veterinary Journal. 2010;**51**:1103-1108

[8] Reid SW, Gettinby G, Fowler JN, Ikin P. Epidemiological observations on sarcoids in a population of donkeys (*Equus asinus*). The Veterinary Record. 1994;**134**:207-211. DOI: 10.1136/

C2009-0-61955-3

evj.12421

epigenetic analysis. BMC Veterinary Research. 2012;**8**:218. DOI: 10.1186/1746-6148-8-218

[30] Pawlina K, Gurgul A, Szmatola T, Koch C, Mahlmann K, Witkowski M, et al. Comprehensive characteristics of microRNA expression profile of equine sarcoids. Biochimie. 2017;**137**:20-28. DOI: 10.1016/j.biochi.2017.02.17

[31] Unger L, Gerber V, Pacholewska A, Leeb T, Jagannathan V. MicroRNA fingerprints in serum and whole blood of sarcoid-affected horses as potential non-invasive diagnostic biomarkers. Veterinary and Comparative Oncology. 2019;**17**:107-117. DOI: 10.1111/vco.12451

[32] Unger L, Jagannathan V, Pacholewska A, Leeb T, Gerber V. Differences in miRNA differential expression in whole blood between horses with sarcoid regression and progression. Journal of Veterinary Internal Medicine. 2019;**33**:241-250. DOI: 10.1111/jvim.15375

[33] Bogedale K, Jagannathan V, Gerber V, Unger L. Differentially expressed microRNAs, including a large microRNA cluster on chromosome 24, are associated with equine sarcoid and squamous cell carcinoma. Veterinary and Comparative Oncology. 2019;**17**(2):155-164. DOI: 10.1111/ vco.12458

[34] Semik-Gurgul E, Zabek T, Formal A, Wnuk M, Pawlina-Tyszko K, Gurgul A, et al. DNA methylation patterns of the S100A14, POU2F3 and SFN genes in equine sarcoid tissues. Research in Veterinary Science. 2018;**119**:302-307. DOI: 10.1016/j. rvsc.2018.07.006

[35] Bogaert L, Martens A, Van Poucke M, Ducatelle R, De Cock H, Dewulf J, et al. High prevalence of bovine papillomaviral DNA in the normal skin of equine sarcoid-affected and healthy horses. Veterinary

Microbiology. 2008;**129**:58-68. DOI: 10.1016/j.vetmic.2007.11.008

[36] Nasir L, Campo MS. Bovine papillomaviruses: Their role in the aetiology of cutaneous tumours of bovids and equids. Veterinary Dermatology. 2008;**19**(5):243-254. DOI: 10.1111/j.1365-3164.2008.00683.x

[37] Bogaert L, Martens A, Kast WM, Van Marck E, De Cock H. Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors. Veterinary Microbiology. 2010;**146**:269- 275. DOI: 10.1016/j.vetmic.2010.05.032

[38] Brandt S, Halarambus R, Schoster A, Kirnbauer R, Stanek C. Peripheral blood mononuclear cells represent a reservoir of bovine papillomavirus DNA in sarcoid-affected equines. The Journal of General Virology. 2008;**89**:1390-1395. DOI: 10.1099/vir.0.83568-0

[39] Yuan Z, Gallagher A, Gault EA, Campo MS, Nasir L. Bovine papillomavirus infection in equine sarcoids and in bovine bladder cancers. Veterinary Journal. 2007;**174**:599-604. DOI: 10.1016/j.tvjl.2006.10.012

[40] Halarambus R, Burgstaller J, Klukowska Roetzler J, Steinborn R, Buchinger S, Gerber V, et al. Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease. Equine Veterinary Journal. 2010;**42**(4):327-331. DOI: 10.1111/j. 2042-3306.2010.00078.x

[41] Finlay M, Yean Z, Burden F, Trawford A, Morgan IM, Campo MS, et al. The detection of bovine papillomavirus type 1 DNA in flies. Virus Research. 2009;**144**:315-317. DOI: 10.1016/j. virusres.2009.04.015

[42] Haspeslagh M, Vlaminck L, Martens A. The possible role of *Stomoxys calcitrans* in equine sarcoid transmission. Veterinary Journal.

**131**

tb02498.x

*Equine Sarcoid*

2017.11.009

2052.1988.tb00833.x

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

[50] Lazary S, Marti E, Szalai G, Gaillard C, Gerber H. Studies on the frequency and associations of equine leucocyte antigens in sarcoid and summer dermatitis. Animal Genetics. 1994;**25**(S1):75-80. DOI: 10.1111/j.1365-

[51] Knottenbelt DC, Schumacher J, Toth F. Sarcoid transformation at wound sites. In: Theoret C, Schumacher J, editors. Equine Wound Management. 3rd ed. Iowa: Wiley; 2017. pp. 490-507.

DOI: 10.1002/9781118999219

[52] Knottenbelt DC, Edwards S, Daniel E. Diagnosis and treatment of the equine sarcoid. In Practice. 1995;**17**(3):123-129. DOI: 10.1136/

Wohlfender FD, Burger D, Koch C. Clinical course of sarcoids in 61 Franches-Montagnes horses over a 5-7 year period. The Veterinary Quarterly.

[54] Knottenbelt DC. A suggested clinical classification for the equine sarcoid. Clinical Techniques in Equine Practice. 2005;**4**:278-295. DOI: 10.1053/j.

[55] Knottenbelt DC, Patterson-Kane JC, Snalune KL, editors. Tumours of the skin. In: Clinical Equine Oncology. Elsevier; 2015. pp. 544-584. DOI:

[56] Knottenbelt DC. The approach to the equine dermatology case in practice.

[57] Taylor S, Haldorson G. A review of equine sarcoid. Equine Veterinary Education. 2013;**25**(4):210-216. DOI: 10.1111/j.2042-3292.2012.00411.x

[58] Bogaert L, Martens A, Depoorter P, Gasthuys F. Equine sarcoids—Part 1:

The Veterinary Clinics of North America. Equine Practice. 2012;**28**:131- 153. DOI: 10.1016/j.cveq.2012.01.004

2052.1994.tb00406.x

inpract.17.3.123

[53] Berruex F, Gerber V,

2016;**36**(4):189-196. DOI: 10.1080/01652176.2016.1204483

10.1016/C2009-0-61955-3

ctep.2005.10.008

[44] Angelos J, Oppenheim Y, Rebhun W, Mohammed H, Antczak DF. Evaluation of breed as a risk factor for sarcoid and uveitis in horses. Animal Genetics. 1988;**19**(4):417-425. DOI: 10.1111/j.1365-

[45] Mohammed HO, Renhun WC, Antczak DF. Factors associated with the risk of developing sarcoid tumours in horses. Equine Veterinary Journal. 1992;**24**(3):165-168. DOI: 10.1111/ j.2042-3306.1992.tb02808.x

[46] Goodrich L, Gerber H, Marti E, Antczak DF. Equine sarcoids. The Veterinary Clinics of North America. Equine Practice. 1998;**14**(3):607-623. DOI: 10.1016/S0749-0739(17)30189-X

[47] Meredith D, Elser AH, Wolf B, Soma LR, Donawick WJ, Lazary S. Equine leukocyte antigens: Relationships with sarcoid tumors and laminitis in two pure breeds. Immunogenetics. 1986;**23**(4):221-225. DOI: 10.1007/

[48] Broström H, Fahlbrink E, Dubath ML, Lazary S. Association between equine leucocyte antigens (ELA) and equine sarcoid tumors in the population of Swedish

Veterinary Immunology and

Halfbreds and some of their families.

Immunopathology. 1988;**19**:215-223. DOI: 10.1016/0165-2427(88)90109-2

[49] Lazary S, Gerber H, Glatt PA, Straub R. Equine leucocyte antigens in sarcoid-affected horses. Equine Veterinary Journal. 1985;**17**(4):

283-286. DOI: 10.1111/j.2042-3306.1985.

bf00373016

2018;**231**:8-12. DOI: 10.1016/j.tvjl.

[43] Christen G, Gerber V, Dolf G, Burger D, Koch C. Inheritance of equine sarcoid disease in Franches-Montagnes horses. Veterinary Journal. 2014;**199**:68- 71. DOI: 10.1016/j.tvjl.2013.09.053

*Equine Sarcoid DOI: http://dx.doi.org/10.5772/intechopen.91013*

2018;**231**:8-12. DOI: 10.1016/j.tvjl. 2017.11.009

*Equine Science*

epigenetic analysis. BMC Veterinary

Microbiology. 2008;**129**:58-68. DOI:

Dermatology. 2008;**19**(5):243-254. DOI: 10.1111/j.1365-3164.2008.00683.x

[37] Bogaert L, Martens A, Kast WM, Van Marck E, De Cock H. Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors. Veterinary Microbiology. 2010;**146**:269- 275. DOI: 10.1016/j.vetmic.2010.05.032

papillomavirus DNA in sarcoid-affected

[38] Brandt S, Halarambus R, Schoster A, Kirnbauer R, Stanek C. Peripheral blood mononuclear cells represent a reservoir of bovine

equines. The Journal of General Virology. 2008;**89**:1390-1395. DOI:

[39] Yuan Z, Gallagher A, Gault EA,

Campo MS, Nasir L. Bovine papillomavirus infection in equine sarcoids and in bovine bladder cancers. Veterinary Journal. 2007;**174**:599-604.

DOI: 10.1016/j.tvjl.2006.10.012

[40] Halarambus R, Burgstaller J, Klukowska Roetzler J, Steinborn R, Buchinger S, Gerber V, et al.

Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease. Equine Veterinary Journal. 2010;**42**(4):327-331. DOI: 10.1111/j. 2042-3306.2010.00078.x

[41] Finlay M, Yean Z, Burden F,

[42] Haspeslagh M, Vlaminck L, Martens A. The possible role of *Stomoxys calcitrans* in equine sarcoid transmission. Veterinary Journal.

virusres.2009.04.015

Trawford A, Morgan IM, Campo MS, et al. The detection of bovine papillomavirus type 1 DNA in flies. Virus Research. 2009;**144**:315-317. DOI: 10.1016/j.

10.1099/vir.0.83568-0

10.1016/j.vetmic.2007.11.008

[36] Nasir L, Campo MS. Bovine papillomaviruses: Their role in the aetiology of cutaneous tumours of bovids and equids. Veterinary

[30] Pawlina K, Gurgul A, Szmatola T, Koch C, Mahlmann K, Witkowski M, et al. Comprehensive characteristics of microRNA expression profile of equine sarcoids. Biochimie. 2017;**137**:20-28. DOI: 10.1016/j.biochi.2017.02.17

[31] Unger L, Gerber V, Pacholewska A, Leeb T, Jagannathan V. MicroRNA fingerprints in serum and whole blood of sarcoid-affected horses as potential non-invasive diagnostic biomarkers. Veterinary and Comparative Oncology. 2019;**17**:107-117. DOI: 10.1111/vco.12451

[32] Unger L, Jagannathan V, Pacholewska A, Leeb T, Gerber V. Differences in miRNA differential expression in whole blood between horses with sarcoid regression and progression. Journal of Veterinary Internal Medicine. 2019;**33**:241-250.

DOI: 10.1111/jvim.15375

vco.12458

rvsc.2018.07.006

[33] Bogedale K, Jagannathan V, Gerber V, Unger L. Differentially expressed microRNAs, including a large microRNA cluster on chromosome 24, are associated with equine sarcoid and squamous cell carcinoma.

[34] Semik-Gurgul E, Zabek T,

[35] Bogaert L, Martens A, Van Poucke M, Ducatelle R, De Cock H, Dewulf J, et al. High prevalence of bovine papillomaviral DNA in the normal skin of equine sarcoid-affected

and healthy horses. Veterinary

Veterinary and Comparative Oncology. 2019;**17**(2):155-164. DOI: 10.1111/

Formal A, Wnuk M, Pawlina-Tyszko K, Gurgul A, et al. DNA methylation patterns of the S100A14, POU2F3 and SFN genes in equine sarcoid tissues. Research in Veterinary Science. 2018;**119**:302-307. DOI: 10.1016/j.

Research. 2012;**8**:218. DOI: 10.1186/1746-6148-8-218

**130**

[43] Christen G, Gerber V, Dolf G, Burger D, Koch C. Inheritance of equine sarcoid disease in Franches-Montagnes horses. Veterinary Journal. 2014;**199**:68- 71. DOI: 10.1016/j.tvjl.2013.09.053

[44] Angelos J, Oppenheim Y, Rebhun W, Mohammed H, Antczak DF. Evaluation of breed as a risk factor for sarcoid and uveitis in horses. Animal Genetics. 1988;**19**(4):417-425. DOI: 10.1111/j.1365- 2052.1988.tb00833.x

[45] Mohammed HO, Renhun WC, Antczak DF. Factors associated with the risk of developing sarcoid tumours in horses. Equine Veterinary Journal. 1992;**24**(3):165-168. DOI: 10.1111/ j.2042-3306.1992.tb02808.x

[46] Goodrich L, Gerber H, Marti E, Antczak DF. Equine sarcoids. The Veterinary Clinics of North America. Equine Practice. 1998;**14**(3):607-623. DOI: 10.1016/S0749-0739(17)30189-X

[47] Meredith D, Elser AH, Wolf B, Soma LR, Donawick WJ, Lazary S. Equine leukocyte antigens: Relationships with sarcoid tumors and laminitis in two pure breeds. Immunogenetics. 1986;**23**(4):221-225. DOI: 10.1007/ bf00373016

[48] Broström H, Fahlbrink E, Dubath ML, Lazary S. Association between equine leucocyte antigens (ELA) and equine sarcoid tumors in the population of Swedish Halfbreds and some of their families. Veterinary Immunology and Immunopathology. 1988;**19**:215-223. DOI: 10.1016/0165-2427(88)90109-2

[49] Lazary S, Gerber H, Glatt PA, Straub R. Equine leucocyte antigens in sarcoid-affected horses. Equine Veterinary Journal. 1985;**17**(4): 283-286. DOI: 10.1111/j.2042-3306.1985. tb02498.x

[50] Lazary S, Marti E, Szalai G, Gaillard C, Gerber H. Studies on the frequency and associations of equine leucocyte antigens in sarcoid and summer dermatitis. Animal Genetics. 1994;**25**(S1):75-80. DOI: 10.1111/j.1365- 2052.1994.tb00406.x

[51] Knottenbelt DC, Schumacher J, Toth F. Sarcoid transformation at wound sites. In: Theoret C, Schumacher J, editors. Equine Wound Management. 3rd ed. Iowa: Wiley; 2017. pp. 490-507. DOI: 10.1002/9781118999219

[52] Knottenbelt DC, Edwards S, Daniel E. Diagnosis and treatment of the equine sarcoid. In Practice. 1995;**17**(3):123-129. DOI: 10.1136/ inpract.17.3.123

[53] Berruex F, Gerber V, Wohlfender FD, Burger D, Koch C. Clinical course of sarcoids in 61 Franches-Montagnes horses over a 5-7 year period. The Veterinary Quarterly. 2016;**36**(4):189-196. DOI: 10.1080/01652176.2016.1204483

[54] Knottenbelt DC. A suggested clinical classification for the equine sarcoid. Clinical Techniques in Equine Practice. 2005;**4**:278-295. DOI: 10.1053/j. ctep.2005.10.008

[55] Knottenbelt DC, Patterson-Kane JC, Snalune KL, editors. Tumours of the skin. In: Clinical Equine Oncology. Elsevier; 2015. pp. 544-584. DOI: 10.1016/C2009-0-61955-3

[56] Knottenbelt DC. The approach to the equine dermatology case in practice. The Veterinary Clinics of North America. Equine Practice. 2012;**28**:131- 153. DOI: 10.1016/j.cveq.2012.01.004

[57] Taylor S, Haldorson G. A review of equine sarcoid. Equine Veterinary Education. 2013;**25**(4):210-216. DOI: 10.1111/j.2042-3292.2012.00411.x

[58] Bogaert L, Martens A, Depoorter P, Gasthuys F. Equine sarcoids—Part 1:

Clinical presentation and epidemiology. Vlaams Diergeneeskundig Tijdschrift. 2008;**77**:2-9

[59] Martano M, Corteggio A, Restucci B, De Biase ME, Borzacchiello G. Extracellular matrix remodeling in equine sarcoid: An immunohistochemical and molecular study. BMC Veterinary Research. 2016;**12**:24. DOI: 10.1186/ s12917-016-0648-1

[60] Pascoe RR, Knottenbelt DC. Neoplastic conditions. In: Pascoe RR, Knottenbelt DC, editors. Manual of Equine Dermatology. London: Saunders; 1999. pp. 244-252

[61] Martens A, De Moor A, Demeulemeester J, Ducatelle R. Histopathological characteristics of five clinical types of equine sarcoid. Research in Veterinary Science. 2000;**69**:295-300. DOI: 10.1053/ rvsc.2000.0432

[62] Mauldin E, Peters-Kennedy J. Integumentary system. In: Maxie MG, editor. Jubb, Kennedy, and Palmer's Pathology of Domestic Animals. 6th ed. Vol. 1. Philadelphia, PA: Elsevier; 2016. pp. 707-710 ISBN: 978-0-7020-5317-7

[63] Scott DW, Miller WH. Neoplastic and non-neoplastic tumors. In: Scott SDW, Miller HM, editors. Equine Dermatology. St Louis, MO: Saunders; 2003. pp. 698-795. ISBN: 0-7216-2571-1

[64] Zahra SN, Abu-Ahmed HM, Korritum AA, Elkhenany HA, Kaifa H. Equine sarcoids: Distribution sites, common types, and diagnosis. Alexandria Journal of Veterinary Sciences. 2019;**60**:123-128. DOI: 10.5455/ajvs.19531

[65] Bogaert L, Van Heerden M, De Cock HEV, Martens A, Chiers K. Molecular and immunohistochemical distinction of equine sarcoid from schwannoma. Veterinary Pathology. 2011;**48**:737-741. DOI: 10.1177/0300985810377070

[66] Epperson ED, Castleman WL. Bovine papillomavirus DNA and S100 profiles in sarcoids and other cutaneous spindle cell tumors in horses. Veterinary Pathology. 2017;**54**:44-52. DOI: 10.1177/0300985816653169

[67] Gaynor AM, Zhu KW, Dela Cruz FN, Affolter VK, Pesavento PA. Localization of bovine papillomavirus nucleic acid in equine sarcoids. Veterinary Pathology. 2016;**53**:567-573. DOI: 10.1177/0300985815594852

[68] Wobeser B. Making the diagnosis: Equine sarcoid. Veterinary Pathology. 2017;**54**:9-10. DOI: 10.1177/0300985816664793

[69] Martens A, De Moor A, Vlaminck L, Pille F, Steenhaut M. Evaluation of excision, cryosurgery and local BCG vaccination for the treatment of equine sarcoids. The Veterinary Record. 2001;**149**:665-669. DOI: 10.1136/ vr.149.22.665

[70] Haspeslagh M, Vlaminck LEM, Martens AM. Treatment of sarcoids in equids: 230 cases (2008-2013). Journal of the American Veterinary Medical Association. 2016;**249**:311-318. DOI: 10.2460/javma.249.3.311

[71] Carstanjen B, Jordan P, Lepage OM. Carbon dioxide laser as a surgical instrument for sarcoid therapy—A retrospective study on 60 cases. The Canadian Veterinary Journal. 1997;**38**:773-776

[72] Compston PC, Turner T, Wylie CE, Payne J. Laser surgery as a treatment for histologically confirmed sarcoids in the horse. Equine Veterinary Journal. 2016;**48**:451-456. DOI: 10.1111/evj.12456

[73] Théon AP, Pusterla N, Magdesian KG, Wittenburg L, Marmulak T, Wilson WD.A pilot phase II study of the efficacy and biosafety of doxorubicin chemotherapy in

**133**

*Equine Sarcoid*

jvim.12144

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

neoplasia in Equidae: 59 cases (2000- 2004). Journal of the American Veterinary Medical Association. 2006;**229**:1617-1622. DOI: 10.2460/

[81] Knottenbelt DC, Watson AH, Hotchkiss JW, Chopra S, Higgins A. A pilot study on the use of ultradeformable liposomes containing bleomycin in the treatment of equine sarcoid. Equine Veterinary Education. 2018. Early View. DOI: 10.1111/eve.12950

[82] Tozon N, Kramaric P, Kos Kadunc V, Sersa G, Cemazar M. Electrochemotherapy as a single treatment or adjuvant treatment to surgery of cutaneous sarcoid tumours in horses: A 31-case retrospective study. The Veterinary Record. 2016;**179**: 627. DOI: 10.1136/vr.103867

[83] Souza C, Villarino NF,

tvjl.1999.0392

Farnsworth K, Black ME. Enhanced cytotoxicity of bleomycin, cisplatin, and carboplatin on equine sarcoid cells following electroporation-mediated delivery *in vitro*. Journal of Veterinary Pharmacology and Therapeutics. 2017;**40**:97-100. DOI: 10.1111/jvp.12331

[84] Martens A, De Moor A, Waelkens E, Merlevede W, De Witte P. *In vitro* and *in vivo* evaluation of hypericin for photodynamic therapy of equine sarcoids. Veterinary Journal. 2000;**159**:77-84. DOI: 10.1053/

[85] Buchholz J, Heinrich W. Veterinary photodynamic therapy: A review. Photodiagnosis and Photodynamic Therapy. 2013;**10**:342-347. DOI: 10.1016/j.pdpdt.2013.05.009

[86] Sellera FP, Nascimento CL, Ribeiro MS, editors. Photodynamic Therapy in Veterinary Medicine: From Basics to Clinical Practice. Switzerland: Springer; 2016. p. 228. DOI: 10.1007/978-3-319-45007-0

javma.229.10.1617

tumor-bearing equidae. Journal of Veterinary Internal Medicine. 2013;**27**:1581-1588. DOI: 10.1111/

[74] Knottenbelt DC, Kelly DF. The diagnosis and treatment of periorbital

Ophthalmology. 2000;**3**:169-191. DOI: 10.1046/j.1463-5224.2000.00119.x

[75] Stewart AA, Brush B, Davis E. The efficacy of intratumoral 5-fluorouracil for the treatment of equine sarcoids. Australian Veterinary Journal. 2006;**84**:101-106. DOI: 10.1111/j.1751-

sarcoid in the horse: 445 cases from 1974 to 1999. Veterinary

0813.2006.tb12239.x

3164.2006.00526.x

DOI: 10.1136/vr.c5430

s12917-017-1215-0

[76] Nogueira SAF, Torres SMF,

[77] Stadler S, Kainzbauer C,

[78] Haspeslagh M, Garcia MJ,

of occult and verrucose equine sarcoids: A double-blinded placebocontrolled study. BMC Veterinary Research. 2017;**13**:296. DOI: 10.1186/

Vlaminck LEM, Martens A. Topical use of 5% acyclovir cream for the treatment

[79] Théon AP. Intralesional and topical chemotherapy and immunotherapy. Veterinary Clinics of North America. Equine Practice. 1998;**14**:659-671. DOI:

10.1016/S0749-0739(17)30191-8

[80] Hewes CA, Sullins KE. Use of cisplatin-containing biodegradable beads for treatment of cutaneous

Halarambus R, Brehm W, Hainish E, Brandt S. Successful treatment of equine sarcoids by topical acyclovir application. The Veterinary Record. 2011;**168**:187.

Malone ED, Diaz SF, Jessen C, Gilbert S. Efficacy of imiquimod 5% cream in the treatment of equine sarcoids: A pilot study. Veterinary Dermatology. 2006;**17**:259-265. DOI: 10.1111/j.1365*Equine Sarcoid DOI: http://dx.doi.org/10.5772/intechopen.91013*

tumor-bearing equidae. Journal of Veterinary Internal Medicine. 2013;**27**:1581-1588. DOI: 10.1111/ jvim.12144

*Equine Science*

2008;**77**:2-9

s12917-016-0648-1

1999. pp. 244-252

rvsc.2000.0432

Clinical presentation and epidemiology. Vlaams Diergeneeskundig Tijdschrift.

[66] Epperson ED, Castleman WL. Bovine papillomavirus DNA and S100 profiles in sarcoids and other cutaneous spindle cell tumors in horses. Veterinary

Pathology. 2017;**54**:44-52. DOI: 10.1177/0300985816653169

[67] Gaynor AM, Zhu KW, Dela Cruz FN, Affolter VK, Pesavento PA. Localization of bovine papillomavirus nucleic acid in equine sarcoids.

[68] Wobeser B. Making the

vr.149.22.665

Veterinary Pathology. 2016;**53**:567-573. DOI: 10.1177/0300985815594852

diagnosis: Equine sarcoid. Veterinary Pathology. 2017;**54**:9-10. DOI: 10.1177/0300985816664793

[69] Martens A, De Moor A, Vlaminck L, Pille F, Steenhaut M. Evaluation of excision, cryosurgery and local BCG vaccination for the treatment of equine sarcoids. The Veterinary Record. 2001;**149**:665-669. DOI: 10.1136/

[70] Haspeslagh M, Vlaminck LEM, Martens AM. Treatment of sarcoids in equids: 230 cases (2008-2013). Journal of the American Veterinary Medical Association. 2016;**249**:311-318. DOI:

[71] Carstanjen B, Jordan P, Lepage OM. Carbon dioxide laser as a surgical instrument for sarcoid therapy—A retrospective study on 60 cases. The Canadian Veterinary Journal.

[72] Compston PC, Turner T, Wylie CE, Payne J. Laser surgery as a treatment for histologically confirmed sarcoids in the horse. Equine Veterinary Journal. 2016;**48**:451-456. DOI: 10.1111/evj.12456

Marmulak T, Wilson WD.A pilot phase II study of the efficacy and biosafety of doxorubicin chemotherapy in

10.2460/javma.249.3.311

[73] Théon AP, Pusterla N, Magdesian KG, Wittenburg L,

1997;**38**:773-776

Restucci B, De Biase ME, Borzacchiello G. Extracellular matrix remodeling in equine sarcoid: An immunohistochemical and molecular study. BMC Veterinary Research. 2016;**12**:24. DOI: 10.1186/

[59] Martano M, Corteggio A,

[60] Pascoe RR, Knottenbelt DC. Neoplastic conditions. In: Pascoe RR, Knottenbelt DC, editors. Manual of Equine Dermatology. London: Saunders;

[61] Martens A, De Moor A, Demeulemeester J, Ducatelle R. Histopathological characteristics of five clinical types of equine sarcoid. Research in Veterinary Science. 2000;**69**:295-300. DOI: 10.1053/

[62] Mauldin E, Peters-Kennedy J. Integumentary system. In: Maxie MG, editor. Jubb, Kennedy, and Palmer's Pathology of Domestic Animals. 6th ed. Vol. 1. Philadelphia, PA: Elsevier; 2016. pp. 707-710 ISBN: 978-0-7020-5317-7

[63] Scott DW, Miller WH. Neoplastic and non-neoplastic tumors. In:

Scott SDW, Miller HM, editors. Equine Dermatology. St Louis, MO: Saunders; 2003. pp. 698-795. ISBN: 0-7216-2571-1

Kaifa H. Equine sarcoids: Distribution sites, common types, and diagnosis. Alexandria Journal of Veterinary Sciences. 2019;**60**:123-128. DOI:

[65] Bogaert L, Van Heerden M, De Cock HEV, Martens A, Chiers K. Molecular and immunohistochemical

distinction of equine sarcoid from schwannoma. Veterinary Pathology. 2011;**48**:737-741. DOI: 10.1177/0300985810377070

[64] Zahra SN, Abu-Ahmed HM, Korritum AA, Elkhenany HA,

10.5455/ajvs.19531

**132**

[74] Knottenbelt DC, Kelly DF. The diagnosis and treatment of periorbital sarcoid in the horse: 445 cases from 1974 to 1999. Veterinary Ophthalmology. 2000;**3**:169-191. DOI: 10.1046/j.1463-5224.2000.00119.x

[75] Stewart AA, Brush B, Davis E. The efficacy of intratumoral 5-fluorouracil for the treatment of equine sarcoids. Australian Veterinary Journal. 2006;**84**:101-106. DOI: 10.1111/j.1751- 0813.2006.tb12239.x

[76] Nogueira SAF, Torres SMF, Malone ED, Diaz SF, Jessen C, Gilbert S. Efficacy of imiquimod 5% cream in the treatment of equine sarcoids: A pilot study. Veterinary Dermatology. 2006;**17**:259-265. DOI: 10.1111/j.1365- 3164.2006.00526.x

[77] Stadler S, Kainzbauer C, Halarambus R, Brehm W, Hainish E, Brandt S. Successful treatment of equine sarcoids by topical acyclovir application. The Veterinary Record. 2011;**168**:187. DOI: 10.1136/vr.c5430

[78] Haspeslagh M, Garcia MJ, Vlaminck LEM, Martens A. Topical use of 5% acyclovir cream for the treatment of occult and verrucose equine sarcoids: A double-blinded placebocontrolled study. BMC Veterinary Research. 2017;**13**:296. DOI: 10.1186/ s12917-017-1215-0

[79] Théon AP. Intralesional and topical chemotherapy and immunotherapy. Veterinary Clinics of North America. Equine Practice. 1998;**14**:659-671. DOI: 10.1016/S0749-0739(17)30191-8

[80] Hewes CA, Sullins KE. Use of cisplatin-containing biodegradable beads for treatment of cutaneous

neoplasia in Equidae: 59 cases (2000- 2004). Journal of the American Veterinary Medical Association. 2006;**229**:1617-1622. DOI: 10.2460/ javma.229.10.1617

[81] Knottenbelt DC, Watson AH, Hotchkiss JW, Chopra S, Higgins A. A pilot study on the use of ultradeformable liposomes containing bleomycin in the treatment of equine sarcoid. Equine Veterinary Education. 2018. Early View. DOI: 10.1111/eve.12950

[82] Tozon N, Kramaric P, Kos Kadunc V, Sersa G, Cemazar M. Electrochemotherapy as a single treatment or adjuvant treatment to surgery of cutaneous sarcoid tumours in horses: A 31-case retrospective study. The Veterinary Record. 2016;**179**: 627. DOI: 10.1136/vr.103867

[83] Souza C, Villarino NF, Farnsworth K, Black ME. Enhanced cytotoxicity of bleomycin, cisplatin, and carboplatin on equine sarcoid cells following electroporation-mediated delivery *in vitro*. Journal of Veterinary Pharmacology and Therapeutics. 2017;**40**:97-100. DOI: 10.1111/jvp.12331

[84] Martens A, De Moor A, Waelkens E, Merlevede W, De Witte P. *In vitro* and *in vivo* evaluation of hypericin for photodynamic therapy of equine sarcoids. Veterinary Journal. 2000;**159**:77-84. DOI: 10.1053/ tvjl.1999.0392

[85] Buchholz J, Heinrich W. Veterinary photodynamic therapy: A review. Photodiagnosis and Photodynamic Therapy. 2013;**10**:342-347. DOI: 10.1016/j.pdpdt.2013.05.009

[86] Sellera FP, Nascimento CL, Ribeiro MS, editors. Photodynamic Therapy in Veterinary Medicine: From Basics to Clinical Practice. Switzerland: Springer; 2016. p. 228. DOI: 10.1007/978-3-319-45007-0

[87] Golding JP, Kemp-Symonds JG, Dobson JM. Glycolysis inhibition improves photodynamic therapy response rates for equine sarcoids. Veterinary and Comparative Oncology. 2017;**15**:1543-1552. DOI: 10.1111/ vco.12299

[88] Dobson J, de Queiroz GF, Golding JP. Photodynamic therapy and diagnosis: Principles and comparative aspects. Veterinary Journal. 2018;**233**: 8-18. DOI: 10.1016/j.tvjl.2017.11.012

[89] Lavach JD, Sullins KE, Roberts SM, Severin GA, Wheeler C, Lueker DC. BCG treatment of periocular sarcoid. Equine Veterinary Journal. 1985;**17**:445-448. DOI: 10.1111/ j.2042-33-06.1985.tb02552.x

[90] Vanselow BA, Abetz I, Jackson ARB. BCG emulsion immunotherapy of equine sarcoid. Equine Veterinary Journal. 1988;**20**(6):444-447. DOI: 10.1111/j.2042-3306.1988.tb01571.x

[91] Hainisch EK, Harnacker J, Shafti-Keramat S, Kirnbauer R, Brandt S. Vaccination with virus-like particles induces long lasting protection from experimentally induced sarcoidlike tumours in horses. Equine Veterinary Journal. 2014;**46**(S47):15-16. DOI: 10.1111/evj.12323\_33

[92] Rothacker CC, Boyle AG, Levine DG. Autologous vaccination for the treatment of equine sarcoids: 18 cases (2009-2014). The Canadian Veterinary Journal. 2015;**56**(7):709-714

[93] Théon AP. Radiation therapy in the horse. Veterinary Clinics of North America: Equine Practice. 1998;**14**(3):673-688. DOI: 10.1016/ S0749-0739(17)30192-X

[94] Théon AP, Pascoe JR. Iridium-192 interstitial brachytherapy for equine periocular tumours: Treatment results and prognostic factors in 115 horses. Equine Veterinary Journal.

1995;**27**(2):117-121. DOI: 10.1111/j.2042- 3306.1995.tb03046.x

[95] Byam-Cook KL, Henson FMD, Slater JD. Treatment of periocular and non-ocular sarcoids in 18 horses by interstitial brachytherapy with iridium-192. The Veterinary Record. 2006;**159**:337-341. DOI: 10.1136/ vr.159.11.337

[96] Hollis AR, Berlato D. Initial experience with high dose rate brachytherapy of periorbital sarcoids in the horse. Equine Veterinary Education. 2018;**30**(8):444-449. DOI: 10.1111/ eve.12782

[97] Christen-Clottu O, Klocke P, Burger D, Straub R, Gerber V. Treatment of clinically diagnosed equine sarcoid with a mistletoe extract (*Viscus album austriacus*). Journal of Veterinary Internal Medicine. 2010;**24**:1483-1489. DOI: 10.1111/j.1939-1676.2010.0597.x

[98] Knottenbelt DC. Skin disorders of the donkey and mule. Veterinary Clinics of North America: Equine Practice. 2019;**35**:493- 514. DOI: 10.1016/j.cveq.2019.08.006

[99] Löhr VC, Juan-Sallés C, Rosas-Rosas A, Parás García A, Garner MM, Teifke JP. Sarcoids in captive zebras (*Equus burchelli*) association with bovine papillomavirus type 1 infection. Journal of Zoo and Wildlife Medicine. 2005;**36**(1):74-81. DOI: 10.1638/03-126

[100] van Dyk E, Oosthuizen MC, Bosman AM, Nel PJ, Zimmerman D, Venter EH. Detection of bovine papillomavirus DNA in sarcoid-affected and healthy free-roaming zebra (*Equus zebra*) populations in South Africa. Journal of Virological Methods. 2009;**158**:141-151. DOI: 10.1016/j. jviromet.2009.02.008

**135**

*Equine Sarcoid*

*DOI: http://dx.doi.org/10.5772/intechopen.91013*

papillomavirus-DNA in mesenchymal tumour cells and not in the hyperplastic epithelium of feline sarcoids. Veterinary Dermatology. 2003;**14**:47-56. DOI: 10.1046/j.1365-3164.2003.00324.x

et al. Neoplasia in 125 donkeys (*Equus asinus*): Literature review and a survey of five veterinary schools in the United States and Canada. Journal of Veterinary Diagnostic

10.1177/1040638716665659

[108] Abel-Reichwald H,

vetmic.2016.10.021

10.1111/vde.12733

Investigation. 2016;**28**(6):662-670. DOI:

Hainisch EK, Zahalka S, Corteggio A, Borzacchiello G, Massa B, et al. Epidemiologic analysis of a sarcoid outbreak involving 12 of 111 donkeys in Northern Italy. Veterinary Microbiology.

2016;**196**:85-92. DOI: 10.1016/J.

[109] White SD, Bourdeaux PJ, Brément T, Vandenabeele SI, Haspeslagh M, Bruet V, et al. Skin disease in donkeys (*Equus asinus*): A retrospective study from four veterinary schools. Veterinary Dermatology. 2019;**30**:247-e76. DOI:

[110] Marais HJ, Page PC. Treatment of equine sarcoid in seven Cape mountain zebra (*Equus zebra zebra*). Journal of Wildlife Diseases. 2011;**47**(4):917-924. DOI: 10.7589/0090-3558-47.4.917

[102] van Dyk E, Bosman AM, van Wilpe E, Williams JH, Bengis RG, van Heerden J, et al. Detection and characterization of papillomavirus in skin lesions of giraffes and sable antelope in South Africa. Journal of the South African Veterinary Association. 2011;**82**(2): 80-85. DOI: 10.4102/jsava.v82i2.39

[103] Williams JH, van Dyk E, Nel PJ, Lane E, van Wilpe E, Bengis RG, et al. Pathology and immunohistochemistry of papillomavirus-associated cutaneous

[104] Kidney BA, Berrocal A. Sarcoids

Dermatology. 2008;**19**(6):380-384. DOI: 10.1111/j.1365-3164.2008.00698.x

Williams J, Thompson PN. Descriptive study of an outbreak of equine sarcoid in a population of Cape mountain zebra (*Equus zebra zebra*) in the Gariep Nature Reserve. Journal of the South African Veterinary Association. 2006;**77**(4): 184-190. DOI: 10.4102/jsava.v77i4.375

[106] Marais HJ, Nel P, Bertschinger H,

[107] Davis CR, Valentine BA, Gordon E, McDonough SP, Schaffer PA, Allen AL,

Schoemann JP, Zimmerman D. Prevalence and body distribution of sarcoids in South African Cape mountain zebra (*Equus zebra zebra*). Journal of the South African Veterinary Association. 2007;**78**(3):145-148. DOI:

10.4102/jsava.v78i3.306

in two captive tapirs (*Tapirus bairdii*): Clinical, pathological and molecular study. Veterinary

[105] Nel PJ, Bertschinger H,

lesions in Cape mountain zebra, giraffe, sable antelope and African buffalo in South Africa. Journal of the South African Veterinary Association. 2011;**82**(2):97-106. DOI: 10.4102/jsava.

v82i2.42

[101] Teifke JP, Kidney BA, Löhr V, Yager JA. Detection of

#### *Equine Sarcoid DOI: http://dx.doi.org/10.5772/intechopen.91013*

*Equine Science*

vco.12299

[87] Golding JP, Kemp-Symonds JG, Dobson JM. Glycolysis inhibition improves photodynamic therapy response rates for equine sarcoids. Veterinary and Comparative Oncology. 2017;**15**:1543-1552. DOI: 10.1111/

1995;**27**(2):117-121. DOI: 10.1111/j.2042-

[95] Byam-Cook KL, Henson FMD, Slater JD. Treatment of periocular and non-ocular sarcoids in 18 horses by interstitial brachytherapy with iridium-192. The Veterinary Record. 2006;**159**:337-341. DOI: 10.1136/

[96] Hollis AR, Berlato D. Initial experience with high dose rate

[97] Christen-Clottu O, Klocke P,

[98] Knottenbelt DC. Skin disorders of the donkey and mule. Veterinary Clinics of North America: Equine Practice. 2019;**35**:493- 514. DOI: 10.1016/j.cveq.2019.08.006

[99] Löhr VC, Juan-Sallés C, Rosas-Rosas A, Parás García A, Garner MM, Teifke JP. Sarcoids in captive zebras (*Equus burchelli*)

DOI: 10.1638/03-126

jviromet.2009.02.008

[101] Teifke JP, Kidney BA, Löhr V, Yager JA. Detection of

association with bovine papillomavirus type 1 infection. Journal of Zoo and Wildlife Medicine. 2005;**36**(1):74-81.

papillomavirus DNA in sarcoid-affected

[100] van Dyk E, Oosthuizen MC, Bosman AM, Nel PJ, Zimmerman D, Venter EH. Detection of bovine

and healthy free-roaming zebra (*Equus zebra*) populations in South Africa. Journal of Virological Methods. 2009;**158**:141-151. DOI: 10.1016/j.

brachytherapy of periorbital sarcoids in the horse. Equine Veterinary Education. 2018;**30**(8):444-449. DOI: 10.1111/

Burger D, Straub R, Gerber V. Treatment of clinically diagnosed equine sarcoid with a mistletoe extract (*Viscus album austriacus*). Journal of Veterinary Internal Medicine. 2010;**24**:1483-1489. DOI: 10.1111/j.1939-1676.2010.0597.x

3306.1995.tb03046.x

vr.159.11.337

eve.12782

[88] Dobson J, de Queiroz GF,

Golding JP. Photodynamic therapy and diagnosis: Principles and comparative aspects. Veterinary Journal. 2018;**233**: 8-18. DOI: 10.1016/j.tvjl.2017.11.012

[89] Lavach JD, Sullins KE, Roberts SM, Severin GA, Wheeler C, Lueker DC. BCG treatment of periocular sarcoid. Equine Veterinary Journal. 1985;**17**:445-448. DOI: 10.1111/ j.2042-33-06.1985.tb02552.x

[90] Vanselow BA, Abetz I, Jackson ARB. BCG emulsion immunotherapy of equine sarcoid. Equine Veterinary Journal. 1988;**20**(6):444-447. DOI: 10.1111/j.2042-3306.1988.tb01571.x

[91] Hainisch EK, Harnacker J, Shafti-Keramat S, Kirnbauer R, Brandt S. Vaccination with virus-like particles induces long lasting protection from experimentally induced sarcoidlike tumours in horses. Equine

DOI: 10.1111/evj.12323\_33

[92] Rothacker CC, Boyle AG, Levine DG. Autologous vaccination for the treatment of equine sarcoids: 18 cases (2009-2014). The Canadian Veterinary Journal. 2015;**56**(7):709-714

[93] Théon AP. Radiation therapy in the horse. Veterinary Clinics of North America: Equine Practice. 1998;**14**(3):673-688. DOI: 10.1016/

[94] Théon AP, Pascoe JR. Iridium-192 interstitial brachytherapy for equine periocular tumours: Treatment results and prognostic factors in 115 horses. Equine Veterinary Journal.

S0749-0739(17)30192-X

Veterinary Journal. 2014;**46**(S47):15-16.

**134**

papillomavirus-DNA in mesenchymal tumour cells and not in the hyperplastic epithelium of feline sarcoids. Veterinary Dermatology. 2003;**14**:47-56. DOI: 10.1046/j.1365-3164.2003.00324.x

[102] van Dyk E, Bosman AM, van Wilpe E, Williams JH, Bengis RG, van Heerden J, et al. Detection and characterization of papillomavirus in skin lesions of giraffes and sable antelope in South Africa. Journal of the South African Veterinary Association. 2011;**82**(2): 80-85. DOI: 10.4102/jsava.v82i2.39

[103] Williams JH, van Dyk E, Nel PJ, Lane E, van Wilpe E, Bengis RG, et al. Pathology and immunohistochemistry of papillomavirus-associated cutaneous lesions in Cape mountain zebra, giraffe, sable antelope and African buffalo in South Africa. Journal of the South African Veterinary Association. 2011;**82**(2):97-106. DOI: 10.4102/jsava. v82i2.42

[104] Kidney BA, Berrocal A. Sarcoids in two captive tapirs (*Tapirus bairdii*): Clinical, pathological and molecular study. Veterinary Dermatology. 2008;**19**(6):380-384. DOI: 10.1111/j.1365-3164.2008.00698.x

[105] Nel PJ, Bertschinger H, Williams J, Thompson PN. Descriptive study of an outbreak of equine sarcoid in a population of Cape mountain zebra (*Equus zebra zebra*) in the Gariep Nature Reserve. Journal of the South African Veterinary Association. 2006;**77**(4): 184-190. DOI: 10.4102/jsava.v77i4.375

[106] Marais HJ, Nel P, Bertschinger H, Schoemann JP, Zimmerman D. Prevalence and body distribution of sarcoids in South African Cape mountain zebra (*Equus zebra zebra*). Journal of the South African Veterinary Association. 2007;**78**(3):145-148. DOI: 10.4102/jsava.v78i3.306

[107] Davis CR, Valentine BA, Gordon E, McDonough SP, Schaffer PA, Allen AL,

et al. Neoplasia in 125 donkeys (*Equus asinus*): Literature review and a survey of five veterinary schools in the United States and Canada. Journal of Veterinary Diagnostic Investigation. 2016;**28**(6):662-670. DOI: 10.1177/1040638716665659

[108] Abel-Reichwald H, Hainisch EK, Zahalka S, Corteggio A, Borzacchiello G, Massa B, et al. Epidemiologic analysis of a sarcoid outbreak involving 12 of 111 donkeys in Northern Italy. Veterinary Microbiology. 2016;**196**:85-92. DOI: 10.1016/J. vetmic.2016.10.021

[109] White SD, Bourdeaux PJ, Brément T, Vandenabeele SI, Haspeslagh M, Bruet V, et al. Skin disease in donkeys (*Equus asinus*): A retrospective study from four veterinary schools. Veterinary Dermatology. 2019;**30**:247-e76. DOI: 10.1111/vde.12733

[110] Marais HJ, Page PC. Treatment of equine sarcoid in seven Cape mountain zebra (*Equus zebra zebra*). Journal of Wildlife Diseases. 2011;**47**(4):917-924. DOI: 10.7589/0090-3558-47.4.917

**137**

Section 3

Digestive System, Diet and

Behavior

## Section 3
