**3. Recent advances in hormonal control of estrous cycle**

In mares, the natural breeding season extends from spring to early autumn. Until now, various methods have been used to advance the onset of the breeding season or to synchronize the estrus during the reproductive season. Ovulation induction protocols have also been developed for use in artificial insemination or embryo transfer programs [41, 42].

**9**

with E2 [51].

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy…*

Seasonal reproductive inactivity in mares is due to reduced synthesis and storage in the hypothalamus of GnRH and decreased amounts of FSH and LH in the anterior pituitary gland [27]. Taking this physiological basis into account, it would be expected that the administration of gonadotropins to anestrous mares will restart

The administration of a single dose of GnRH to mares causes an increase in the circulating concentrations of FSH and LH [43]. However, constant infusions result in a continuous release of both hormones [44]. An experience conducted in the late 1980s reported that 50% of mares treated during the seasonal anestrous had fertile estrous after infusion of GnRH for 28 days (100 ng/kg; SC). However, this same experiment showed that mares with transitional anestrous were more likely to

In another study, daily but not continuous administration of GnRH to induce ovulation in anestrous mares only induced the development of preovulatory follicles [46]. Also, another report [47] showed that the administration of 0.5 mg GnRH three times daily for 7 or 7.5 days induced normal follicular maturation and normal luteinization in anestrous mares. From these studies, it has been demonstrated that the administration of GnRH in diverse protocols is not profitable and requires a lot of manpower. It also results in variable response to treatment among mares, espe-

GnRH is known to be responsible for the secretion of FSH and LH, but studies performed to evaluate the efficacy of GnRH-agonists are conflicting. GnRH agonists were used as injections or slow-releasing implants to induce estrus and ovulation in anestrous and transitional mares. The GnRH agonists available for mares

According to Allen et al. [49] two injections of GnRH agonists each day or continuous administration of GnRH agonists were able to induce follicular development and ovulation in acyclic mares. In the same way, Bergfelt and Ginther [26], demonstrate the same result where mares where about 60% of treated mares with

In a study conducted in transition mares for 28 days, Harrison et al. [50] administered buserelin twice daily (40 μg, IM, q 12 h) for 28 days, or as SC implants releasing 100 μg/day. 45% of the mares ovulated between the 10th and 25th day after the start of treatment, in response to the two daily injections. However, 60% of the mares ovulated between 4 and 30 days after implant treatment. The same results were observed when the GnRH agonist was combined

Deslorelin has also been used to induce cycle and ovulation in mares. Slow liberation subcutaneous deslorelin implants are effective in increasing LH and

It is important to indicate that the response is in correlation with the follicular size at the beginning of the treatment and the depth of anestrus. This means that due to the insensitivity of GnRH, mares that are already in the transition period are more likely to respond to the treatment compared to those who are in deep anestrus [54]. Another negative aspect of GnRH treatment in anestrous mares is the risk of

*DOI: http://dx.doi.org/10.5772/intechopen.90387*

**3.1 Gonadotropin releasing hormone**

respond to GnRH than mares with deep anestrous [45].

include deslorelin, buserelin, and historelin [48].

accelerating ovulation in mares [52, 53].

GnRH-agonist ovulated within a 21-day long treatment.

early pregnancy losses due to inadequate luteal function [26].

reproductive capacity.

cially deep anestrous mares.

**3.2 GnRH agonists**

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy… DOI: http://dx.doi.org/10.5772/intechopen.90387*

### **3.1 Gonadotropin releasing hormone**

*Animal Reproduction in Veterinary Medicine*

as previously referred [31].

*2.3.5 Progesterone*

*2.3.6 Prostaglandin F2<sup>α</sup>*

therefore secretion P4 [39, 40].

embryo transfer programs [41, 42].

ing basal levels at day 5 post-ovulation [13, 19].

fact which promotes the mare returns to estrus [19, 36].

**3. Recent advances in hormonal control of estrous cycle**

the preovulatory release of LH. After ovulation, E2 levels begin to decrease, reach-

Although estrogen levels are directly related to the degree of ovarian activity, sexual receptivity and reproductive tract changes [4, 6, 13, 31, 35] there is no evidence of a direct relationship between the intensity of endometrial edema and E2 concentration. This situation is much clearer on P4. Swelling occurs when P4 levels are <1 ng/ml, so this hormone could be responsible in principle on the intensity of edema, among other behavioral and morphological changes of the cervix and uterus [35]. However, at the time of ovulation inverse correlations are established between E2 and FSH levels associated with the negative feedback effect of inhibin,

The steroidogenic activity of P4 depends on the action of LH on theca cells. As noted above, levels of P4 are <1 ng/ml during estrus [19, 36]. After ovulation, it increases progressively and significantly to the 5th or 6th day, with values similar to those of pregnant mares during the first 14 days of gestation. At this time the CL is fully functional and P4 levels remain high until day 9 [35, 37], consistent with the maximum diameter reached by the CL [7, 20, 35, 37]. However, peripheral concentrations of P4 are highly variable between mares. This variability is associated with secretory capacity CL and hormonal catabolic rate. Perhaps this fact may explain the differences in P4 levels between different breeds during the first 5 days of the luteal period*,* despite the similarity in length of estrous cycles. Among other factors related to variations in levels of P4 highlights the number of ovulations. In fact, double ovulations induce higher concentrations of P4 compared to simple ones [35]. P4 inhibits the secretion and pulsatile release of GnRH and LH but does not modify the pattern of FSH [7, 13, 15]. This event, unlike what happens in other species, enabling a new wave of follicular growth and in some cases the presence of ovulation during diestrus related to high levels of this hormone [18, 22, 38]. After lysis of the CL at the end of diestrus, P4 is drastically reduced to levels <1 ng/ml, a

In the absence of pregnancy, the average life span of the CL is controlled by the release of endometrial PGF2α source, establishing a bimodal pattern of discharge around day 13–16 of diestrus. While the first 4-h peak precedes the decline of P4, the second occurs during and after luteolysis. Luteolysis involves decreased blood supply, leukocyte infiltration, cell disruption and loss of lutein steroidogenic capacity by apoptotic or non-apoptotic mechanisms intended to disintegrate the CL and

In mares, the natural breeding season extends from spring to early autumn. Until now, various methods have been used to advance the onset of the breeding season or to synchronize the estrus during the reproductive season. Ovulation induction protocols have also been developed for use in artificial insemination or

**8**

Seasonal reproductive inactivity in mares is due to reduced synthesis and storage in the hypothalamus of GnRH and decreased amounts of FSH and LH in the anterior pituitary gland [27]. Taking this physiological basis into account, it would be expected that the administration of gonadotropins to anestrous mares will restart reproductive capacity.

The administration of a single dose of GnRH to mares causes an increase in the circulating concentrations of FSH and LH [43]. However, constant infusions result in a continuous release of both hormones [44]. An experience conducted in the late 1980s reported that 50% of mares treated during the seasonal anestrous had fertile estrous after infusion of GnRH for 28 days (100 ng/kg; SC). However, this same experiment showed that mares with transitional anestrous were more likely to respond to GnRH than mares with deep anestrous [45].

In another study, daily but not continuous administration of GnRH to induce ovulation in anestrous mares only induced the development of preovulatory follicles [46]. Also, another report [47] showed that the administration of 0.5 mg GnRH three times daily for 7 or 7.5 days induced normal follicular maturation and normal luteinization in anestrous mares. From these studies, it has been demonstrated that the administration of GnRH in diverse protocols is not profitable and requires a lot of manpower. It also results in variable response to treatment among mares, especially deep anestrous mares.

#### **3.2 GnRH agonists**

GnRH is known to be responsible for the secretion of FSH and LH, but studies performed to evaluate the efficacy of GnRH-agonists are conflicting. GnRH agonists were used as injections or slow-releasing implants to induce estrus and ovulation in anestrous and transitional mares. The GnRH agonists available for mares include deslorelin, buserelin, and historelin [48].

According to Allen et al. [49] two injections of GnRH agonists each day or continuous administration of GnRH agonists were able to induce follicular development and ovulation in acyclic mares. In the same way, Bergfelt and Ginther [26], demonstrate the same result where mares where about 60% of treated mares with GnRH-agonist ovulated within a 21-day long treatment.

In a study conducted in transition mares for 28 days, Harrison et al. [50] administered buserelin twice daily (40 μg, IM, q 12 h) for 28 days, or as SC implants releasing 100 μg/day. 45% of the mares ovulated between the 10th and 25th day after the start of treatment, in response to the two daily injections. However, 60% of the mares ovulated between 4 and 30 days after implant treatment. The same results were observed when the GnRH agonist was combined with E2 [51].

Deslorelin has also been used to induce cycle and ovulation in mares. Slow liberation subcutaneous deslorelin implants are effective in increasing LH and accelerating ovulation in mares [52, 53].

It is important to indicate that the response is in correlation with the follicular size at the beginning of the treatment and the depth of anestrus. This means that due to the insensitivity of GnRH, mares that are already in the transition period are more likely to respond to the treatment compared to those who are in deep anestrus [54]. Another negative aspect of GnRH treatment in anestrous mares is the risk of early pregnancy losses due to inadequate luteal function [26].

#### **3.3 Progesterone and progestins**

The administration of P4 suppresses the release of LH from the anterior pituitary gland. Once P4 supplementation ceases, the so-called "rebound effect" induces follicular maturation and ovulation. Its use in equine reproduction is a common practice and the available protocols include progestogens administered orally or parenterally. However, its use in mares with seasonal anestrous is questionable.

Different studies indicate that mares in deep anestrous or early transition do not anticipate the first ovulation of the year with P4 treatments [30, 55]. However, it has been shown that, if treatment is carried out at the end of the transition period and the mares have at least one follicle of more than 20 mm in diameter in the ovaries, they show regular post-treatment cycles [56].

Intravaginal devices containing P4 (CIDR, PRID, and intravaginal sponges) have been used in mares. Indeed, Hanlon and Firth [57] examined the effect of intravaginal devices placed during 10 days in transitional Thoroughbred mares. The results of the experiment showed that the use of P4 has a positive effect in bringing forward the first estral cycle of the breeding season. Compared to control mares, in the first 21 days of the season, 95.2% treated mares were served and conceived sooner after the start of the breeding season.

Regumate is the most commonly used orally administered progestogen. Its active ingredient is allyl trenbolone, also called Altrenogest. Allen et al. [55] evaluated the effect of oral P4 treatment in mares with seasonal anestrous. Within 8 days, 88% of the treated mares showed estrous behavior and within 18 days of treatment interruption, 84% had ovulated. Based on these figures, the treatment gave a positive result in the acceleration of cyclicity in mares, but its response depends on the depth of the anestrus.

#### **3.4 Recombinant equine FSH (reFSH) and LH (reLH)**

The use of recombinant equine FSH (reFSH) has been reported to induce follicular growth in cyclic mares [58, 59]. A study reviewed in 2013 however determined the efficacy of it in deep anestrous mares to be very successful with ovulation rate of 76.7% in response to FSH treatment followed by human chorionic gonadotropin (hCG) administration [60].

Mares in deep anestrous treated with reFSH alone or reFSH and reLH in combination under natural photoperiod showed a significant increase in follicular development within 6 days on average and all of them ovulated within 10 days. In comparison, the control group needed a significantly longer time for follicular growth and only 30% of the control mares had ovulated at the end of the 14 days used for the experiment [61].

#### **3.5 Dopamine antagonists and prolactin**

Studies in sheep found that dopamine antagonists are effective in increasing LH secretion during estrus by inhibiting the release of dopamine in the brain [62]. In mares, the increased release of dopamine during winter anestrous has been confirmed in studies measuring a higher concentration of dopamine in the cerebrospinal fluid during deep anestrous. It has also been shown that inhibition of dopamine D2 receptors may accelerate the onset of the ovulatory season in mares. Sulpiride, domperidone, and perphenazine have been studied [63].

Mari et al. [64] compared the efficacy of sulpiride and domperidone, two longacting dopamine antagonists, to induce ovarian activity in mares with deep anestrous. The results showed that sulpiride administration was effective in accelerating the transition period and first ovulation in mares with deep anestrous.

**11**

[29, 75, 76].

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy…*

On the other hand, as daylight increases, the concentration of prolactin (PRL) also increases. Dopamine is an inhibitor of PRL release, and it has been suggested that the administration of this hormone may help stimulate cycling in mares in anestrus [65]. Various studies have confirmed that the administration of recombinant prolactin from different animal species (equine, porcine and ovine) has a stimulating effect on mares in anestrus. Thompson et al. [66] examined the effect of subcutaneous administration of recombinant porcine prolactin (rpPRL) pony mares for 45 days. About 17 days after the start of treatment, a high percentage of treated mares showed signs of heat and ovulation accelerated by more than 1 month. However, another study examined the effect of a single dose of recombinant ovine prolactin (ovPRL). As a result, significant stimulation of follicular develop-

A reliable ovulation-inducing drug is one that can trigger ovulation within a certain "fixed" period of time. This pharmacological action can provide enormous advantages in anticipating the right time for artificial insemination. Several pharmacological agents such as GnRH and GnRH agonist, hCG, recombinant equine LH, and equine pituitary extracts, prostaglandins and kisspeptin have been used to

The frequency of GnRH pulses is the main regulator of LH secretion by the adenohypophysis [69]. Because of this stimulation, they can be used as an ovulatory agent and therefore can be used to induce ovulation in mares. On the other hand, due to its natural origin, it does not cause an immune response after being administered in several sessions. There is also little risk of contamination as GnRH is a synthetic product. In the 1990s, several experiments were conducted to evaluate the efficacy of GnRH in ovulation induction in cyclic mares [70, 71]. In one of them, the effect of a single administration of 2 mg of synthetic GnRH was tested but did not affect ovulation induction. However, daily injections of the same compound from day 2 of heat to ovulation resulted in a shortening of the duration of heat and the time for ovulation [72]. Likewise, Duchamp et al. [73] conducted a study to try to identify a more suitable ovulatory agent. To do that, they compared the effect of an intramuscular injection of 2.500 i.u. hCG and 2 mg GnRH (not synthetic). The use of hCG, injected when the follicle reached 35 mm in diameter, induced ovulation in 24 or 48 h. However, GnRH was not effective in shortening ovulation time

On the other hand, the pulsatile infusion of endogenous GnRH was effective in advancing ovulation time in cyclic mares [70]. Treatments with low doses of endogenous GnRH (2.5 μg) continuous infusion for 14 days demonstrated increased

Deslorelin is a potent GnRH agonist and is marketed as a controlled-release subcutaneous implant under the trade name Ovuplant™. In the past, several authors have investigated the efficacy of Deslorelin in inducing ovulation in mares

LH and ovulation in all treated mares compared to controls [74].

*3.6.2.1 Deslorelin (ovuplant and other products)*

*DOI: http://dx.doi.org/10.5772/intechopen.90387*

ment was observed, but only one mare ovulated [67].

determine their efficacy in ovulation induction [68].

**3.6 Induction of ovulation in mares**

compared to the control group.

*3.6.2 GnRH-agonist*

*3.6.1 GnRH*

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy… DOI: http://dx.doi.org/10.5772/intechopen.90387*

On the other hand, as daylight increases, the concentration of prolactin (PRL) also increases. Dopamine is an inhibitor of PRL release, and it has been suggested that the administration of this hormone may help stimulate cycling in mares in anestrus [65]. Various studies have confirmed that the administration of recombinant prolactin from different animal species (equine, porcine and ovine) has a stimulating effect on mares in anestrus. Thompson et al. [66] examined the effect of subcutaneous administration of recombinant porcine prolactin (rpPRL) pony mares for 45 days. About 17 days after the start of treatment, a high percentage of treated mares showed signs of heat and ovulation accelerated by more than 1 month. However, another study examined the effect of a single dose of recombinant ovine prolactin (ovPRL). As a result, significant stimulation of follicular development was observed, but only one mare ovulated [67].
