**2.1.7 Inadequate follicular growth**

It has also been proposed that failure of pregnancy in RBCs could be associated to dysfunction in the recruitment of large follicles during the second half of the estrous cycle, resulting in absent or abnormal ovulation, or early cow-embryo asynchrony, occurring EED and RBC. The critical period in the follicular recruitment occurs as from 10 days before ovulation (Staigmiller & England, 1982; Thibier et al., 1985). Dominant follicle continues its growth when progesterone levels are subluteal (or suprabasal), in which case follicular function is compromised and oocyte quality is reduced, affecting negatively the fertility (Odde, 1990; Stock & Fortune, 1993).

## **2.1.8 Effect of nutrition on RBC syndrome**

The importance of nutrition in all vital processes is indisputable, and the qualitative and quantitative differences in the ration in dairy cattle may cause reproductive dysfunctions (Roberts, 1971; Casida, 1961; Lagneau, 1981; Pedroso & Roller, 1996; Dovensky et al., 1996). Nutritional deficiency and increment of services per cow are linked, because the decrease in food intake, weight and body condition causes endocrine imbalances that affect fertility and other organs or systems. The mentioned nutritional deficiency can also affect the postpartum period, causing delay in the uterine involution and increasing the number of

obtained after embryo transfer in RBC suggest that uterine environment is the most important factor in the EED, reporting abnormal concentrations of ions and proteins in the uterus. Intrinsic embryo factors are also related with this syndrome. Linares (1982) observed that blastocysts collected in RBCs had more abnormalities than those of control cows (3:1), and the oocyte collection rate was also lower, possibly due to a degenerative process initiated after fertilization. It was determined that the reduction of embryo viability could be associated with the reduction of nutrients exchange capacity and other regulatory substances. They could disrupt the process of cell differentiation, initiate degenerative processes, and slightly reduce the number of cilliar cells in the endometrium. High and early endometrial progesterone receptors have been detected 3 days after estrus in RBC that

Abnormal embryonic development by hormonal asynchrony during estrus and metaestrus have been involved in the RBC syndrome. Inadequate levels of estrogen and abnormal interactions with gonadotrophins injure the oocyte maturation, resulting in abnormal embryonic development. The altered hormonal pattern in RBCs could cause the incompetence of oocytes that will suffer abnormal embryonic development (Gustafsson et al., 1986; Albihn et al., 1991). However, other authors consider that the majority of embryonic abnormalities occur during the way through the oviducts, although those abnormalities are not evident until 6-7 days post-AI (i.e. in blastocyst stage). As oocytes of RBCs are competent to reach the blastocyst stage and to continue the development, embryo production (IVM-IVF-IVC-ET) could improve the reproductive success in these animals.

EED has usually no effect on the normal length of the estrous cycle. In occasions, anamnesis in RBC shows normal estrous cycle and fertilization rate, and the "apparent infertility" is due to earlier embryo mortality (day 7). No significant differences have been detected in these animals regarding estrus duration, ovulation time and the incidence of anovulation or silent

It has also been proposed that failure of pregnancy in RBCs could be associated to dysfunction in the recruitment of large follicles during the second half of the estrous cycle, resulting in absent or abnormal ovulation, or early cow-embryo asynchrony, occurring EED and RBC. The critical period in the follicular recruitment occurs as from 10 days before ovulation (Staigmiller & England, 1982; Thibier et al., 1985). Dominant follicle continues its growth when progesterone levels are subluteal (or suprabasal), in which case follicular function is compromised and oocyte quality is reduced, affecting negatively the fertility

The importance of nutrition in all vital processes is indisputable, and the qualitative and quantitative differences in the ration in dairy cattle may cause reproductive dysfunctions (Roberts, 1971; Casida, 1961; Lagneau, 1981; Pedroso & Roller, 1996; Dovensky et al., 1996). Nutritional deficiency and increment of services per cow are linked, because the decrease in food intake, weight and body condition causes endocrine imbalances that affect fertility and other organs or systems. The mentioned nutritional deficiency can also affect the postpartum period, causing delay in the uterine involution and increasing the number of

estrus (Linares et al., 1984).

**2.1.7 Inadequate follicular growth** 

(Odde, 1990; Stock & Fortune, 1993).

**2.1.8 Effect of nutrition on RBC syndrome** 

suggest hormonal and cellular changes in the uterus (Almeida et al., 1987, 1995).

open days. McClure (1995) reported that reproductive disorders could occur at 3 levels: the synthesis and release of LH from hypothalamus, at ovarian function, or at ovulation, fertilization and development of the fertilized egg, embryo and fetus.

Homeostasis of nutritional elements can be maintained through correct animal diet (deficit or excess depends on the production status). Nutritional variations may be due to an excess or deficiency of certain elements or imbalances of their concentrations in diet, and provoke alterations in their absorption or utilization. Nutritional deficiencies are more significant in high production animals, as occur in dairy cattle. In the 70's, Payne et al. (1973) developed the theory of metabolic profiles, which aimed to monitor the metabolic status and health of the herd.

#### **2.2 Bull factors involved in the RBC syndrome**

Factors related to the bull and sperm quality must also be taken into account when RBCs are evaluated. It is essential to evaluate the sperm function in both natural mating and AI. Frozen semen straws should be carefully stored and managed. Repeat pregnancy failure could be linked to the mentioned bull factors, in which estrus is repeated and interestrus interval has normal duration. Some relevant aspects are mentioned below.

### **2.2.1 Influence of bull fertility and semen quality on repeat breeding**

Optimal bull fertility (by natural breeding or AI) is necessary to achieve high pregnancy rate and normal calving interval. Semen doses for AI must contain at least 6 million of motile sperm after thawing, and fertility drops if sperm concentration is reduced (Foote & Parks, 1993). Currently, frozen semen doses are packed with 15-25 million of motile sperm prefreezing, because around 50% of spermatozoa recover motility after thawing. However, despite semen doses may fulfill all requirements, there are donors of sperm with erratic fertility. Eid et al. (1994) found that embryos formed from high fertile bulls reached earlier the S phase of DNA synthesis and the 2-cell phase, showing high blastocyst viability. Embryos from low fertile bulls showed a longer G2 phase associated with sperm DNA damage or DNA replication failure during the S3 phase (Eid et al., 1995). The strict selection of semen donors based on the semen quality after thawing and sexual behavior, among other variables, has reduced defects in bulls used for the creation of frozen semen banks.

At natural breeding, it is necessary to assess the reproductive performance of the bulls at least twice a year, carrying out semen assessment (macroscopic and microscopic) and physical evaluations. It is also interesting to evaluate libido and the sexual behavioral pattern for mating to diagnose reproductive failures in bulls. On the other hand, it is essential to maintain an appropriate male:female ratio for mating. For guaranteeing optimal ejaculates, bulls should do 10 mating throughout a week.

#### **2.2.2 Site of semen deposition and estrus return**

Sperm that has just been ejaculated into the vagina by the bull or placed into the uterus by the inseminator should reach the oviducts, where fertilization occurs. Sperm begins its upward through the tubular tract of the female and reaches, within minutes, the fallopian tubes to find the oocyte. It has been reported that the utero-tubal isthmus acts as a reservoir

Clinical Approach to the Repeat Breeder Cow Syndrome 347

endocrine mechanisms (reduced intensity of estrus, decreased preovulatory LH peak, etc)

However, other authors (Gonzalez, 1981) observed that higher progesterone concentrations was present in fertile and anestrus females during warm periods, attributing it to increased activity of the CL or the adrenals by heat stress. Photoperiod length and temperature variations are linked to the season, and could influence the endocrine regulation of the estrous cycle. Other factors as body condition at calving, feeding level and calvinginsemination interval are also season-related, and can be modified through management practices (Fulkerson & Dickens, 1985). GnRH-based treatments used in RBCs reveal a marked seasonal response, and it has been demonstrated that RBCs and infertility increase during summer (hot) months (BonDurant et al., 1991; Gonzalez-Stagnaro et al., 1993).

The poor heat detection is one of the main causes of RBC and infertility in cattle (Lafi & Kaneene, 1988; Perez-Marin & España, 2007). Computer simulation models have been implemented to improve the estrus detection and it has been showed that they increase the frequency of heat detection and reduce the services/conception and days open (Bailie, 1982). Reproductive failure may be observed after mis-timed inseminations or in cows without evident estrus. Pregnant cows could show estrous signs and they could be inseminated. This intrauterine procedure could induce early or late embryonic death, or abortion in pregnant cows. Between 3 and 10% of pregnant cows can be mounted by other cows, and early pregnancy diagnosis reduces these mis-timed insemination. Some authors recommend making intracervical insemination if reproductive status of cows in heat is doubtful (Lopez-

It is essential to act carefully and prevent infections at reproductive tract. During artificial insemination, the catheter is inserted into the uterus, and may act as a disseminator of pathogenic bacteria that alter the normal uterine environment and provoke subacute, acute

On the other hand, is essential to care the peripartum period. In fact, many authors consider this period, and particularly the postpartum period, the most critical in the reproductive activity, observing that animals showing problems during this period are finally RBCs (Erb

RBCs have a higher predisposition to be stressed (Bage, 2002), as evidenced by the increased capacity to produce cortisol from adrenal glands after induced stress. Adrenal progesterone in induced-stress cows reaches suprabasal levels. Thus, stress can be considered as a

Heat is one of the stressful causes in cattle. It has been shown that heat-stressed cows reduce the duration and intensity of estrus, and alter hormone levels by increasing circulating

and/or the embryo, reducing the fertility.

Gatius & Camón-Urgel, 1991; Bullman & Lamming, 1978).

**2.3.3 Hygiene at artificial insemination and parturition** 

or chronic infectious diseases, with harmful effects on reproduction.

**2.3.2 Estrus detection** 

et al., 1985).

**2.3.4 Stress** 

potential factor in the RBC syndrome.

of sperm, making these cells to go up gradually towards the ampulla, preventing polyspermy and ensuring that sperm finds the oocyte into the oviduct (Hunter, 2002). The interaction of sperm with the reproductive female tract results in a drastic decline of spermatozoa. Thus, the female reproductive tract may alter the mechanism of sperm transport and induces infertility. In addition, defective inseminations (e.g. deposition of semen at the entrance of the cervix) may provoke RBC and affect negatively the fertility rates. Higher conception rates are obtained when sperm is deposited into the uterus (Gwazdauskas et al., 1986), but the incidence of uterine disease in RBCs ranges between 36 and 89% (Ellington & Schlafer, 1993). It has been suggested that intraperitoneal insemination may be an alternative to uterine insemination (López-Gatius, 1995) in cows with uterine diseases.

#### **2.2.3 Time of semen deposition**

Time of semen deposition is closely related with the detection of estrus and with all those factors that may affect the preovulatory LH. Other cows or a bull mounting the cow are recognized as signs of the cow heat. This behavior could last between 13-17 h. on average, and ovulation will take place approximately 30 h. after starting the heat. It must be ensured that semen is deposited in the female tract as close as possible to that time. The AM/PM rule is the most common practice in dairy cattle farms: it consists of making insemination at morning if the cow was in heat the previous evening, or being inseminated in the afternoon if heat was detected in the morning. If the technician palpates ovaries before insemination and considers that the follicle texture is far to ovulation, double insemination is recommended separated by 12 hours. Similar pregnancy rates have been reported after one or two inseminations per day (Wahome et al., 1985). As noted above, semen is normally deposited into the uterus, and the uterine environment will be adequate depending on the stage of the estrous cycle. This is one of the most common problems in RBCs, in which any change of pH, endometrial stroma and glands, endometrial flora, etc. could affect the reproduction (Ohtani & Okuda, 1995).

#### **2.3 Environmental and management factors affecting the RBC syndrome**

These factors are very miscellaneous. Hewett (1968) suggests a correlation between RBC and the following variables: lactational stress, size of livestock (the number of cows with repeat estrus increases in bigger livestocks), milk yield or calving season (a greater number of RBCs are detected in autumn calves). Similarly, Gustafsson & Emmanuelson (2000) considered as risk factors for RBC syndrome the first lactation, dystocia or calving difficulties, the first insemination at winter, reproductive disorders before the first insemination, high milk yield or clinical history of repeat estrus. Therefore, environmental and animal management factors should be considered in order to reduce the incidence of RBC syndrome.

#### **2.3.1 Season**

Barlett et al. (1986) reported that there is no association between breeding season and RBC syndrome. However, ovarian follicular growth and development of the dominant follicle can be altered during the summer months, and heat stress exerts a depressant effect on the endocrine mechanisms (reduced intensity of estrus, decreased preovulatory LH peak, etc) and/or the embryo, reducing the fertility.

However, other authors (Gonzalez, 1981) observed that higher progesterone concentrations was present in fertile and anestrus females during warm periods, attributing it to increased activity of the CL or the adrenals by heat stress. Photoperiod length and temperature variations are linked to the season, and could influence the endocrine regulation of the estrous cycle. Other factors as body condition at calving, feeding level and calvinginsemination interval are also season-related, and can be modified through management practices (Fulkerson & Dickens, 1985). GnRH-based treatments used in RBCs reveal a marked seasonal response, and it has been demonstrated that RBCs and infertility increase during summer (hot) months (BonDurant et al., 1991; Gonzalez-Stagnaro et al., 1993).

### **2.3.2 Estrus detection**

346 A Bird's-Eye View of Veterinary Medicine

of sperm, making these cells to go up gradually towards the ampulla, preventing polyspermy and ensuring that sperm finds the oocyte into the oviduct (Hunter, 2002). The interaction of sperm with the reproductive female tract results in a drastic decline of spermatozoa. Thus, the female reproductive tract may alter the mechanism of sperm transport and induces infertility. In addition, defective inseminations (e.g. deposition of semen at the entrance of the cervix) may provoke RBC and affect negatively the fertility rates. Higher conception rates are obtained when sperm is deposited into the uterus (Gwazdauskas et al., 1986), but the incidence of uterine disease in RBCs ranges between 36 and 89% (Ellington & Schlafer, 1993). It has been suggested that intraperitoneal insemination may be an alternative to uterine insemination (López-Gatius, 1995) in cows with uterine

Time of semen deposition is closely related with the detection of estrus and with all those factors that may affect the preovulatory LH. Other cows or a bull mounting the cow are recognized as signs of the cow heat. This behavior could last between 13-17 h. on average, and ovulation will take place approximately 30 h. after starting the heat. It must be ensured that semen is deposited in the female tract as close as possible to that time. The AM/PM rule is the most common practice in dairy cattle farms: it consists of making insemination at morning if the cow was in heat the previous evening, or being inseminated in the afternoon if heat was detected in the morning. If the technician palpates ovaries before insemination and considers that the follicle texture is far to ovulation, double insemination is recommended separated by 12 hours. Similar pregnancy rates have been reported after one or two inseminations per day (Wahome et al., 1985). As noted above, semen is normally deposited into the uterus, and the uterine environment will be adequate depending on the stage of the estrous cycle. This is one of the most common problems in RBCs, in which any change of pH, endometrial stroma and glands, endometrial flora, etc. could affect the

**2.3 Environmental and management factors affecting the RBC syndrome** 

factors should be considered in order to reduce the incidence of RBC syndrome.

These factors are very miscellaneous. Hewett (1968) suggests a correlation between RBC and the following variables: lactational stress, size of livestock (the number of cows with repeat estrus increases in bigger livestocks), milk yield or calving season (a greater number of RBCs are detected in autumn calves). Similarly, Gustafsson & Emmanuelson (2000) considered as risk factors for RBC syndrome the first lactation, dystocia or calving difficulties, the first insemination at winter, reproductive disorders before the first insemination, high milk yield or clinical history of repeat estrus. Therefore, environmental and animal management

Barlett et al. (1986) reported that there is no association between breeding season and RBC syndrome. However, ovarian follicular growth and development of the dominant follicle can be altered during the summer months, and heat stress exerts a depressant effect on the

diseases.

**2.2.3 Time of semen deposition** 

reproduction (Ohtani & Okuda, 1995).

**2.3.1 Season** 

The poor heat detection is one of the main causes of RBC and infertility in cattle (Lafi & Kaneene, 1988; Perez-Marin & España, 2007). Computer simulation models have been implemented to improve the estrus detection and it has been showed that they increase the frequency of heat detection and reduce the services/conception and days open (Bailie, 1982). Reproductive failure may be observed after mis-timed inseminations or in cows without evident estrus. Pregnant cows could show estrous signs and they could be inseminated. This intrauterine procedure could induce early or late embryonic death, or abortion in pregnant cows. Between 3 and 10% of pregnant cows can be mounted by other cows, and early pregnancy diagnosis reduces these mis-timed insemination. Some authors recommend making intracervical insemination if reproductive status of cows in heat is doubtful (Lopez-Gatius & Camón-Urgel, 1991; Bullman & Lamming, 1978).

#### **2.3.3 Hygiene at artificial insemination and parturition**

It is essential to act carefully and prevent infections at reproductive tract. During artificial insemination, the catheter is inserted into the uterus, and may act as a disseminator of pathogenic bacteria that alter the normal uterine environment and provoke subacute, acute or chronic infectious diseases, with harmful effects on reproduction.

On the other hand, is essential to care the peripartum period. In fact, many authors consider this period, and particularly the postpartum period, the most critical in the reproductive activity, observing that animals showing problems during this period are finally RBCs (Erb et al., 1985).

#### **2.3.4 Stress**

RBCs have a higher predisposition to be stressed (Bage, 2002), as evidenced by the increased capacity to produce cortisol from adrenal glands after induced stress. Adrenal progesterone in induced-stress cows reaches suprabasal levels. Thus, stress can be considered as a potential factor in the RBC syndrome.

Heat is one of the stressful causes in cattle. It has been shown that heat-stressed cows reduce the duration and intensity of estrus, and alter hormone levels by increasing circulating

Clinical Approach to the Repeat Breeder Cow Syndrome 349

Vaginoscopy is helpful to visualize the vaginal cavity and cervix. Feces are removed from rectum before the perineal area, vestibule and clitoral fossa were cleaned and finally dried. The speculum -which must be clean, dry, sterile, and lubricated- is inserted in direction of the pelvic canal. Once crossed the vestibule, the negative pressure inside allows air to penetrate. Hyperemia may result in 30-60 sec, which hampers the assessment of the vaginal mucosa coloration. The vestibule acts as a defensive barrier of the female genital tract, and hinders the entry of the speculum. If this does not occurs, dysfunctions can be present (e.g. in pneumovagina). Similarly, if material is accumulated into the vagina, speculum helps to

The examination of internal organs (uterus, oviducts and ovaries) demands other

Rectal palpation is a widely used diagnostic method in cattle with high accuracy, easy to be implemented and at low cost in comparison with other sophisticated techniques. Plastic gloves are lubricated and then feces are withdrawn. Air should not be present into the rectum to get a more relaxed mucosa and easily manipulate the structures beneath. The cervix is presented as a solid structure, tubular, fibrous, with 3-4 folds projected inside and localized on pelvis floor in normal non-pregnant cows. It is cylindrical, with a length of 5-10 cm and a diameter of 1,5-7,0 cm. Cranially the uterus can be palpated. At heat, uterus is turgid, erect and coiled. However, it is soft and flaccid during luteal phase and palpation is a bit more difficult; it is a consequence of the progesterone action, released from CL.

After that, it is interesting to palpate the ovaries. They are located ventrolaterally to the pelvis floor, and sometimes placed under the bone. During anestrus, ovary size ranges from 2 to 3 cm approximately. Follicles (at different stages of growth) and CLs (hemorrhagic, mature or/and albicans) are developed at the ovaries and its size could suggest some

The first description of cattle pregnancy by real-time ultrasound was conducted by Chaffeux et al. (1982). Later, Pierson & Ginther (1984) showed ultrasound images of normal ovarian structures in superovulated heifers. Also Reeves et al. (1984) described the echogenic cystic CL. More recently, ultrasound technology has been used for developing more effective superovulation, embryo collection and recipient´s synchronization. Foetal sex can be determined by ultrasonography (US) from day 50 onward, emphasizing that it can be accurately established around day 60 of pregnancy. Ultrasonographic anatomy of foetal sex organs, from genital tubercle (GT) to fully developed organs, has been extensively described in their entire progression. The external genitalia are initially formed on the caudoventral surface of the abdominal wall, between the hind limbs. These primordial structures are seen as poorly defined elevations and they constitute the GT, the urogenital fold and the genital swelling, which are developed into the male or female gonads. In males, the GT becomes elongated to form penis, the urogenital folds enclose the penis to form the prepuce and the genital swelling become enlarged to develop into the scrotum. In females, the GT forms the

localize its origin.

diseases.

**3.3 Ultrasonography (US)** 

**3.2 Rectal palpation in cow** 

techniques, as manual palpation and ultrasound.

Palpation helps to diagnose anomalies such as uterine infections.

corticosteroids and by reducing the concentration of progesterone. Uterine environment is altered and viability of the pre-implanted embryo is reduced. It is recommended to use sprinklers, fans cooling, etc., to handle this stress (Ferreira et al., 2011).
