**3.2 Rectal palpation in cow**

348 A Bird's-Eye View of Veterinary Medicine

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

It is necessary to diagnose the aethiology of reproductive failures in cows having an apparently normal clinical history and then, reduce the economical impact. However, although many diagnostic tools are available, it is usually difficult to get a correct diagnostic

First of all, a complete clinical history should be obtained at herd and individual level. Age, parity, milk yield, previous diseases, reproductive indexes, estrous cycles characteristics, insemination schedule, bulls, estrus detection, hormones, food and farm hygiene should be registered. Now, anatomy, morphology and function of cows should be inspected. The reproductive status of animals must be according to their production. Sexual behavior must be evaluated to detect disorders, as muscle or claw lameness. Similarly, it is necessary to examine the behavior of bull and bull-cow interactions when natural breeding is carried out. Vulva, vagina, cervix, uterus, fallopian tubes and ovaries must be evaluated to diagnoses

External inspection can identify congenital or acquired anatomical defects as pneumovagina, vulvar defects, tumors or injuries. The anatomy of the area, secretions

In vaginal palpation, vulvar and vestibular functionality are evaluated though the pressure of these structures around the clinician's hand. It also assesses the presence of adhesions, abnormal structures and cervical defects. The vaginal contents should be inspected to detect urine (if urovagina), pus (if endometritis, vaginitis ...), blood (post-ovulation or some

Fig. 4. Transparent and abundant vaginal mucus indicates optimal heat and good uterine

around vulva or tail, and vulvar and vaginal coloration should be evaluated.

diseases) or clear and clean mucus (associated with heat) (Figure 4).

sprinklers, fans cooling, etc., to handle this stress (Ferreira et al., 2011).

**3. Diagnosis of RBC syndrome** 

because sometimes it is unprofitable.

**3.1 External inspection and vaginal evaluation** 

reproductive defects.

environment.

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 Plastic gloves are lubricated and then feces are withdrawn.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. Palpation helps to diagnose anomalies such as uterine infections.

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 diseases.

#### **3.3 Ultrasonography (US)**

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

Clinical Approach to the Repeat Breeder Cow Syndrome 351

urine turns to reddish if oviduct is normal. After 4 hours, the test can be repeated in the

Another diagnostic technique to check the oviductal patency is collecting oocytes or embryos, either with or without superovulation treatments. In addition, if they are collected, it is possible to transfer them to cows without reproductive problems, in which case it also

other oviduct (Figure 5).

becomes a therapeutic tool to overcome this syndrome.

Fig. 5. Phenolsulphonfthalein test to investigate the infertility in cow.

Numerous pathogens could be localized at the female reproductive tract, affecting the success of fertilization. Infectious diseases could provoke vulvitis, vaginitis, cervicitis or endometritis, and it is interesting to diagnose these disorders. Usually, uterine inflammatory disorders begin with bacterial contamination into the uterine lumen, and continue with adhesion of pathogens to the mucosa, colonization or penetration of the epithelium, and/or release of endotoxins. Uterine inflammation, even in the absence of active bacterial infection, may disrupt embryonic survival and provoke RBC syndrome. The endometrial bacteriological diagnosis is interesting to detect pathogens implicated in infertility. In cattle, especially due to the cervical anatomy, samples can be taken using a catheter connected to a syringe containing 30-60 ml of sterile saline. It is deposited into the uterus and then it is removed and cultured. Clinical or subclinical endometritis could be diagnosed. The sample

**3.6 Endometrial cytology and uterine bacterial culture** 

clitoris, the genital swellings completely disappear and the urogenital folds develop to enclose the GT, forming the labia (Quintela et al., 2011).

Other ultrasound application is the aspiration of oocytes, or "ovum pick-up" (OPU) (Pieterse et al., 1988). It is less traumatic and invasive than laparoscopy, does not affect the ovarian activity and can be given many other utilities. Among them the treatment of RBCs syndrome is the most notable, since it allows collecting oocytes that will be in vitro maturated, fertilized and cultured.

Transrectal ultrasound diagnosis has improved our ability to assess the reproductive organs in cattle and to follow the dynamic interactions between ovarian follicular cohorts. Even 2-3 mm follicles can be seen, quantified and sequentially monitored, allowing the development of superovulation regimens, an essential practice for the embryo transfer industry. US practical uses include routine assessment of follicular and luteal development, and differential diagnosis of cystic ovaries, ovarian abscesses and tumors, which can be considered factors associated with RBC syndrome. US helps to evaluate the uterus by detecting changes in shape and echotexture related with circulating hormone concentrations during the estrous cycle, and thereby detecting hormonal imbalances. It is also useful for detecting pathological conditions such as metritis, pyometra, maceration or mummification, and it is an important tool for diagnosing ovarian cystic disease (Rajamahendran, 1994).

#### **3.4 Hormonal function tests**

Sex hormones, neurotransmitters and other substances are involved in the regulation of the sexual cycle in cow. Synthesis, release, actuation or interaction imbalance of these substances could be related to reproductive alterations as repeat estrus and reduced fertility. Progesterone can be considered as a sensor of the reproductive capacity, both for its information about the estrous cycle and for its easy determination. Progesterone assay is an objective and accurate test to evaluate the ovarian function and to diagnose certain diseases that otherwise could not be correctly determined, such as delayed ovulation, persistent luteal activity, ovarian cysts or suprabasal progesterone levels (Lamming et al., 1989; Waldmann et al., 2000). When cows are in estrus, this assessment could confirm that the hormonal environment is within the correct hormonal framework. Then, if progesterone is measured around day 19-21 after AI, it could be an effective method to detect females with pregnancy failure. Radioimmunoassay (RIA) and enzyme immunoassay (ELISA) are the usual analytical techniques for determining steroids in biological fluids.

#### **3.5 Oviductal patency**

The determination of oviductal permeability is interesting, although it is difficult to carry out. An injection of 500 ml of sterile solution containing 30 gr of starch has been described by Kessy & Noakes (1979). It is intraperitoneally injected and starch reaches the oviduct and descends to the cervical mucus if the fallopian tubes are permeable. It takes about 12 hours to arrive at the cervical mucus, where it will remain between 2 and 4 days. A sample of mucus is collected, stained with lugol and observed under the microscope. Other procedure to study the oviducts individually is infusing phenolsulphonfthalein into the uterine horn. A Foley catheter is inserted through the uterus and the balloon is inflated on the end of the horn before dye is infused. If the oviduct is normal, the dye will cross it, reach the abdominal cavity, and is eliminated by urine. Bladder is catheterized 20 minutes later and

clitoris, the genital swellings completely disappear and the urogenital folds develop to

Other ultrasound application is the aspiration of oocytes, or "ovum pick-up" (OPU) (Pieterse et al., 1988). It is less traumatic and invasive than laparoscopy, does not affect the ovarian activity and can be given many other utilities. Among them the treatment of RBCs syndrome is the most notable, since it allows collecting oocytes that will be in vitro

Transrectal ultrasound diagnosis has improved our ability to assess the reproductive organs in cattle and to follow the dynamic interactions between ovarian follicular cohorts. Even 2-3 mm follicles can be seen, quantified and sequentially monitored, allowing the development of superovulation regimens, an essential practice for the embryo transfer industry. US practical uses include routine assessment of follicular and luteal development, and differential diagnosis of cystic ovaries, ovarian abscesses and tumors, which can be considered factors associated with RBC syndrome. US helps to evaluate the uterus by detecting changes in shape and echotexture related with circulating hormone concentrations during the estrous cycle, and thereby detecting hormonal imbalances. It is also useful for detecting pathological conditions such as metritis, pyometra, maceration or mummification, and it is an important tool for diagnosing ovarian cystic disease (Rajamahendran, 1994).

Sex hormones, neurotransmitters and other substances are involved in the regulation of the sexual cycle in cow. Synthesis, release, actuation or interaction imbalance of these substances could be related to reproductive alterations as repeat estrus and reduced fertility. Progesterone can be considered as a sensor of the reproductive capacity, both for its information about the estrous cycle and for its easy determination. Progesterone assay is an objective and accurate test to evaluate the ovarian function and to diagnose certain diseases that otherwise could not be correctly determined, such as delayed ovulation, persistent luteal activity, ovarian cysts or suprabasal progesterone levels (Lamming et al., 1989; Waldmann et al., 2000). When cows are in estrus, this assessment could confirm that the hormonal environment is within the correct hormonal framework. Then, if progesterone is measured around day 19-21 after AI, it could be an effective method to detect females with pregnancy failure. Radioimmunoassay (RIA) and enzyme immunoassay (ELISA) are the

The determination of oviductal permeability is interesting, although it is difficult to carry out. An injection of 500 ml of sterile solution containing 30 gr of starch has been described by Kessy & Noakes (1979). It is intraperitoneally injected and starch reaches the oviduct and descends to the cervical mucus if the fallopian tubes are permeable. It takes about 12 hours to arrive at the cervical mucus, where it will remain between 2 and 4 days. A sample of mucus is collected, stained with lugol and observed under the microscope. Other procedure to study the oviducts individually is infusing phenolsulphonfthalein into the uterine horn. A Foley catheter is inserted through the uterus and the balloon is inflated on the end of the horn before dye is infused. If the oviduct is normal, the dye will cross it, reach the abdominal cavity, and is eliminated by urine. Bladder is catheterized 20 minutes later and

usual analytical techniques for determining steroids in biological fluids.

enclose the GT, forming the labia (Quintela et al., 2011).

maturated, fertilized and cultured.

**3.4 Hormonal function tests** 

**3.5 Oviductal patency** 

urine turns to reddish if oviduct is normal. After 4 hours, the test can be repeated in the other oviduct (Figure 5).

Another diagnostic technique to check the oviductal patency is collecting oocytes or embryos, either with or without superovulation treatments. In addition, if they are collected, it is possible to transfer them to cows without reproductive problems, in which case it also becomes a therapeutic tool to overcome this syndrome.

Fig. 5. Phenolsulphonfthalein test to investigate the infertility in cow.

#### **3.6 Endometrial cytology and uterine bacterial culture**

Numerous pathogens could be localized at the female reproductive tract, affecting the success of fertilization. Infectious diseases could provoke vulvitis, vaginitis, cervicitis or endometritis, and it is interesting to diagnose these disorders. Usually, uterine inflammatory disorders begin with bacterial contamination into the uterine lumen, and continue with adhesion of pathogens to the mucosa, colonization or penetration of the epithelium, and/or release of endotoxins. Uterine inflammation, even in the absence of active bacterial infection, may disrupt embryonic survival and provoke RBC syndrome. The endometrial bacteriological diagnosis is interesting to detect pathogens implicated in infertility. In cattle, especially due to the cervical anatomy, samples can be taken using a catheter connected to a syringe containing 30-60 ml of sterile saline. It is deposited into the uterus and then it is removed and cultured. Clinical or subclinical endometritis could be diagnosed. The sample

Clinical Approach to the Repeat Breeder Cow Syndrome 353

In conclusion, it is important to consider the nutritional imbalance in RBCs, moreover in dairy cattle with higher nutritional requirements. However, it is not practical to analyze the mineral or chemical composition in blood. It is more reasonable to monitor the food rations

Abnormal implantation and transport of gametes are associated with endometrial defects, resulting in RBC syndrome. Certain assisted reproduction techniques, as in vitro production of embryos or intraperitoneal insemination, have been proposed to solve this syndrome.

Intraperitoneal insemination could be an alternative procedure to the normal deposition of semen in the genital tract of the cow (Lopez-Gatius, 1995). It is considered that between 36 and 89% of RBCs show uterine diseases but are often difficult to detect in field conditions. A bypass from vagina to peritoneal area (around the ovary) is possible to avoid the negative

Other authors (Tanaka et al., 1994) consider that the majority of embryonic abnormalities occur in the oviducts, but are not apparent until 6-7 days post-breeding. Then, oocytes of RBCs are competent to reach the blastocyst stage. IVM, IVF, IVC and ET techniques are

Disorders related to reproductive tract infections may be related to RBC syndrome and very often overlooked by clinicians. Prophylactic practices have been used, as the uterine administration of antiseptic solutions (lugol) 24 h. after mating/AI (Tanaka et al., 1994),

proposed to improve the reproductive success in these animals.

although fertility results are poor (Huszenicsa et al., 1994).

in order to avoid disruptions to the reproductive function.

effects of altered uterine environment on the sperm quality (Figure 6).

**4.2 Assisted reproduction techniques** 

Fig. 6. Intraperitoneal insemination.

**4.3 Intrauterine treatment** 

can also be taken by cytology brushes (65 cm, 4 mm Ø) protected by a sterile metal tube (50 cm, 5 mm Ø). Endometrial cytology is a practical technique to diagnose subclinical endometritis, when clinical signs are absent. The number of neutrophils indicates the type and grade of endometrial inflammation.
