ARS - Get ready for the breeding season: reproductive evaluation of the stallion

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Get ready for the breeding season: reproductive evaluation of the mare

By Patrick McCue, DVM, PhD, Diplomate American College of Theriogenologists
Colorado State University

A reproductive evaluation is recommended for all broodmares before they are turned out in a pasture breeding program, before they go to the breeding shed in a live-cover program, or before they are inseminated or semen is ordered in an artificial insemination program. It is common sense to make sure that the mare is cycling and does not have any reproductive issues that would potentially limit her fertility before the breeding season starts.

The goal of a mare reproductive evaluation is to identify known or potential reproductive abnormalities and to evaluate the potential for a mare to become pregnant and carry a foal successfully to term.

Depending on the reproductive history, clinical situation, value of the horse and/or goals of the owner, a reproductive evaluation may very basic (i.e. just an ultrasound examination with or without a uterine culture) or may include a battery of diagnostic tests (Table 1).

Table 1. Diagnostic procedures that may be included in a reproductive evaluation on a mare.

Urospermia

Diagnostic Procedures
Reproductive History	Vaginal Speculum Examination	Hysteroscopy
General Physical Examination	Digital Cervical Exam	Endocrine Profile
Perineal Conformation	Uterine Culture	Karyotype
Teasing Behavior	Endometrial Cytology	Laparoscopy
Palpation	Endometrial Biopsy	Oviductal Patency Test
Ultrasonography	PCR for Bacterial/Fungal DNA	Test Breed

Reproductive History

All mares should be properly identified, and the registration name, registration number, microchip number, horse breed and birth date recorded. Accurate drawings of markings and tattoos should be recorded and photographs taken (if needed) to identify the horse.

A complete breeding history should be obtained, including current reproduc¬tive status (maiden, barren, pregnant or foaling), number of cycles bred during the last season, date of last breeding, breeding technique used (artificial insemination, natural cover or pasture breeding), number of stallions utilized previously, date of last foal, number of previous foals and any previous history of abnormal estrous cycles, uterine infections, embryonic loss or abortion. Any and all information that may be pertinent to the physical health or reproductive health of the mare should be obtained and recorded in the medical record.

Physical Examination

A general physical examination should be performed in addition to the reproductive examination to assess whether or not the mare has the capacity to carry a foal to term. The evaluation should include, but is not limited to, examination of the oral cavity, eyes, and respiratory, cardiac and musculoskeletal systems. In addition, diet and body condition should be evaluated. Brood¬mares do not have to be fit for athletic competition, but they should not be severely over¬weight or underweight. It may be prudent to screen older mares with reproductive problems for pituitary pars intermedia dysfunction (PPID; Equine Cushing’s Disease).

Perineal Conformation

The external genitalia (vulva) should be evaluated for conformation and muscular tone. The vulva is the first physical barrier for prevention of contamination of the reproductive tract by pathogenic organisms. The optimal perineal conformation consists of a vulva in a nearly vertical position with at least 70% of the vulva ventral to the brim of the pelvis. Horizontal sloping of the vulva dorsal to the pelvic brim secondary to recession of the anus and/or poor muscular tone to the labia of the vulva may predispose the mare to an ascending infection of the uterus.

The muscular tone of the vulva should be sufficient to prevent aspiration of air into the vestibule or vagina, a condition commonly referred to as windsucking. Muscular tone of the vulva is evaluated by placing fingers on each side of the vulva and providing firm continuous pressure laterally. Application of additional lateral pressure can be used to evaluate the integrity and tone of the vestibulo-vaginal seal. A mare with a vulva that opens easily with minimal digital pressure (i.e. poor muscular tone) may tend to aspirate air and bacterial organisms into the reproductive tract.

Prevention of bacterial contamination of the reproductive tract due to abnormal perineal conformation or poor muscular tone of the vulva may be attained by performing a minor surgical technique termed an episioplasty or Caslick procedure.

Estrus Detection

The mare should be teased with a stallion that exhibits good libido in order to successfully evaluate estrous cycle stage. Adequate time should be taken to allow shy or nervous mares to express behavioral estrus. Maiden mares may not show heat well and foaling mares may not show heat at all unless the foal is restrained and safely away from the stallion.

Teasing mares in a group may seem easy, but is often not an accurate means of determining correct behavioral status and cycle stage of all mares in the paddock or pasture. Often the only mares that come to the fence or tease rail are assertive mares in heat or mares that want to attack the stallion. One may not be able to determine the heat status of mares that remain a distance from the stallion. It is generally more effective, but certainly more time consuming to tease mares individually.

There are many systems used for teasing mares, including chutes, rails, fences, pens and paddocks. Keys to successful teasing are patience, persistence and knowing the behavioral characteristics of each mare.

Estrus. Estrus refers to the period during which a mare is receptive to the advances of the stallion. Estrus typically lasts 5 to 7 days and is stimulated by elevated estrogen levels produced by developing follicles. Common behavioral responses of mares in estrus when teased with a stallion include raising of the tail, passive urination, repeated eversion of the labia with exposure of the clitoris (winking), and assuming a mating posture with the back legs in a base-wide stance)

Diestrus. Diestrus refers to the period during which a mare rejects the advances of the stallion. Progesterone produc¬tion by the corpus luteum controls the behavioral responses of the mare during the 14 to 16 day diestrous period. When teased to a stallion, mares in diestrus will pin their ears back, kick, switch their tail, vocalize and attempt to either actively move away from the stallion or to aggressively attack the stallion.

Transrectal Palpation

The goal of manual transrectal palpation is to identify significant features of the reproductive tract, determine stage of the estrous cycle and identify potential problems. Palpation is often performed in conjunction with transrectal ultrasonography. It should be noted that manual palpation can identify some features of the tract that cannot be detected by ultrasonography. These include tone in the uterus and cervix, softness of an ovarian follicle and sensitivity of the ovary to touch and pressure. In addition, manual palpation is useful to confirm the presence of a parovarian cyst, which may have the appearance of an ovarian follicle on ultrasonography.

Ovarian structures that may be noted during palpation include developing and preovulatory follicles, a fresh or recent ovulation, and an early developing corpus hemorrhagicum. It should be noted that a mature corpus luteum cannot be detected by transrectal palpation in the mare. The uterus is generally large and edematous during estrus and has a characteristic ‘doughy’ feel. The cervix of a mare in heat is generally soft and relaxed. After ovulation, the increase in progesterone causes the uterus and cervix to increase in tone.

Ultrasonography

Ultrasound is used in broodmares to visualize structures in the reproductive tract that cannot be discerned on transrectal palpation, early diagnosis of pregnancy, diagnosis and management of twins and detection of ovarian or uterine pathology. Ultrasonography is also an excellent technique for monitoring follicular development, predicting and confirming ovulation, and determining the presence or absence of a corpus luteum.

As with transrectal palpation, the technique of transrectal ultrasonography should be consistent and thorough. One method would be to begin at the bifurcation of the uterus, examine one uterine horn, then examine the ovary adjacent to that horn, move back along the same horn to the bifurcation, examine the opposite uterine horn, followed by the adjacent ovary, then back again along the uterine horn and finally examine the uterine body.

Abnormalities detected on ultrasonography of the uterus include the presence of free fluid within the uterine lumen, endometrial cysts, twins, pregnancy loss, and occasionally foreign bodies (i.e. tip of a uterine culture instrument, marble, etc.) and other issues.

Vaginal Speculum Examination

A vaginal speculum examination is performed to evaluate the vaginal vault and the external os of the cervix, determine the stage of the estrous cycle, and to detect pathologic conditions such as urine pooling, cervical discharge and trauma.

The external os of the cervix is identified and notations made regarding location, shape, tone, color and presence of secretions. The presence or absence of urine in the cranial vagina is also recorded. The visual examination is continued as the speculum is withdrawn to identify abnormalities of the caudal vagina and vestibule.

Digital Examination of the Cervix

After the speculum examination is completed, the cervix may be examined manually for patency and the presence of adhesions, lacerations or other abnormalities.

Endometrial Culture

Culture of the uterine lumen is usually done in conjunction with cytology for the diagnosis of uterine infection. Endometritis can be suspected in a mare that exhibits an abnormally short duration between estrous periods (i.e. short-cycling), has a vaginal or cervical discharge, an inflamed cervix on speculum examination, echogenic fluid in the uterus detected on ultrasound, and possibly failure to become pregnant when bred to a fertile stallion. Ultimately, infectious endometritis is a significant cause of reduced fertility in broodmares.

Cultures may be performed prior to the onset of the breeding season, during the breeding season, at the conclusion of the breeding season or at any time as part of a breeding soundness examination. In many instances, a stallion manager may require that a mare be cultured and determined to be ‘clean‘ (i.e. have a negative culture) prior to allowing a live cover or prior to shipping semen. An important reason for culturing a mare prior to breeding is that the odds are very low that a mare will become pregnant or stay pregnant with an active uterine infection.

Cultures may be collected using a double guarded swab or by low-volume uterine lavage. The bacteria most commonly recovered from the uterus of mares are presented in Table 2. Streptococcus equi subsp. zooepidemicus is the most common bacterium isolated from the uterus of mares, with E. coli the second most common organism cultured. Approximately 30 to 40 % of samples yield growth of more than one bacterial organism.

Table 2. Prevalence of bacteria recovered from the uterus of mares.

Organism	Prevalence (%)
Streptococcus equi subsp. zooepidemicus	40 to 60
Escherichia coli	20 to 40
Pseudomonas aeruginosa	2 to 5
Klebsiella pneumoniae	2 to 5
Miscellaneous organisms	5 to 10

Fungal infections of the equine uterus are not common. The yeast Candida albicans is the most common fungal organism cultured and often associated with older mares with a history of chronic reproductive problems.

As a final note, culture results prior to the breeding season are not necessarily representative of the same mare months later after she has been bred multiple times over several estrous cycles. It may be necessary to re-culture a mare periodically during a breeding season to accurately monitor uterine health.

Endometrial Cytology

Cytologic evaluation of the uterus involves the collection and interpretation of cells lining the uterus (endometrium) and within the uterine lumen. Cytology is used in conjunction with culture and biopsy in the diagnosis of endometritis. Advantages of endometrial cytology for diagnosis of endometritis include the ease of sample collection and rapid availability of results. Endometrial cytology is one of the least expensive and most useful techniques routinely used to detect the presence of endometritis. Samples for cytologic evaluation can be obtained using a double guarded uterine brush or uterine swab or by low-volume uterine lavage.

Endometrial Biopsy

The endometrial biopsy technique involves collection of a small sample of the uterine lining (endometrium) for histologic evaluation. It is primarily used as an aid in the diagnosis of uterine disease and as a prognostic indicator of the ability of a mare to carry a foal to term.

The primary indications for the collection of an endometrial biopsy include:

§ Prepurchase fertility evaluation of a potential broodmare.
§ Repeat breeders or barren mares.
§ Mares with a history of pregnancy loss or abortion.

Other indications for collection of an endometrial biopsy include mares with abnormalities of the repro¬duc¬tive tract detected on palpation or ultra¬sound, mares that fail to cycle during the physiologic breeding season, mares requiring urogenital surgery, and mares recovering from severe uterine infections.

Endometrial biopsy samples are examined for the presence of inflammatory and degen¬erative changes. Inflammation is recog¬nized by the accumulation of inflammatory cells, primarily lymphocytes, in the endometrial tissue. Inflammation represents a potentially treatable pathologic condition. Endometrial glandular degeneration is most often recognized by a deposition of collagen in the form of fibrosis or scar tissue around the endometrial glands. In contrast to inflammation, fibrosis represents a permanent, untreatable pathologic condition.

The endometrium is classified on a I-III grading scale based on biopsy characteristics (Table 3). Grade I endometrium is essentially normal, with minimal inflammation or fibrosis. Grade III endometrium includes severe inflammatory and/or fibrotic changes. Grade II is a broad category encompassing all pathologic levels between Grades I and III. It is possible for an improvement in endometrial grade if the inflammation present is reduced or eliminated with proper treatment.

The endometrial biopsy is often used to determine the prognosis for a mare to carry a foal to term. Expected foaling rates for each endometrial biopsy category are given in the following table. It must be emphasized that many additional factors, such as stallion and broodmare management, also play critical roles in conception and maintenance of pregnancy.

Table 3. Expected foaling rates of mares according to endometrial biopsy grade.

Category	Degree of Change	Expected Foaling Rate (%)
1	Absent	> 80
2A	Mild	50 to 80
2B	Moterate	10 to 50
3	Severe	< 10

Hysteroscopy

Hysteroscopy is a technique of direct visualization of the interior of the uterus performed using a videoendoscope. The endoscope may be used to detect intrauterine adhesions, cysts, and foreign bodies, as well as inflammation, fibrosis and other abnormalities.

The major obstacle preventing widespread use of this technique is the high cost of the equipment. Consequently, endoscopy is used primarily in specialty practices and referral hospitals. Most pathologic changes, such as inflammation, infection and fibrosis, can be diagnosed based on a combination of cytology, culture and biopsy. Endometrial cysts can be visualized utilizing ultrasound. Uterine adhe¬sions, however, are difficult to diagnose with any procedure other than endoscopy of the uterine lumen.

Endocrinology

Analysis of reproductive hormones in the non-pregnant mare is most commonly performed to evaluate corpus luteum function and to diagnose ovarian abnormalities (Table 4). Progesterone concentrations of < 1 ng/ml indicate an absence of luteal tissue, while concentrations > 1 ng/ml indicate the presence of an active corpus luteum. Anti-Müllerian hormone (AMH), inhibin and testosterone may be measured to determine if an ovarian granulosa cell tumor (GCT) is present. Concentrations of equine chorionic gonadotropin (eCG) may be evaluated to determine if retained endometrial cups are present in a mare with abnormal estrous cycles.

Table 4. Endocrine values in normal non-pregnant mares.

Hormone	Normal Range
Progesterone	< 1.0 ng/ml in estrus or anestrus > 1.0 ng/ml in diestrus (i.e. corpus luteum present)
Testosterone	20 to 45 pg/ml
Inhibin	0.1 to 0.7 ng/ml
Estradiol	5 to 45 pg/ml
Anti-Müllerian Hormone	0.1 to 3.8 ng/ml

Other Tests. The standard examination procedures in the mare breeding soundness examination may not identify the cause of subfertility. Consequently, other examinations may be indicated (Table 5).

Table 5. Diagnostic tests that may be performed in addition to the standard tests in a mare reproductive evaluation.

Test	Indication
Karyotype	Analysis of the chromosome number and structure; may be performed on blood, hair or tissue samples by a cytogenetics laboratories. Performed on mares with a history of primarily infertility or repeated embryonic loss.
Laparoscopy	Direct visualization of the serosal surface of the ovary, oviduct and uterus and the abdominal cavity. Also used in ovarian biopsy, evaluation of oviductal patency and the application of prostaglandin E2 to the oviductal surface.
Oviductal flush	Performed by laparotomy or laparoscopy; used both diagnostically and therapeutically in suspected cases of oviductal blockage.
Oviductal patency test	Deposition of fluorescent microbeads or starch granules within the infundibulum and subsequent examination of the uterine lumen for passage of the test material is used diagnostically to evaluate oviductal patency.
Oviductal PGE₂ application	Direct application of prostaglandin E₂can be used diagnostically and therapeutically in suspected cases of oviductal blockage
Ovarian Biopsy	Laparoscopic collection of an ovarian biopsy sample for histologic evaluation may be used in the diagnosis of ovarian pathology.
Test breed	Breeding to a highly fertile stallion can be used diagnostically to help determine if the mare is a cause of subfertility or infertility

Summary. Interpretation of the results should take into account the mare age, reproductive history, breed, breeding management and other factors. Ultimately, the goals are detect abnormalities that may be associated with reduced fertility, provide information for treatment or management options and to determine the potential for fertility in the future. It is important to emphasize that a mare reproductive examination is only an evaluation of potential fertility and that the true assessment of fertility is based on the ability of a mare to conceive and carry a foal to term.