How do our breeding soundness exams compare? (Proceedings)

1. The basic reproductive examination

A basic reproductive examination includes obtaining a history, conducting a physical examination, evaluating the perineal conformation, and performing a rectal/ultrasonographic exam of the reproductive tract, vaginal speculum examination and digital examination of the cervix. Ancillary aids include uterine cytology and culture, endometrial biopsy and endoscopic examination of the reproductive tract. The latter diagnostics are chosen on a case by case basis. The importance of obtaining a complete reproductive and performance history can not be over stated. Clinical signs are frequently repeated from one cycle to the next and these findings provide important information as to the primary cause of the infertility. If a reproductive case is a referral from another veterinarian, discussing previous diagnostics, treatments performed and the veterinarian's opinion about the case provides valuable information on what should or should not be pursued. All clinical findings and treatment protocols should be recorded so if the mare does not become pregnant the data can be reviewed. A one page check off sheet that includes space (history, perineal conformation, physical exam etc.) to write one's findings is helpful in ensuring that a complete examination is performed.

External exam

The general physical condition of the mare and farm management should be appraised before transrectal evaluation of the reproductive tract as systemic problems such as poor body condition, laminitis or pain may adversely affect fertility. Mares with laminitis may accumulate uterine fluid because they have limited mobility. Abnormal estrous cycle length or lack of estrous cycles during the mare's natural cyclic season may be associated with dramatic drops in ambient temperature and subsequent weight loss, pain, uterine infection, or systemic endocrine abnormality such as Cushing's Disease or metabolic syndrome. Feeding or management changes, dental care or parasite control may be needed. Race mares brought directly from the track or performance mares completing their career may not handle group dynamics of a brood mare band. Placing a maiden race mare in a small paddock with another mare may improve her ability to cope. Mares housed in northern temperate climates require more energy in January, February and March as they expend more calories in the cold. Mares that lose weight in the early spring tend not to cycle properly even when placed under 14.5 or more hours of artificial light in December.

Because most uterine infections are due to bacteria or yeast ascending through the vagina, perineal anatomy must be evaluated critically. Conformational characteristics that correlate with high fertility include a long sloping hip, a sacroiliac joint located 3 or more inches dorsal to the tail setting (as viewed from the side) and a vulva that is no more that 10 to 20° off of vertical. Mares that pool intra-uterine fluid after breeding often are flat over their croup with a tail setting level with the sacral iliac joint and a sunken anus. The perineum is best evaluated during estrus, when relaxation and elongation of the vulvar lips are greatest. The integrity of the vulvar lips, the angulation of the vulva and the location of the dorsal commissure of the vulva in relation to the pelvis need to be evaluated. If the dorsal aspect of the vulva is more than 4 cm above the pelvis, or the vulvar lips are more than 20° off vertical, air and/or feces may be aspirated into the vestibule during estrus. The lips of the vulva should be parted to determine the integrity of the vestibulo-vaginal sphincter. The sphincter is intact when the labia can be spread slightly without air entering the cranial vagina. By parting the labia, the color and moisture of the vestibular walls can be assessed. Estrus produces a glistening pink to slightly red mucosa that is uniform in color and wet. Anestrus generally is reflected by a pale, dry mucosa; dark-red, muddy color or a splotchy color suggests inflammation. A white, tacky mucosa indicates progesterone dominance.

The perineal body may be defective in older, pluriparous mares. Damage may occur after repeated foalings, delivering a large foal, after a dystocia, or from poor reproductive conformation. The integrity of the perineal body can be assessed by placing one finger into the rectum (usually the second finger) and the thumb into the vestibule. A vestibuloplasty or "deep caslick's" may be required to prevent contamination of the anterior reproductive tract especially if the vestibulovaginal seal is lost.

Rectal/ultrasonographic examination of the reproductive tract

Repeated ultrasonographic evaluation of the reproductive tract during the estrus cycle is routine in many reproductive practices. Correlations between uterine edema, cervical dilation and follicle dynamics should be made and inconsistencies such as excessive edema early in estrus (lack of cervical dilation or endometritis), a narrow long cervix during estrus or free intra-luminal fluid (uterine drainage problem or endometritis) need to be noted. With the advent of ultrasonography, veterinarians can time breeding more closely with ovulation and can identify pathologies such as anovulatory follicles, granulosa cell tumors, intra-luminal fluid and uterine cysts. However, ultrasonography does not replace good manual skills. The tone and consistency of the uterus and dominant follicle and the length and width of the cervix can only be identified by a veterinarian skilled in rectal palpation. Inconsistencies such as a turgid, firm uterus during estrus or a narrow, long cervix in a mare on the day of ovulation indicate problems such as endometritis and cervical incompetence. A grading system for uterine edema, fluid accumulation and follicular dynamics should be developed so veterinarians within a practice can follow up on each other's cases if needed. Reviewing the ultrasonographic findings over a number of cycles in an infertile mare can provide valuable information on possible causes of the infertility.

Vaginal speculum

Vaginal speculum examination is an integral part of the breeding soundness examination. Findings include determining the integrity of the vestibulo-vaginal sphincter, identifying abnormal fluids in the vaginal vault such as pus or urine, finding a discrepancy between cervical relaxation and stage of estrous cycle or finding a cervical tear or cervical adhesion to the vaginal fornix. In addition, vaginal varicosities or an imperforate hymen may be identified. Mares with endometritis may have an edematous red cervix that is located midway up the vaginal wall. Maiden mares with cervical incompetence may have a pale, puckered, closed cervix that is located high on the vaginal wall during estrus. Color of the vaginal walls and cervix should be noted as color is correlated with stage of cycle or inflammation.

Cervical examination

Cervical tears or fibrosis. Proper cervical function is needed for a successful pregnancy. Trauma that results in a cervical tear, fibrosis or adhesions of the external os to the vaginal fornix can render a mares infertile. Afflicted mares frequently accumulate intra-uterine fluid and have micro-organisms isolated repeatedly from their uterus, even after repeated treatment with uterine lavage, intra-uterine antibiotic or anti-mycotic drugs. Some mares can clear the infection during winter anestrus, only to have the infection flair in the spring once the mare starts to exhibit estrous behavior. Cervical defects or adhesions between 4 and 8 o'clock are the most difficult to identify as vaginal exams are most commonly conducted during estrus when the cervix is lying on the vaginal floor. These lesions tend to be associated with chronic uterine infections as the cervix is pulled open ventrally during diestrus allowing bacteria access into the cervical canal and uterus. If a cervical defect is suspected during estrus, the cervix should be re-examined when the mare is under the influence of progesterone. A digital exam of the external os and the entire length of the cervical canal should be conducted. Fibrotic lesions associated with a previous foaling injury may be identified during rectal palpation of the cervix and reproductive tract. The cervical canal may be bent laterally to the right or left, likely as a result of tearing and subsequent scarring of the muscle on one side of the cervical canal during foaling. A common history in mares with cervical fibrosis is delivery of a large foal, manual assistance during foaling, dystocia or abortion in late gestation.

Determining what lesions need to be repaired surgically is based on clinical history-persistent uterine infections or pregnancy loss-and the clinical experience of the veterinarian. Cervical tears longer than one third the length of the canal usually need to be repaired to ensure an adequate seal. Treatment for cervical adhesions (adhesion of the external os to the vaginal fornix) must be determined on an individual basis as their removal may result in additional adhesion formation. The length of time needed between surgical repair and breeding varies. If the tear has occurred within the last 60 days, fibrosis and chronic infection are less severe than if the lesion is 2 to 3 years old. The former group of mares may be bred 3 to 4 weeks after surgery, whereas, the latter group will require 45 or more days for the edema and inflammation to subside. In addition, long standing cervical lesions are commonly associated with chronic bacterial or fungal endometritis. The infection may flair after a cervical repair because air and blood contaminate the uterus during surgery. The cervix tends to lose its pliability after surgery and a stricture may form within the canal at the cranial aspect of the repair. These complications can delay physical drainage of the uterus. Therefore, mares that have undergone a cervical repair should be treated after breeding with uterine lavage, ecbolics and possibly intra-uterine antibiotics. Mares mated by natural service should be re-enforced with semen after the horse dismounts. A surgically repaired cervix will likely tear again when the mare foals so cesarean section is recommended.

Inadequate cervical dilation during estrus. Incomplete dilation of the cervical canal during estrus is most commonly seen in older (> 10 yrs of age) maiden mares retired from a performance career. Clinical impression is that it occurs more commonly in Saddlebreds and Morgan mares. In some cases, mares may present with a partial relaxed external os but the cranial aspect of the canal and the internal os will be tightly closed. In other cases, the entire cervical canal is tight and it may be difficult to pass a finger through the cervix. Incomplete dilation results in chronic endometritis, fluid retention in the uterine body cranial to the cervix, and decreased pregnancy rates. As most mares are bred artificially with cooled or frozen semen, an adequate amount of semen can be deposited in the uterus as there is not great difficulty in passing an artificial insemination pipette through the cervical canal. However, these mares tend to retain intra-uterine fluid and if not treated promptly after breeding for fluid retention, they can have decreased embryo recovery rates, poor quality embryos recovered or decreased 14 day pregnancy rates. If mares are bred by natural service, there is the additional problem of decreased semen deposition within the uterus.

Cervical fibrosis results from trauma at foaling, severe metritis or infusion of a caustic drug or chemical into the uterus. Diagnosing cervical fibrosis can be difficult and many times requires a number of examinations before the diagnosis can be made. A fibrotic cervix is narrow, not pliable and is snug around a finger. In some mares, the canal can be tortuous and may bend laterally, dorsally, or ventrally. Some defects can be identified on rectal palpation when the mare is in diestrus as the cervix may bend laterally by 20 to 45 degrees. The reproductive history may include delivery of a large foal with or without assistance. The mare may deliver a few foals after the initial trauma before she experiences chronic endometritis. The repeated stretching of the cervix at foaling will cause tearing of the fibrotic tissue and additional scarring. Repeated embryo transfer attempts over a number of years may also result in cervical fibrosis. Histological descriptions of cervical pathology are limited because it is difficult to properly prepare cervical tissue for histological evaluation.

Mares with cervical fibrosis may develop uterine infections that require prolonged intra-uterine and systemic treatment, especially if the lesion is more than a year old and the mare has been bred repeatedly before the defect is diagnosed. Once the infection is cleared, sexual rest for 30 or more days is recommended so that the chronic uterine inflammation can resolve. Aggressive post breeding management may be required to assist with physical uterine clearance. We recommend uterine lavage between 4 and 8 hrs after breeding, repeated use of ecbolics and intra-uterine antibiotics in the first 48 hrs after breeding. Systemic antibiotics around the time of breeding and a single dose of dexamethasone (40 mg IV) at the time of breeding, or infusion of 30 ml of N-acetylcysteine 24 h before breeding have also been used.

2. Laboratory analyses

Introduction

In addition to the physical examination, laboratory diagnostics: uterine cytology, culture and endometrial biopsy, are extremely helpful in identifying the cause of infertility. Swabbing the endometrium on the 1st or 2nd day of estrus is the most commonly used method for identifying Thoroughbred mares with endometritis, however, recent work from Denmark indicates that a uterine culture swab may not identify all uterine pathogens. Neilsen (2005) reported that only 38 of 84 mares (45%) with bacteria isolated from the surface of an endometrial biopsy, had bacteria isolated from a uterine culture swab.

Uterine culture swab and endometrial cytology

Evaluation of cytological specimens in conjunction with endometrial swabs has been promoted since the early 1980s as a technique that enhances the accuracy of endometrial culture in identifying mares with endometritis. Correlation between the presence of neutrophils in cytological specimens and the isolation of uterine pathogens is high when a sample is collected with a guarded uterine swab. Isolation of a microorganism from the uterus of a mare during estrus in the absence of cytological evidence of endometrial inflammation has been considered to be a contaminant. However, recent work indicates that some of these tenets may not be correct. Riddle et al evaluated 2123 endometrial cytology and culture specimens collected concurrently with a guarded uterine culture instrument from 970 mares (738 barren, 1230 foaling and 155 maiden mares) during the 2001-2004 breeding seasons and compared findings to 28 day pregnancy rates.

Fifteen hundred and thirty seven of the 2123 (72.4%) paired cytology and culture samples evaluated were normal (0-2 neutrophils and no bacteria isolated). Four hundred twenty three cytology samples were positive for inflammation (> 2 neutrophils) while 231 cultures had microorganisms recovered. Sixty four percent of the positive cultures (147/231) had inflammation on cytology smears. Pregnancy rates per cycle in mares with positive cytology or culture results were 50 or more percent lower than mares with normal cytologies and no bacteria isolated. Lowest pregnancy rates were recorded for mares with severe endometrial inflammation (23%). Isolation of gram positive organisms was associated with more positive cytologies than isolation of gram negative organisms.

Based on these data, positive cytologies (set at >2 neutrophils) were twice as common as positive cultures. Degree of inflammation was more important in diagnosing infertility than mere presence or absence of inflammation (set at > 2 neutrophils). Isolation of microorganisms was associated with reduced pregnancy rates, even in the apparent absence of inflammation. Our findings indicate that if one solely relies on culture results 55% of mares with decreased fertility due to inflammation may be missed. Conversely if one solely relies on cytology results, 17% of mares with decreased fertility due to inflammation may be missed. Our findings contrast with the dogma that if bacteria are isolated from the uterus of mares with normal cytological findings, the organism should be considered a non- pathogen as mares in our clinical investigation had lower pregnancy rates than control mares.

Small volume uterine flush for uterine cytology and culture

Obtaining a uterine swab from the uterine body during estrus may not be the most accurate method for evaluating the bacterial status of the uterus as the swab only comes in contact with a small area of endometrium. This may be especially true in older mares with a pendulous uterus as fluid pools in the uterine horns. Flushing the uterus with a small volume of sterile saline or lactated ringers identifies a higher number of subclinical infections because the fluid infused into the uterus comes in contact with the majority of the endometrium. Uterine problems can be identified immediately as the efflux collected from mares with infections or severe inflammation is cloudy or has strains of particulate matter. We have modified the technique first described by Dr Barry Ball (1986; Theriogenology) for ease of use in the field. A 150 ml bag of saline is attached to the end of a uterine catheter and the bag is squeezed to express the fluid. The operator then removes his arm from the vagina and performs a rectal examination. After the uterus is sufficiently balloted the fluid is drained back into the bag. Efflux can then be poured into a clear 50 ml conical tube and assessed for fluid clarity. Cloudy efflux or efflux with strains of mucus is associated with isolation of bacteria or inflammation. In order to perform a culture and cytology, the efflux is allowed to settle for 1 or more hrs or the tube can be centrifuged for 10 min. If centrifuged the supernatant is discarded, the pellet at the bottom of the tube is cultured and a cytology smear is made by placing a second sterile swab in the pellet and rolling the swab on a glass slide.

Between June of 2004 through July 2006, we collected 401 small volume uterine flushes from 308 chronically infertile mares to diagnose endometritis. Mares evaluated were either barren after 3 or more breedings or had two or more unsuccessful embryo recovery attempts during consecutive cycles. Culture results were compared with cytological and histological findings, efflux clarity and pH to substantiate that the micro-organisms recovered were truly pathogens. Cytological specimens were evaluated for presence of epithelial and inflammatory cells, bacteria, yeast and debris. Endometrial biopsies (n = 110) were examined for the presence of neutrophils in the stratum compactum. Micro-organisms were recovered in 282/401 (70%) of low-volume flushes; E coli was most frequently isolated (42.2%), followed by β hemolytic Streptococcus (37.6%). Efflux clarity of 318 flushes was clear (n = 109), cloudy (n = 149) or mucoid (n = 60). Isolation of micro-organisms was highly associated with cloudy and mucoid effluxes (P < 0.001), debris on cytological specimens (P < 0.001), increased efflux pH (P < 0.003), and neutrophils on endometrial biopsy (P < 0.01). E coli was associated with debris on cytological smear whereas β hemolytic Streptococcus was associated with increased efflux pH. Using the presence of neutrophils in a tissue specimen as the "best standard" for diagnosing endometritis, the sensitivity of flush culture was 0.71 and for flush cytology was 0.8, whereas the specificity was 0.86 and 0.67, respectively. Neutrophils in uterine flushes under-reported inflammation; only 86/282 positive cultures were positive on cytology. The clinical estimate of a contaminated (false positive) flush culture was 11%, if a false positive was defined as positive culture with clear efflux and no debris or neutrophils on cytology (26/228).

Flush culture was twice as sensitive as swab culture estimated by Neilsen (0.71 versus 0.34) when the same "best standard" was used (presence of neutrophils in a tissue specimen). Therefore, flush culture doubled the ability to detect infected mares based on culture alone. The improved sensitivity appeared to result from improved detection of gram negative organisms as recovery of β hemolytic Streptococcus from uterine flush did not differ from previous reports nor from swab results obtained from Thoroughbred mares in central Kentucky (38% of positive flush cultures; 39% of positive swab cultures from RREH medical laboratory during the years 2005 and 2006). The technique is a rapid, accurate and practical method for diagnosing endometritis in chronically infertile mares based on sensitivity and specificity estimates approaching 0.75 and 0.9, respectively.

Endometrial biopsy

Endometrial biopsies provide valuable information on the integrity of the endometrium. However, endometrial biopsies have lost favor as a diagnostic technique because the current grading system rarely provides a change in management strategy. In addition to evaluating the degree of inflammation, fibrosis and gland density, endometrial biopsies should be evaluated for the degree of endometrial mucus, vacuolization of epithelial cells lining the endometrium, degree of endometrial edema, lymphatic lacunae and/or angeosis as all these findings will influence treatment strategies.

3. Endoscopy

Endoscopy is a helpful tool in identifying mares with uterine adhesions, foreign material in the uterine lumen, endometrial cysts, fetal remnants, endometrial tears and fungal or bacterial plaques. It is reserved for select cases and is usually conducted during an estrous cycle that the mare will not be bred or after the breeding season as infusion of air into the uterus can be irritating. The endoscope should always be properly cold sterilized for a minimum of 20 minutes to prevent infusion of bacteria into the uterus. The outside of the endoscope and the port lines are rinsed with sterile saline before placing the scope into the uterus. The procedure is best performed when the mare is in diestrus as air or fluid will drain into the vagina through the open cervix during estrus making it difficult to visualize the endometrium. The mare should be sedated as the procedure can be painful if air is used to fill the uterus. We have attached an aerator from a fish tank to the endoscope so that the uterine horns and body can be quickly dilated. A line is attached to the aerator and that line is inserted into the port from which instruments are passed. Air is infused into the uterine lumen until the endometrium can be visualized, then the air line is disconnected to prevent over filling. Residual air is removed after the procedure with either a pump or by placing a large bore catheter such as a Bivona catheter in the uterine lumen and allowing the air to be expelled. The uterus should be lavaged with saline or lactated ringers after the procedure as the air is irritating to the endometrium. Antibiotics may be infused in some cases for 2 to 3 days to prevent bacterial endometritis.

References

Blanchard TL, Varner DD, Schumacher J et al. Manual of Equine Reproduction 2nd ed. St Louis, MO: Mosby, 2003; 31-58.

Pycock JF. Breeding management of the problem mare. In: Samper JC, ed. Equine Breeding Management and Artificial Insemination. Philadelphia: WB Saunders Co, 2000; 195-228.

Nielsen JM. Endometritis in the mare: a diagnostic study comparing cultures from swab and biopsy. Theriogenology 2005;64:510-518.

Riddle TW, LeBlanc MM, Pierce SW et al. Relationships between pregnancy rates, uterine cytology, and culture results in a Thoroughbred practice in central Kentucky, in Proceedings. Am Assoc Equine Pract 2005; 51: 198-201.

LeBlanc MM, Magsig J, Stromberg AJ. Use of a low-volume uterine flush for diagnosing endometritis in chronically infertile mares. Theriogenology 2007; In Press.

Kenney RM. Cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early embryonic death. J Am Vet Med Assoc 172:241-262, 1978.