What makes a mare a difficult breeder? (Proceedings)

Horse breeding is the procedure by which an adequate number of sperm are deposited into the uterus of a "healthy" mare at the right time. Although semen deposition is performed in all breeds by natural cover (NC), it is often, and with increasing frequency done artificially. Although the main difference between natural breeding and artificial insemination (AI) is the delivery method of semen into the uterus, many factors determine the success or failure of the breeding. Natural breeding, if performed properly, should not be considered a cheap alternative to artificial insemination. Many horse breeders are of the opinion that if the mare does not become pregnant by AI, turning her out with a stallion to be pasture bred or natural mating will maximize the chances of establishing a pregnancy. In my opinion, a mare that fails to become pregnant by AI with good semen quality and does so by natural cover without human intervention, is an example of poor or inadequate breeding management by the personnel performing the AI. Our ability to differentiate between physiologic and pathologic conditions and to establish appropriate therapies is a hallmark of those of us that practice reproductive medicine, and should be the basis for proper reproductive management.

Breeding management includes: 1) Diagnostic procedures to determine the soundness for breeding of both male and female, 2) Necessary therapies pre and post breeding, and 3) Determination of the optimal time for insemination. This paper will discuss managerial procedures as well as address some common problems that veterinarians encounter when breeding mares by artificial insemination.

Stallion diagnostic procedures

Before deciding what stallion to breed a particular mare, it is important to determine whether the semen will be fresh, cooled or frozen. If cooled semen is chosen, collection and shipping schedules, as well as sperm longevity and stallion fertility need to be determined. If the mare is to be bred with frozen semen, semen quality after thawing as well as historical fertility of the frozen semen should be established. If possible, one should work with experienced laboratories that have a good reputation for freezing stallion sperm. If no information about the semen can be obtained, one may assume that the semen will be of mediocre to poor quality. Unfortunately, mare owners spend weeks, months or even years settling on a stallion that "best suits" their mare with little or no regard for semen quality. Because of this habit, veterinarians must educate owners on the pitfalls of using cooled or frozen semen, explain the added costs of these techniques and clearly present realistic pregnancy rates per attempt.

Due to the processing of semen, some of the heterogeneity of the raw ejaculate is lost, and this relatively physiologically homogeneous sample, coupled with the reduction in sperm numbers requires that the semen be deposited in the uterus close to the time of ovulation. Fertility of cooled and frozen semen from stallions ranges greatly,1,2 although the quality of frozen semen has been improving steadily over the last few years. The veterinarian serving the mare owner rarely has control of the quality of the shipped cooled or frozen semen so the breeding management prior to and after insemination is the most important tool that a veterinarian has to establish a pregnancy in a given mare.

Mare diagnostic procedures

Age and reproductive history are probably the most important pieces of information that a veterinarian needs when examining a mare. Many mare owners unrealistically expect that that their mare should become pregnant at the first cycle regardless of her age or reproductive history. These expectations are extremely common when the mare has had a long and successful performance career, is 10 years or older and is still a maiden. Clinical findings common to the older maiden mare include accumulation of uterine fluid during estrus, wide spread endometrial glandular dilation, anovulatory hemorrhagic follicles and cervical incompetence resulting in a failure to relax either due to fibrotic changes or adhesions.

Mares in their mid-teen years start to have a reduction in their fertility potential and therefore must be considered potential candidates for repeat breeding.

So how should we define a problem breeder? I consider that a mare is a problem breeder when she has been bred in two consecutive cycles with good quality semen and at the appropriate time. Signs that a mare is a candidate for potential problems could include one or all of the following: 1) Irregular inter-ovulatory intervals (too long or too short), 2) Presence of free fluid in the uterus before and/or after insemination, 3) Increased uterine edema after insemination, or 4) Presence or persistence of marked endometrial edema post-ovulation.

Inter-ovulatory Intervals

Irregular cycles are characterized by either prolonged or short luteal phases. Normal mares ovulate at 19-22 day intervals once they have entered the regular ovulatory season. The uterus releases prostaglandin at or around day 16 post-ovulation if there is no conceptus. However bacteria, microorganisms or foreign material (i.e. urine) can either prevent, delay or stimulate the uterine prostaglandin production thereby altering the normal inter-ovulatory interval. Ultrasonographic examination of the uterus for pregnancy at day 14 or 15 should be performed routinely in older mares as one can evaluate uterine health. A mare that is in estrus with a large follicle and uterine edema at day 14 or 15 post-ovulation is abnormal. Additionally a mare that has a known ovulation date and does not have a dominant follicle or uterine edema by day 18-19 should also be considered abnormal. So veterinarians are encouraged to record ovulation dates and examine the mares at these critical dates as this could be the first and most effective indication of uterine health.

Both physiologic and pathologic conditions can alter the interovulatory interval. Two examples are a) Administration of prostaglandin to shorten the normal 15-day luteal phase3 or b) The development of anovulatory hemorrhagic follicles.

a) Prostaglandin induced estrus

The use of prostaglandin or its analogues is perhaps the most common reproductive managerial tool used by veterinarians and farm managers. However, it is perhaps also the most abused and sometimes misused hormone in brood mare practice. Reduction of fertility, delay of heat, increase in twin rates, are examples of problems after prostaglandin administration. Careful case selection as well as an accurate knowledge of the day of the cycle that prostaglandin is to be given is pertinent to successful use of prostaglandin.

Mares have two and sometimes three follicular waves during the 21 day estrous cycle. A primary follicular wave is characterized by the recruitment of several follicles that eventually results in the development of a dominant ovulatory follicle. This is compared to a secondary wave, where the number of recruited follicles is less and does not necessary results in ovulation due to the progesterone dominance. Therefore at any stage of the cycle there are growing and regressing (atretic) follicles. To distinguish between growing and atretic follicles, repeated ultrasonographic examinations are needed. Even then, atretic follicles from a proceeding wave may linger and confuse the identity of newly emerging follicles. The mechanism associated with follicle selection appears to involve a developmental advantage of one follicle over another. The future dominant follicle has an increased capacity for producing estradiol, is more sensitive to follicle stimulating hormone (FSH), and an increased response to luteinizing hormone (LH) through the induction of granulosa cell LH receptors, while subordinate follicles appear to be insensitive to low systemic concentrations of FSH and have fewer numbers of granulosa cell LH receptors. Hence, subordinate follicles regress because of an insufficient ability to respond to gonadotropins. During the dominance phase (estrus) there is a continuous suppression of FSH due to the combined effect of inhibin and estradiol. The increase in circulating concentrations of both hormones primarily originates from the developing dominant or preovulatory follicle. Apart from its FSH-suppressive effects, high concentrations of estradiol, in the absence of progesterone, appear to exert a stimulatory effect on circulating concentrations of LH.10,11 High concentrations of LH during the latter part of estrus are necessary for continued growth of the dominant follicle, as well as for signaling and preparing the follicle and its contents for ovulation. However in the presence of progesterone during the luteal phase, there is a constant emergence of minor follicular waves. Depending on the mare, the follicles of the minor waves can reach preovulatory size before starting the atretic process.

Random prostaglandin treatment therefore has the potential of finding mares with large growing follicles, large regressing follicles or a population of both growing and regressing medium size follicles. Treatment of mares with a large growing follicle in most instances results in a very short treatment to ovulation interval (24-96 hrs),12 sometimes with no obvious signs of estrus when exposed to a teaser stallion. On the other hand, mares with a large regressing follicle treated with prostaglandin will often have a delayed return to estrus and a long treatment to ovulation interval, due to the length of time that it takes for a new follicle to emerge and reach preovulatory size (8-12 days). Mares that have a mixed population of follicles will display signs of heat within 3-4 days and ovulate between 5-9 days post-treatment. Therefore, in order to maximize the beneficial effects of prostaglandin use, the last ovulation date and the follicular population present at the time of treatment should be known. This is especially important when semen has to be ordered a few days in advance. Failure to monitor a mare may result in an undetected ovulation that will invariably delay her breeding or will result in an inappropriate breeding.

Breeding a mare with a large diestrus follicles is perhaps the most common cause of an untimely breeding. The decision when to breed can be frustrating if there is no means for teasing a mare to determine if she is truly in estrus. Therefore, other signs of estrus should be present before one orders semen. These include edematous folds visualized on ultrasonography, a wide, soft, short cervix and a flaccid uterus on rectal palpation, pink, moist vaginal walls and a cervix that is edematous and relaxed on the vaginal floor. A tight cervix, an acute onset of endometrial edema after breeding as well as the presence of fluid in the uterine lumen post insemination are signs that a mare may have been bred during the luteal phase. This problem is seldom encountered in mares bred by natural cover because the mare will not be receptive to the stallion when not in heat.

b) Development of anovulatory follicles

Around 12 hours prior to ovulation most dominant follicles become less turgid and change from a round to nonspherical shape. Mares become more sensitive to touch on the ovary with the preovulatory follicle. In addition to the drop in follicular pressure, there is a separation of the follicular wall and echogenic material appears in the follicular fluid Color-flow Doppler ultrasonography will permit direct, real-time assessment of the degree of vascularization of the ovulatory follicle in the mare which can be observed with power doppler around the follicular wall as an increase in blood flow.

Ovulation is the rupture of the follicular wall, resulting in a decrease in size of the antrum. In about 50% of ovulations, evacuation of follicular fluid from the dominant follicle is an abrupt process ranging from 5 to 90 seconds, with approximately 15% of the initial fluid remaining in the antrum. In the other 50%, release of follicular fluid is a slow and gradual process taking 6 to 7 minutes to evacuate all but 4% to 17% of the initial volume. Complete loss of detectable fluid from the antrum does not always happen. Ovulation may be confirmed by reexamining several minutes later and by observing a progressive loss of fluid in the follicle. Ovulation may be more difficult to detect in older mares because they have atypical ovulations.11 Highly irregular, semi evacuated, and numerous echogenic particles are common characteristics of an ovulating follicle and are observed most often in old mares

The corpus luteum (CL) formed after ovulation may appear as a uniformly white structure on ultrasonography (about 1/3rd to 1/2 of corpora lutea) or it may contain fluid within the antral cavity (1/2 to 2/3rds). A hemorrhagic CL is one that after oocyte release bleeds abundantly into the antral cavity and is not a pathologic condition. Its formation appears to be a random event and it may occur during successive estrous cycles in mares. The ratio of luteal tissue to nonluteal tissue (fluid within the antral cavity) of a newly formed corpus luteum is minimal during early diestrus and maximal during mid-diestrus The length of an interovulatory interval is similar whether the corpus luteum develops with or without an intraluteal cavity.14

Failure to ovulate properly results in the formation of anovulatory follicles in 8 to 10% of estrous cycles during the physiologic breeding season. Anovulatory hemorrhagic follicles may be large (5 to 15 cm in diameter), persist for up 1 to 7 weeks and can result in a prolonged period of behavioral anestrus and a long interovulatory interval. Specific causes of ovulation failure in the mare are not known but have been suggested to be insufficient pituitary gonadotropin stimulation to induce ovulation, abnormal estrogen production from the follicle itself, or hemorrhage into the lumen of the dominant follicle. Gastal et al16 recently compared endocrine profiles of mares that did and did not develop anovulatory follicles and found that mares with anovulatory follicles had a higher level of estrogen in the follicular fluid than mares that ovulated. No differences in FSH or LH were detected.

The incidence of anovulatory follicles increases with age. Mares 16 to 20 years old were noted to form anovulatory follicles during 13.1% of estrous cycles during the physiologic breeding season, with almost half of the mares having a recurrent anovulatory follicle during the same breeding season.

Prediction of the formation of an anovulatory follicle is difficult. Most mares develop endometrial edema and initial growth pattern of the follicle appears normal. The first clinical indication of possible development of an anovulatory follicle in the author's opinion is the failure to respond to an ovulatory inducing agent within 48-96 hrs. Echogenic particles within the follicular fluid or a strand within the follicular antrum may be visualized ultrasonographically. The follicular wall may also thicken. Anovulatory follicles may contain blood and have consequently also been called hemorrhagic anovulatory follicles. Scattered, free-floating echogenic spots within the follicular fluid detected during ultrasound examination of a dominant follicle may be a result of hemorrhage or the shedding of granulosa cells from the follicular wall. Hemorrhagic follicular fluid may form a gelatinous mass within the follicular lumen.

Over 80% of anovulatory follicles eventually luteinize, although some remain as follicular (nonluteal) structures (15-20%).16 Progesterone levels may be used to determine the luteal status of anovulatory follicles Anovulatory follicles containing a highly echogenic lumen have elevated progesterone levels. Prostaglandin treatment will result in the destruction of the luteal cells in mares with luteinized anovulatory follicles and a return to estrus. Prostaglandin treatment has no apparent effect on non-luteinized anovulatory follicles but in general, these structures are endocrinologically inactive and often do not interfere with subsequent cycles4. The majority of nonluteinized anovulatory follicles regress spontaneously in 1 to 4 weeks. Administration of human chorionic gonadotropin (hCG) or the gonadotropin-releasing hormone (GnRH) agonist deslorelin is generally ineffective in inducing luteinization. Large anovulatory follicles could interfere with the ovulation process if the mare develops a preovulatory follicle on the same ovary on the subsequent cycle.

Pregnancy does not occur if a dominant follicle becomes anovulatory because the egg remains within the follicular cavity. Mares may be bred before it is recognized that a dominant follicle is destined to become anovulatory. In addition, it may be difficult to discern between a partially luteinized anovulatory follicle and a corpus hemorrhagicum or early corpus luteum in mares that are not examined frequently (i.e., daily) by ultrasonography.

The potential of color-flow Doppler for evaluating the vascular dynamics associated with equine reproduction will be an important tool in determining the vascular patterns of primary corpora lutea as well as that of anovulatory hemorrhagic follicles. Vascular perfusion of a dominant-sized follicle can be quantified by the number of colored pixels in an ultrasonographic image so that follicle status (anovulatory or ovulatory) can be assessed and time to ovulation predicted.

Presence of free fluid

Although free uterine fluid is considered by some as a primary pathological condition, in my opinion it should be seen as a clinical sign that could indicate a primary pathology elsewhere in the reproductive tract. Uterine fluid accumulation is detected by ultrasonographic evaluation. The veterinarian's ability to detect the fluid can be influenced by the image quality and resolution of the ultrasound unit, the amount and echogenicity of the fluid as well as the location of the fluid within the uterine lumen. In addition, mares with very heavy edema will have an increased surface area in the uterus resulting in more areas for free fluid to dissipate. The pressure exerted by the veterinarian on the uterus with the ultrasound transducer while examining the mare can also result in the dissipation and inability of free uterine fluid to be detected. There are several conditions or circumstances that could lead to fluid accumulation. Some of these include: a) Inability of the uterus to contract due to age or uterine position, b) Cervical tightness or fibrosis, c) Poor perineal conformation or cervical incompetence leading to urine accumulation.

Older pluiriparous mares tend to have a uterus that becomes more dependent in the abdomen because supporting ligaments stretch from repeated pregnancies. Uterine degeneration results in decreased uterine contractions and the more ventral position of the uterus in the abdomen interferes with physical clearance. Afflicted mares tend to retain uterine fluid. This results in low grade inflammation that may escalate after breeding. Mares with cervical incompetence may also retain uterine fluid. The cervix may fail to relax in maiden mares or the cervix may become fibrotic due to it being traumatized from a previous foaling, from aging, from prolonged, chronic endometritis, or from infusion of caustic substances into the vagina or uterus. As previous discussed, mares with a diestral follicle may be inadvertently bred. These mares will invariably have a tight cervix due to high circulating progesterone concentrations. When bred, these mares will have a sudden increase in uterine edema and will accumulate uterine fluid.

Some mares with poor perineal conformation or with cervical tears may reflux urine into the cranial vagina and uterus. Vesicovaginal reflux also known as urovagina or urine pooling, is the retention of incompletely voided urine in the cranial vagina due to an exaggerated downward cranial slope of the reproductive tract. Pneumovagina from a defective vulval conformation also predisposes to the condition. Transient urine pooling, is sometimes found in postpartum mares and usually resolves after uterine involution has occurred. Clinical signs can include urine dripping from the vulva, urine scalding and a history of failure to conceive. Diagnosis is made by inspecting the vagina with a speculum during estrus. Urovagina must be differentiated from an accumulation of exudate in the vagina secondary to endometritis. Prolonged retention of large quantities of urine in the uterus with result in a thick, mucoid, discharge draining from the uterus. Ultrasonographically, urine sediment in the uterus appears as a bright hyperechoic line in the uterine lumen. Thick (0.5 to 1 cm) hyperechoic plaques in the uterine lumen may be observed ultrasonographically in chronic conditions. Severe cases of urine pooling may require a more extensive surgical correction.