Social signalling and its effect on mare reproduction
In mares, Dr Burger’s research group has shown that mares exposed to a stallion ovulate earlier in the breeding season than mares without male stimulation, and that when in season, mares show consistent preferences towards certain stallions. It is thought that the MHC or linked genes may influence equine female mate choice in a similar manner to previous observations in humans and other species. MHC-dissimilar mares and stallions have been shown to be more compatible and in fact, close proximity to a stallion with similar-MHC at the time of fertilisation; and early gestation can have a negative effect on pregnancy rates even when bred to an MHC-dissimilar stallion. The same group also showed that oxytocin levels have been shown to be significantly elevated in response to teasing, and multiple periovulatory inseminations potentially lead to an increase in fertility results per cycle, without causing increased inflammatory uterine reactions in healthy, fertile females.
Social signalling and its effect on stallion reproduction
In stallions, they found that the MHC-linked signals influence testosterone secretion and ejaculate characteristics, both indicators of male reproductive strategies. Higher testosterone levels and higher sperm numbers were recorded in ejaculates from males exposed to MHC-dissimilar mares than when kept close to MHC-similar mares.
How management strategies can increase reproductive efficiency
Dr Burger concluded firstly that exposing transitional mares to the proximity of a stallion is an easy and safe alternative to, for example, light programs or elaborated hormonal therapies, to start the breeding season earlier and to increase the number of oestrous cycles and possible matings in horses. Secondly, that optimal MHC-linked social signalling around the time of breeding has an effect of cryptic female mate choice and hence leads to better pregnancy results. Thirdly, optimal housing social conditions including the use of MHC-linked social signalling promises potential optimisations regarding sexual behaviour and semen output of stallions, and that introducing optimal MHC-linked social signalling around the time of breeding can lead to an increase of semen quantity and quality in the stallions’ ejaculates.
Applying these new and practicable options in the field may result in improved management and well-being of both breeding stallions and mares as well as a more desirable economic situation for breeders.
Stallion fertility and reproductive efficiency
The second speaker of the day was Dr Charles Love, a professor in the Department of Large Animal Clinical Sciences at Texas A&M. Dr Love is a world-renowned stallion expert and internationally recognised for his extensive and ground-breaking research in stallion fertility. He spoke on his work on record analysis systems for critically studying stallion fertility and ways in which we can improve stallion reproductive efficiency.
“There are many factors to evaluate in addition to the seasonal pregnancy rate and per cycle pregnancy rate. The breeding season (mid-February through the end of June) is a dynamic period such that the conditions (type of mare bred [barren, maiden, foaling], number of mares bred) vary considerably throughout the breeding season. For instance, a stallion’s fertility may decline in the middle of the breeding season when the number of mares he breeds increases, while early in the season he may do well. In contrast, stallions may do poorly early in the breeding season due to reproductively poor-quality barren mares, then improve once they start breeding foaling mares. The fertility of thoroughbred stallions may be affected by the cover (1-4) on which a mare was bred. A mare bred on a later cover (2-4) may receive fewer sperm than the mare on the first cover, therefore, evaluating the effect of cover can provide insight into a stallion’s fertility.”
The importance of monitoring testicular size
Evaluation of testes size and function is a fundamental part of the breeding soundness evaluation and in particular insurance examinations for first-season stallion infertility. The evaluation of sexually immature stallions that have recently retired from racing is challenging, because this can be a very dynamic period for testes growth due to the stress of racing as well as the immaturity of the stallion. Testes size can be measured by ultrasonography, and a volume measure can be attached to each testis. This provides an objective measure of the testes for all the parties involved and identifies those testes that are clearly very small and unlikely to produce sperm at the time of the evaluation. Measurements of the testes also allow the practitioner to re-evaluate a suspect stallion at a later stage to determine if the size is changing.
A common cause of stallion subfertility
Dr Love spoke about one of the most common conditions of the stallion, plugged ampullae or sperm accumulation. “This condition results when sperm back-up in the ampullae (an accessory gland located in the pelvis) and become non-viable (dead). The condition usually occurs in stallions with large testes (they produce more sperm) that have not bred since the end of the previous breeding season. Depending on the severity of the condition, the clean-out period, which requires frequent ejaculation to remove the accumulated sperm, can take from days to weeks. If stallions are bred during this time, subfertility may result because of the deposition of poor-quality sperm into the mare. Semen collection prior to the breeding season will help identify these stallions so they can be cleaned out before the start of the breeding season.”
Does reinforcement breeding improve fertility?
Reinforcement breeding occurs when a stallion finishes his cover, dismounts, and the “dripping” from the penis are collected into a container, mixed with semen extender and then passed (reinforced) into the uterus of the mare that the stallion just covered. Opinions vary as to how often this procedure should be applied (to select mares and stallions, only select covers, or all covers). One factor that plays a role is simply the number of sperm that are recovered in the dismount. The more sperm in the dismount sample, the fewer remain in the mare; thus, unless the dismount sample is reinforced, the mare may not receive an adequate “dose” of sperm. A study from Texas A&M University found that when >200 million sperm were reinforced, fertility increased almost 12% in those mares that were reinforced. Other factors that may affect the decision to reinforce include stallions that are physically limited (e.g., hindlimb/back pain, size disproportion [tall mare, short stallion]), resulting in a premature dismount and sperm deposited in the vagina or even outside the mare rather than the uterus.
Management of the barren mare for optimal fertility
Moving on to the mare, Dr Karen Wolfsdorf—a partner at Hagyard Equine Medical Institute Kentucky—gave the first talk on management of the barren mare for optimal fertility. Karen explained that “a broodmare needs to produce six foals every seven years and consistently produce viable foals to be economically successful. In order to achieve this, the mare needs to be in good physical condition, have regular oestrous cycles, mate, conceive, maintain pregnancy, give birth and raise a foal. If there is a breakdown in any of these areas, she will become considered a “problem” mare. Consideration of breeding practices and fertility of the stallion is important before all the blame of subfertility is placed on the mare. Therefore, to manage the mare appropriately, a complete reproductive examination is imperative in order to identify the potential cause of the mare’s infertility. Once this has been determined, specific therapy can be initiated. Practising appropriate breeding techniques, considering the mare’s inadequacy and then providing post-breeding treatments can aid in optimising pregnancy rates.”
Investigating the problem mare
As with any investigation, taking a thorough history of the past reproductive performance of the mare is essential. This includes age, breed, past and present reproductive status, cycling patterns, previous uterine infections and treatment, hormonal use, foaling problems, abortions and surgery.
Clinical evaluation of the mare starts by external examination. Good body condition and perineal conformation are essential for good fertility. Mares with extreme weight loss or obesity (equine metabolic or Cushing’s disease) have poor reproduction performance. The mare’s reproductive tract is composed of the vulva, vestibule, vagina, cervix, uterus, oviducts and ovaries. All work together to provide the most conducive environment for embryonic development and the birth of a healthy foal. The reproductive tract resides in the caudal portion of the body suspended from the body wall by the broad ligaments. As the number of oestrous cycles and foals produced increases, so does the laxity of these ligaments causing the reproductive tract to lie more cranial and ventral to the pelvis. Three major anatomical barriers protect the uterine environment. These include the vulva labia, vulvovaginal fold (hymen) and the cervix. Failure or compromise of these barriers leads to contamination by particulate matter, pneumovagina (sucking air in the vagina), cervicitis/vaginitis and endometritis. Perineal conformation involves the anatomic relationship between the vulva and anus. The presence of abnormalities and the degree of angular change may predispose to pneumovagina or urine pooling. Several surgical procedures to correct abnormal perineal conformation have been described to include Caslick vulvoplasty and different types of perineal body reconstruction (vestibuloplasty, Gadd and Pouret’s technique).
Palpation of the entire ovarian surface assesses size, consistency, follicular activity and the presence of an ovulation fossa. Small ovaries with little or no follicular activity as in anestrous, transition or genetic abnormalities can be differentiated from normal follicular activity. Normal ovarian consistency can be distinguished from large firm ovaries, which may be cystic in appearance related to ovarian haematomas or tumours. Examination of the uterus allows size, tone and location in the abdomen to be determined. Identifying the presence and amount of endometrial oedema as well as intra-luminal abnormalities such as fluid, hyperechogenicities, endometrial cysts or masses are also important in trying to differentiate the presence of inflammation versus other causes of infertility. Palpation and measurement ultrasonographically of the cervix allows direct correlation of length and width to stage of the oestrous cycle and pregnancy. Cervical tears must be definitively evaluated manually and during dioestrus when the cervix is closed.
Uterine culture and cytology are useful tools in the diagnosis of endometrial inflammation and an indicator of bacterial endometritis. Bacteriological (culture) and cytological results obtained by endometrial biopsy are the most sensitive indicators bearing positive predictive value (a positive result is indicative of endometritis) compared to results obtained by endometrial swab. The use of low-volume lavage is a fast and accurate method to obtain endometrial samples for identification of mares with chronic or subclinical endometritis.
Endometrial biopsies can be indicated in barren mares, repeat breeders, early embryonic death or abortion, pyometra and mucometra, genital surgery and fertility evaluation. They provide an endometrial biopsy score which is an accepted maker of uterine health and predicted fertility. The endometrium is classified according to the presence of inflammation (type and severity), endometrial gland density, peri-glandular fibrosis and nesting, cystic glandular distension, lymphatic lacunae and angiopathies or angiosclerotic changes. As the number and severity of pathologies increase, so does the category classification and thus decreasing the potential of carrying a foal to term. Hysteroscopy (direct visualisation of the uterus) is a valuable tool when uterine abnormalities need further investigation or the cause of infertility cannot be determined by other diagnostic procedures.
Endometritis: a leading cause of subfertility in the mare
One of the most commonly identified problems is inflammation of the uterus or endometritis. This can be caused by acute or chronic infections or persistent mating induced endometritis (PMIE). The most common aetiological agents found to cause uterine infections are Streptococcus zooepidemicus and Escherichia coli as the result of faecal and genital flora contamination. Mares that fail to clear the uterus of bacteria are classified as susceptible to chronic infection and are believed to have a compromised uterine defence system. This is usually identified by the presence of intraluminal fluid on ultrasound for an excessive period of time. Chronic endometritis is a major cause of equine infertility in older and multiparous mares. Normal mares are able to clear their uterus of bacteria with their uterine defence mechanisms, inflammatory response, uterine contraction and lymphatic drainage. In susceptible mares when bacteria infiltrate and contaminate the uterus, a persistent or chronic endometritis persists. In addition, some bacteria are able to utilise methods to survive degradation by the host immune system and antibiotic therapy.
Mares with chronic infectious endometritis will be treated with conventional methods such as appropriate antimicrobials (determined by culture and sensitivity) as well as uterine lavage, oxytocin and cloprostenol to assist the uterus to physically clear contaminants and inflammatory products. Most importantly, correction of perineal conformation and the breakdown of external barriers by Caslick’s vulvoplasty or cervical repair is imperative. With the recognition of increased production of mucus with inflammation and the identification of biofilms, emphasis on treatment has become focused on new alternative therapies that can potentiate the effectiveness of antimicrobials. BActivate has recently been described as aiding clearance of dormant B. strep, allowing for identification and enhanced antibiotic therapy and treatment. Treatment is usually systemic for 10–14 days as well as intrauterine during oestrus. Chemical curettage with kerosene has been demonstrated to produce glandular activation in mares, with improved conception rates in treated versus control mares as biopsy grades increased from I to III.
After identifying and resolving the cause of infertility, specific breeding strategies should be considered. A problem mare should be bred once within the oestrus period so inflammation is kept to a minimum.
Modulating the mare’s immune response in cases of PMIE
Most therapies in the past for PMIE have been directed at aiding uterine contractions and uterine clearance; however, as more research is directed toward the abnormal immune response of susceptible mares, new therapeutics are starting to address the modulation of the inflammatory response.
A significant improvement of pregnancy rates was observed when prednisolone acetate was administered to mares with a history of PMIE. When dexamethasone was administered within one hour of breeding in combination with traditional post-breeding therapies, mares with a history of fluid accumulation had increased pregnancy rates when three or more risk factors for susceptibility to endometritis were identified.
Enhancement of cell-mediated immunity by a cell-wall extract of Mycobacterium phlei intrauterine or IV (Settle; Bioniche Animal Health - Bogard, Georgia) or Proprionibacterium acnes IV (EqStime; Neogen Corp - Lexington, Kentucky) also aid in clearance of inflammation experimentally and clinically increased pregnancy rates respectively.
Intrauterine autologous plasma has been used to aid the immune response post-mating to clear spermatozoa and bacteria. More recently treatment with platelet rich plasma (PrP) has been shown to reduce the inflammatory response and improve pregnancy rates after breeding particularly in mares susceptible to endometritis.
Biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs) have been used in human and veterinary medicine for immunomodulation for over 10 years. Additional studies need to determine the effects of MSCs on problem mares affected by PMIE.
Dr Wolfsdorf concluded that in order to manage the “problem” mare we first need to determine the cause of her problem. Specific treatments have been illustrated for varying diseases however it is important to remember for best results or pregnancy it is imperative to have a non-infected, un-inflamed uterine environment into which the semen is deposited.
Managing the high-risk pregnancy
Dr Wolfsdorf’s second talk was on managing the high-risk pregnancy. A recent report estimated that the proportion of annual lost pregnancies in Thoroughbred mares ranges from 7.9% to 15.2% in the UK, Ireland and Kentucky. A recent study showed that the most common causes of pregnancy loss worldwide from 1960–2020 include EHV-1, placentitis, leptospirosis, twinning, congenital abnormalities, EHV-4, umbilical cord torsion and equine amnionitis/mare reproductive loss syndrome. When a mare becomes stressed, debilitated or her reproductive tract is compromised, inflammatory cytokines initiate prostaglandin release which can induce abnormal uterine irritability and potential pregnancy loss. Reproductive problems that arise during gestation, when diagnosed early and treated appropriately, can be overcome; and foetal viability increases, producing a live foal.
Transrectal ultrasound monitoring
In mares that are considered to be high-risk for pregnancy loss, monitoring of the uterus, placenta and foetus by screening monthly with transrectal and transabdominal ultrasonography is a viable method for detecting abnormalities early. Foetal presentation, combined utero-placental thickness at the cervical star and orbital diameter and blood flow of foetuses in anterior presentation can be assessed. Normal values for the utero-placental thickness at 271–300, 301–330, and >330 days of gestation are <8, <10 and <12 mm respectively. Qualitative and quantitative assessment of foetal fluids can be monitored by trans-rectal ultrasonography. Fluids that have increased echo density are likely to have increased cellularity due to infection or inflammation and therefore should be noted. Increased foetal movement can also falsely increase cellularity of the fluid at that period of time due to the stirring affect. Integrity of the utero-placental unit at the cervical star region can also be monitored, helping to identify opening of the cervix, placental separation, placental or uterine oedema or the presence of exudate. Since ascending placentitis is one of the most common abnormalities identified, examination of this area is imperative.
Transabdominal ultrasound monitoring
Transabdominal ultrasonography is extremely useful in evaluating for multiple foetuses, foetal growth, activity, mobility, presentation, viability as well as placental abnormalities and foetal fluid volume and echogenicity. Normal values for heart rate and rhythm, foetal activity, size, stomach measurements, cervical pole and foetal fluid depth have been determined and therefore comparisons can be made. The foetal presentation can be identified by finding the ribs and thorax usually midline and cranial to the mammary gland. Foetal heart rates can vary depending on activity, ranging from 70–100bpm with consistently low or high heart rates indicative of foetal stress or distress. Identification and examination of the umbilical cord can sometimes be assessed, depending on positioning of the foetus. It is important to always examine both sides of the abdomen completely to eliminate the possibility of twins (a differential for premature mammary gland development). Foetal activity and tone can be determined when monitoring heart rate and reaction to ultrasonography.
Biomarkers of foetal well-being
In the normal physiology of pregnancy, progesterone (P4) is synthesised by the ovaries until about 150 days of gestation. From then until 320–360 days, P5 is supplied by the foetus, which is converted into P4 by the placenta. During the second half of pregnancy, little if any P4 is present because it is rapidly metabolised into progestagens. These progestagens increase gradually during the last few weeks prior to parturition (>300 days) but decline within a few days or even hours of delivery. In the first trimester impending abortion is preceded by declining or low P4 levels. However, foetal losses or premature deliveries in late gestation, particularly those caused by placental abnormalities especially placentitis are associated with high concentrations of total progestagens. In mares where there was acute foetal distress (colic, uterine torsion) production of progestagens was dramatically reduced, indicating the importance of a healthy foetal-placental unit for progestagen formation.
In general, the predominant oestrogens during pregnancy in mares in order of magnitude are oestrone, equilin, equilenin and oestradiol-17B. These hormones are produced by precursors from the foetus, metabolised by the placenta and act primarily on the maternal uterus. Measuring total serum oestrogens or more specifically estradiol-17B should help predict foetal viability and potentially help determine if therapy is successful. Supplementing with oestrogens when total oestrogens are lower than normal is controversial at this time. Relaxin, a placental hormone, can be used as a biochemical marker of placental function and foetal well-being and as a predictor of pregnancy outcome in the horse. Serum Amyloid A has been identified to be increased in studies in which ascending placentitis has been induced. Unfortunately, this has not been translated into the farm situation in which subclinical placentitis or ultrasonographic changes are identified. In recent studies, pro- and anti-inflammatory cytokines have been identified within the different compartments of the foetal placental unit and serum of the mare. Especially oestradiol 17B and IL-6 may be of interest when commercial testing is available and not too cost prohibitive. Further studies are needed to evaluate uterine blood flow in the compromised pregnancy as well as identify a potential “diagnostic panel” for pre-clinical placentitis specific for inflammation and for the placenta.
Treating the high-risk pregnancy to improve outcomes
Treatment to help manage and support the high-risk pregnancy is directed at resolving the microbial invasion if present, inflammation, providing good or improved blood flow to the uterus, diminishing uterine contractility and improving viability of the foetus. Systemic treatment can include antimicrobials, exogenous progestagens, anti-inflammatories and rheostatic agents. If a mare has vaginal discharge and the cervix open, speculum examination and culture of the exudate yields identification and sensitivity of the organism and allows for appropriate local treatment. Specific antibiotics have been documented as crossing the placenta and achieving therapeutic values in the foetus and foetal fluids. Anti-inflammatories use consists of Flunixin Meglamine and Firocoxib. Administration of acetylsalicylic acid (aspirin) improves uterine and ovarian perfusion and increases plasma progesterone concentration, but whether this results in increased uterine or placental perfusion remains to be determined. Extrapolation and use of pentoxifylline has also been utilised for similar reasons.
Hopefully in the future, using a combination of these markers and techniques, the clinician will be able to identify abnormalities and initiate treatment early in the course of disease.”
Genetics of early pregnancy loss
Dr Mandi De Mestre, a reader in Reproductive Immunology from The Royal Veterinary College, London, spoke on her research into the genetics of early pregnancy loss. “Embryonic and foetal loss remain one of the greatest challenges in reproductive health with 5–10% of established day-15 pregnancies failing in the first two months. If they make it to two months, 5% of these pregnancies also fail to produce a viable foal. The underlying reason for these losses is variable, but ultimately most cases will be attributed to either a pathology of the mare (such as endometrial pathology, hormone function, aberrations in the immune response to pregnancy and egg characteristics) or the pregnancy itself (embryo/foetal-placental unit). Defects in the embryo can be derived from the stallion (sperm) or mare (eggs) or acquired during early embryonic development or in the case of abortion, later in pregnancy. In both cases, external factors such as pathogens, nutrients and environmental contaminants play a role, too. Whilst previous research has focused on factors that impact the environment in which the embryo develops, surprisingly little is known about defects of the embryo and foetus and how and why they occur.
In early pregnancy, the most common reason for a pregnancy to fail is an abnormal number of chromosomes. When an embryo has either one extra or one less chromosome, the whole genome of the developing foal is disrupted which impacts critical events in early development. A number of outcomes can follow, ranging from loss of the embryo before it is even detected at two weeks post cover, to development of an embryo with no foetus, or development of a foetus with an abnormal cardiovascular or central nervous system incompatible with life. This condition can also be associated with abortion and stillbirth, but it is less common in later stages of pregnancy. Other genetic changes in the embryo such as single nucleotide polymorphisms and microdeletions are well described in other species and are likely to be revealed to be important for equine foetal health in the next couple of years.
What we have learned about umbilical cord torsion
Dr DeMestre also spoke about umbilical cord torsion (UCT), which is the number one cause of abortion in the United Kingdom. “A recent study of a large population of mares in the UK and Ireland found for every 200 pregnant mares, there are three mares that lose a pregnancy due to UCT. The underlying cause of umbilical cord torsion is still being investigated, but its impact on foetal health and viability is likely related to the length of the amniotic portion of the umbilical cord and the degree of movement of the foetus between 100 and 200 days. In contrast, other causes of abortion are much less common with just three mares losing a pregnancy due to placentitis (infection of the placenta) for every 1,000 pregnant mares. A similar number of pregnancies are lost due to equine herpes virus infection and developmental defects of the foetus.
Genetics and late-term abortion
Whilst genetic causes of abortion are less common than during the early pregnancy period, they still contribute. Indeed, a single base pair change in DNA within the procollagen-lysine, 2-oxoglutarate 5-dioxygenase1 (PLOD1) gene leads to foals being born with extensive skin lesions due to abnormally thin and fragile skin, and other significant musculoskeletal abnormalities. A foal will only be affected by the disorder if it has two copies of the mutation, therefore it can be avoided with mating selection.”
