Advances in Stallion and Mare Fertility - EquiManagement on Audio

0:00All the articles you have come to love in Equamanagement Magazine are now available on the Equamanagement on Audio podcast. Each article is released as its own separate episode to make them quick and easy to listen to. Just search Equamanagement on Audio on your favorite podcast player to listen to all the latest articles. Advances in stallion and mere fertility. Exploring impaired acrosome exocytosis and uterine fluid to improve our understanding of subfertility. Unlike other livestock species such as cattle, horses intended for breeding purposes aren't typically selected based on their reproductive skills. Instead, they're bred for performance parameters, confirmation and pedigree. Thus, fertility issues can easily arise, making the process of breeding via natural cover or artificial insemination, frustrating. Here, we'll discuss the results of three recent studies on subfertility in both mayors and stallions.

1:04Stallion subfertility. Impaired acrosome exocytosis. Subfertility and stallions can be particularly challenging when the usual sperm parameters. Number, motility and morphology are acceptable, yet pregnancy rates remain suboptimal. According to Lucino et al, 2023, 30-40% of stallions in commercial breeding programs are moderately fertile and 8-12% are subfertil. Of those, 0.5-3% are severely subfertil, defined as less than 10% pregnancy rate per cycle, and up to 20% of subfertility cases are deemed idiopathic. One potential, although rare, cause of stallion infertility, is impaired acrosome exocytosis, first described in 1995. It is now being studied by the research group of Dr. Camilo Hernandez-Avias, assistant professor of equintheriology at Texas A, an M University College of Veterinary Medicine and Biomedical Sciences.

2:08Acrosomal exocytosis is the physiological process by which a spermatozoan undergoes molecular changes that result in the release of enzymes contained at the acrosome, spermatozoan cap, in the vicinity of the mature oocyte, to facilitate the interaction between these gametes, sperm and oocyte, during fertilization, he explains. Acrosom dysfunction, or IAE, occurs when sperm from an affected stallion cannot undergo IAE, at the same rate as sperm from fertile stallions, either under and vivo conditions, resulting in subfertility, or after being stimulated under in vitro conditions. In the 1990s, Dr. Dixon D. Varner, one of Hernandez-Avias' colleagues at Texas A and M, was the first to identify subfertile thoroughbred stallions with excellent sperm quality parameters and adequate breeding management. Transmission electron microscopy revealed the stallions had a much lower proportion of sperm, displaying acrosome vesiculation, only 2-4%, compared to fertile control stallions that displayed over 80% vesiculation.

3:17These vesiculations formed between the plasma membrane of the spermatozoa, an outer acrosome membrane, and are characteristic during acrosome exocytosis. Hernandez-Avias explains. In a subsequent study of seven subfertil thoroughbred stallions with IAE, brinsco and colleagues at Texas A and M University, found these stallions had high cholesterol to phospholipid ratios, about 2-fold higher, in the sperm membranes and seminal plasma compare. Such results established the basis to understand the mechanism behind IAE, as cholesterol removal from the plasma membrane is a necessary step for AE to occur. As Hernandez-Avias, Terrier-Raudseppe, PhD, a molecular geneticist at Texas A and M University, performed a genetic analysis and identified a region on equine chromosome 13 that was highly associated with IAE, gene FKBP6 on Exxon-5.

4:20Her team consistently observed two single nucleotide polymorphisms in subfertil thoroughbred stallions, but not in fertile controls, or stallions from other breed registries. G11,353,372, G2A, and G11,353,436, C2A. Thus, thoroughbred showing A-A-A-A-A in the gene, FKBP6 displayed IAE. Additional studies in clinical cases submitted to Raudseppe's laboratory reported this susceptibility genotype in nine thoroughbred stallions from Central Kentucky, Newmarket, UK, and Australia. In 2022, Hernandez-Avias et al analyzed 1,128 semen samples from subfertil stallions and diagnosed 21 with idiopathic subfertility. Of those, eight had IAE, determined by exposure to the calcium ionophore A23187, seven of which carried the susceptibility phenotype, and were thoroughbreds.

5:31A second study by Castaneda and colleagues at Texas A and M found four of 150 thoroughbred stallions in Central Kentucky were subfertil and carried the IAE genotype. These data show that the overall prevalence of IAE in thoroughbreds occurs only rarely in about 2-3% of the thoroughbred stallion population, but accounts for virtually 100% of cases of idiopathic subfertility in this breed, says Hernandez-Avias. In other words, all thoroughbred stallions diagnosed with idiopathic subfertility had IAE based on analyses using the calcium ionophore A23187 and FKBP6 genotyping. Hernandez-Avias says they do not perform acrosome function and genotyping analyses in every stallion they encounter with subfertility. But they will for stallions that display excellent sperm quality and breeding management yet are highly subfertil, particularly thoroughbreds.

6:35Hernandez-Avias at all are also studying the proteome protein makeup of sperm from fertile and subfertil thoroughbred stallions. Those studies have revealed some potential candidate proteins that could help us understand how this condition results in acrosome dysfunction. Acrosomal exocytosis is still a poorly understood process in horses and conducting studies on how the proteins in sperm from fertile and subfertil stallions differ may help us to know why these FKBP6 positive stallions are subfertil, he explains. We may also learn more about what happens typically in stallion sperm during and after acrosomal exocytosis. Through studies of the sperm proteome in these stallions, Hernandez-Avias at all identified two proteins that were less abundant in sperm from subfertil thoroughbred stallions. These proteins, ARSF and ZPBP2, have relevant roles during interactions between sperm and oocyte during fertilization.

7:39Subfertil thoroughbred stallions had no expression of ARSF at the acrosome region of the sperm it is soon, when compared to fertile stallions, he says. Our current studies focus on understanding the potential relationship between these proteins and the gene FKBP6, as this gene has no known role in acrosome function and is not expressed in sperm. It is only expressed in the developing spermatocyte in the testis. We are also focused on understanding the function of ARSF and ZPBP2 during the acrosomal exocytosis process. In non-therobred stallions, Hernandez-Avias identified three horses with IAE, but none were carriers for the subfertil genotype, FKBP6A slash A-A slash A. While the incidence of acrosome dysfunction is relatively low within the thoroughbred stallion population and other breeds, he says it becomes relevant when trying to identify the potential causes of stallion subfertility in the face of normal conventional sperm quality assays.

8:45From an economic standpoint, if you think about our study in 2021, where we tested 150 stallions in central Kentucky, the fact that we found four of those in one year suggests that every breeding season, there is the potential that one or two of these subfertil stallions may be present within the breeding pool for that region. He adds, although we can now identify IAE, Hernandez-Avias says there are no recommendations for the breeding management of these subfertil thoroughbred stallions. This is something we hope to change in the years to come, he says. Our current focus is on understanding the mechanism behind this condition to shed light on how the acrosome function is affected in these stallions, which genes or proteins can explain this condition, and how we can potentially overcome this condition, he adds. We are also using these stallions as a model to understand which proteins are necessary for sperm to acquire fertilizing competence and, in the future, to develop what we call fertility biomarkers and stallions that can be used to determine potential fertility or identify causes of subfertility in horses.

9:57Take home message. The standard breeding soundness exam might have limited utility and subfertil stallions with normal semen analysis, but low fertility because they provide limited information on sperm's functional competence. Assessing IAE might be useful in subfertility cases with normal sperm motility, morphology, DNA quality, breeding exam findings, and adequate breeding management, mere subfertility, uterine fluid analyses. In mayors, endometritis is the leading cause of subfertility, given the various ideologies, postbreeding induced versus infectious versus chronic endometritis, and despite the gamut of treatment options, the condition continues to pose clinical challenges. Reproductive immunology in LVL fluid samples. Endometrial biopsy and cytology slash culture are widely employed during breeding soundness exams, particularly in the face of subfertility. However, they have their limitations.

11:01Dr. Jenny Leccian, assistant professor of theoriogenology at North Carolina State University's College of Veterinary Medicine, and colleagues at Cornell University suggest reproductive immunology might provide an alternative technique for differentiating between a healthy endometrium, acute endometritis, and chronic endometrial fibrosis. Here, the term reproductive immunology refers to using monoclonal antibodies against equine cytokines and chemokines as a non-invasive tool for assessing acute and chronic endometritis. In their study, Leccian at All, 2023, recruited 46 mayors presenting for breeding management with various levels of endometrial inflammation, ranging from healthy to chronic inflammation, all mayors underwent a breeding soundness examination, a uterine low-volume lavage, LVL, and uterine biopsy analyzed using the Kenny Doig scale. The LVL fluid was centrifuged and the pellet stained with modified gymsa for endometrial cytology.

12:05Mayors were classified as healthy, N equals 20, acute endometritis, N equals 9, or chronic endometrial fibrosis, CEF, N equals 17, based on cytology and biopsy results. 12 inflammatory markers in LVL fluid were assayed using monoclonal antibodies. Interferon alpha was not detected in any samples, while interferon Y was lower in healthy mayors than mayors with acute endometritis and chronic endometrial fibrosis. Further, interleukin, 17A, 10, 1 beta, tumor necrosis factor alpha, and the chemokines CCL2, 3, 5, and 11 were significantly elevated in CEF compared to healthy mayors. Further analysis of these markers found that CCL2 concentrations greater than or equal to 550 pg per milliliter identified mayors with acute or chronic endometritis.

13:06LVL fluid analysis allows for sampling of the entire endometrium rather than just one location provided by a biopsy, election explains. Though biopsies are consistently performed in equine reproductive practice, our method is considered to be less invasive. Our technique offers a complementary diagnostic tool that can screen mayors prior to breeding or identify mayors that would benefit from uterine biopsy. Further, this technique can be performed within a few hours, is economic, and does not require a pathologist to interpret the results. The question is whether it can currently be used in clinical practice. I would start submitting samples for mayors who are difficult to get in full, says election. While we typically rely on culture, cytology, and biopsy as diagnostics, we are able to further characterize the inflammatory response based on this multiplex assay. We have since published a second study looking at inflammatory cytokine determination from endometrial swabs and cytobrushes to make the diagnostic test even more user-friendly to equine veterinarians who commonly use those techniques to take samples.

14:17LVL, uterine fluid, proteome. The different types of endometritis require distinct diagnostic testing and therapeutics due to their unique idiopathogenesis. The research group of Dr. Mariana Machado-Neves recently explored using proteomics on uterine fluid as a non-invasive means of understanding the molecular events of equine pregnancy establishment and maintenance, as well as pathologic processes such as endometritis. In that study, take Shira Suarez had all recruited 24 Manga Larga Marchador horses that underwent artificial insemination. LVL mayors subsequently had routine transrectal ultrasound exams, following insemination to assess uterine fluid, as well as LVL, cytology, and uterine biopsies. Based on the results, mayors were divided into the following groups, healthy mayors, n equals 8, mayors diagnosed with infectious endometritis, n equals 8, mayors diagnosed with postbreeding endometritis, n equals 8.

15:23LVL fluid was analyzed via electrophoresis and mass spectroscopy with the following proteins differentially expressed. Albumin was the most abundant protein in the uterine fluid of mayors with infectious endometritis and healthy mayors compared to mayors with postbreeding endometritis. The polymeric immunoglobulin receptor, PIGR, was predominantly found in mayors with postbreeding endometritis, 11.32%, compared to healthy mayors, 5.95%, and mayors with infectious endometritis, 3.05%. This protein promotes IgA transport from plasma into the endometrial mucosa. Utero-globins were highest in mayors with infectious endometritis, 12.4%, and healthy mayors, 11.85%, compared to mayors with postbreeding endometritis, 7.25%. Type 1 and 2 vannins were higher in healthy mayors, 6.8%, then in mayors with postbreeding, 3.03%, and infectious, 5.79% endometritis.

16:36Lipocolins were more abundant in mayors with infectious endometritis, 11.88%, then mayors with postbreeding endometritis, 3.65%, and healthy mayors, 6.67%. Take Cheris Suarez said, this is the first description of the protein profile of uterine fluid from mayors with either postbreeding or infectious endometritis. They noted clear differences in the profiles, and said these results provided valuable insights into the molecular alterations during the establishment and progression of endometritis, contributing to further identification of potential biomarkers. Take Home, Message, Advances in uterine fluid analyses might provide a more comprehensive view of the uterus than relying on biopsies representing a limited portion of the uterus. Reproductive immunology and proteomics might yield valuable information regarding uterine pathology, potentially differentiating between types of endometritis and guiding treatment protocols,