The role of impaired acrosomal exocytosis (IAE) in stallion subfertility: A retrospective analysis of the clinical condition, and an update on its diagnosis by high throughput technologies.

Abstract: Acrosomal dysfunction has been considered as a cause of subfertility in males of different species, including stallions. A subset of subfertile stallions with acrosomal dysfunction is unique because they have normal sperm quality (motility, morphology, viability, and DNA quality). The current work aims to describe the clinical characteristics of subfertile stallions that were diagnosed with Impaired Acrosomal Exocytosis (IAE) by using two high throughput methods: flow cytometry and molecular genetic analysis, and to identify the prevalence of subfertility due to IAE in stallions evaluated at Texas A&M University. Clinical data from 1,128 stallions evaluated during 17 years at a Veterinary Teaching Hospital was retrospectively analyzed. Only stallions with a history of subfertility not explained following a breeding soundness examination and/or conventional semen analysis, were included. For those stallions, the acrosomal exocytosis test (AE test), in which sperm is incubated at 37 °C for up to 2 h in the presence of the calcium ionophore A23187, was used to determine IAE. The difference in AE-Rate (AE-Diff) between each pair of fertile control stallion and subfertile stallion was categorized as: Normal: AE-Diff < 14%; Questionable: AE-Diff 15-29%; Abnormal: AE-Diff > 30%. In selected cases, blood or hair was procured for identification of the susceptibility genotype for IAE, A/A-A/A, in the FKBP6 gene, exon 5. Twenty-one (21) stallions (1.86% total population analyzed) had reduced fertility despite having acceptable sperm quality. Sperm from these stallions were subjected to the AE Test. Of these, 8 stallions had reduced sperm AE-rate, based on the AE Test (8/21; 38.1%). Subsequently, blood or hair samples from these 8 stallions which had either questionable (AE-Diff 15 - 29%; n = 5) or abnormal (AE-Diff > 30%; n = 3) responses to the AE Test were analyzed for the susceptibility genotype for IAE, A/A-A/A (FKBP6 gene, exon 5). Seven out of the eight (7/8) stallions carried this susceptibility genotype. All of these were Thoroughbreds. After 2 h of incubation, the viability in fertile stallion sperm was lower than in A/A-A/A stallions (4% vs. 25%, respectively; P < 0.05), while the AE-rate was higher for fertile than for A/A-A/A stallions (85% vs. 56%, respectively; P < 0.05). The use of two high throughput tests (i.e., flow cytometry and molecular genetic analysis) may complement each other in the diagnosis of IAE in breeding stallions. In this study, 5/7 subfertile stallions diagnosed with the IAE susceptibility genotype would have been diagnosed as normal with the AE Test. This study introduces a subset of stallions with the IAE genotype with fertility higher than has been previously reported (i.e., <15% per-cycle pregnancy rate), suggesting that IAE manifests as a broader range of subfertility.