51 Comparison of proAKAP4 concentrations with motility parameters between fresh and post-thaw Asturcon ponies semen cryopreserved with two different extenders

Cryopreservation of sperm is crucial for safeguarding endangered species, conserving genetic diversity, and preserving unique breeds like the Asturcon ponies. In stallions, there are variations in semen cryotolerance among individuals, primarily assessed by post-thaw sperm motility. Nevertheless, a sperm-specific molecular marker evaluates sperm quality and sustained motility. ProAKAP4, the precursor of AKAP4, is a specialised protein found in the sperm flagellum structure, essential for maintaining efficient sperm motility over time. Across species such as bulls, dogs, rams, bucks, donkeys, and even humans, the quantity of proAKAP4 correlates with both total and progressive motility. In humans, proAKAP4 serves as an infertility marker, as men with unexplained infertility exhibit reduced levels of this protein. Our study aimed to assess the impact of cryopreservation on equine semen, using proAKAP4 sperm concentration and motility as indicators. We compared two different extenders for the cryopreservation process. Our animal cohort included eight Asturcon ponies, from which three ejaculates per pony were collected and frozen. For each ejaculate, 2 mL of fresh raw semen was directly frozen in liquid nitrogen for proAKAP4 measurement. The computer-aided sperm analysis was conducted before dilution, and the remaining ejaculate was divided and frozen in two distinct extenders, namely BotuCrio^® and FAO^®. The post-thaw semen from these two groups underwent CASA analysis, while proAKAP4 levels were quantified using the commercial Horse 4MID^® Kit (4BioDx). Preliminary findings indicate a significant reduction in proAKAP4 levels during the freezing process: 83.27 ng/10 M spz in the fresh ejaculate, 22.95 ng/10 M spz in the thawed BotuCrio group, and 21.59 ng/10 M spz in the thawed FAO group. The average total motility in freshly collected ejaculation was 39.75%. Post-thaw, total motility dropped to 17.38% for the BotuCrio group and 14.79% for the FAO group. Progressive motility decreased from 24.96% in fresh ejaculate to 5.92% and 4.50% in the BotuCrio and FAO groups. A statistically significant difference (P < 0.05) was observed between fresh ejaculate and post-thaw semen for both extenders, emphasising the impact of cryopreservation on sperm quality. When comparing the two commercialized extenders in post-thaw conditions, there was no significant difference (P > 0.05) in proAKAP4 concentrations, progressive motility, or total motility. In conclusion, despite the relatively low cryotolerance of the ejaculates utilised in this study, the cryopreservation procedure led to a substantial reduction in proAKAP4 levels, aligning with the observed decline in sperm motility post-thaw. Our experimental conditions did not reveal significant disparities between the BotuCrio and FAO extenders.