# Influence of Cryopreservation on the Acrosome Reaction in Hucul Stallion Spermatozoa

## TL;DR

Cryopreservation increases the acrosome reaction in Hucul stallion sperm, potentially reducing fertility.

## Contribution

This study is the first to investigate the effect of cryopreservation on acrosome reaction in Hucul stallion sperm.

## Key findings

- Frozen/thawed semen showed a significantly higher proportion of acrosome-reacted spermatozoa compared to chilled semen.
- Cryopreservation was associated with reduced sperm motility and increased acrosome reaction, suggesting a negative impact on sperm functionality.

## Abstract

The acrosome reaction is one of the most important physiological events associated with the fertilization process. The acrosome becomes more labile under reduced temperature conditions, and the contents of the acrosomal cap may be lost. The effect of cryopreservation on the acrosomal status in stallion spermatozoa, particularly for Hucul stallions, has not been studied. The aim of this study was to determine the level of acrosome reaction in chilled and frozen/thawed Hucul stallion semen. Our results showed a significantly higher proportion of acrosome-reacted spermatozoa in frozen/thawed semen compared to chilled semen. In chilled semen, most of the acrosomes remained intact, suggesting the preserved potential for fertilization. In cryopreserved semen, a greater number of spermatozoa had reacted acrosomes, which in turn causes a significant reduction in sperm functionality. These findings suggest that cryopreservation may negatively affect acrosomal integrity, and further studies using appropriate controls are necessary.

The Hucul horse is a Polish primitive breed with a small population size, which highlights the importance of preserving the genetic resources. The cryopreservation of semen is essential for creating gene banks, but its effect on the acrosome reaction in Hucul stallions has not yet been investigated. The acrosome reaction is one of the most important physiological events associated with the fertilization process. Therefore, our goal was to determine the level of acrosome reaction in chilled and frozen/thawed Hucul stallion semen using the FluoAcro test and the SCA® semen analysis system. We found that semen cryopreservation significantly reduced sperm motility and was associated with an increased percentage of acrosome-reacted spermatozoa. It should be noted, however, that, in this case, there was no negative control, and the results may reflect acrosomal damage rather than the elicited responses. Further validation of the methods with equine sperm and the inclusion of a control are recommended.