Impact of different cryopreservation protocols on epididymal sperm quality and subsequent in vitro embryo production in domestic cats

Abstract

Cryopreservation of epididymal spermatozoa is a pivotal strategy in the conservation of genetic resources from endangered felids. This study aimed at the standardization of both evaluation and cryopreservation protocols in the domestic cat in field conditions as a starting point to endangered felids. To this purpose, routine field conditions evaluation and cooling methods were compared to more refined ones. Two commercial cryopreservation extenders were included in this study: one supplemented with whole egg yolk (WEY) (Caniplus Freeze, Minitube®), and one containing egg yolk plasma (EYP) (Canifreeze, IMV Technologies®), combined with two different cooling methods prior to freezing) on epididymal sperm parameters and subsequent embryo in vitro production (IVP). Epididymal spermatozoa were cryopreserved, thawed and submitted to density gradient prior to in vitro fertilization (IVF). Sperm samples were analyzed at three processing steps: fresh, post-thaw, and post-gradient. Assessment included total and progressive motility, membrane and acrosomal integrity, and mitochondrial activity. For IVP, in vitro matured oocytes (n = 755 in 9 replicates) were coincubated with frozen-thawed samples and cultured in vitro for 10 days. Cleavage and blastocyst rates were compared. While cryopreservation significantly reduced sperm quality, gradient selection effectively restored parameters to near-fresh levels. EYP extender yielded superior motility parameters post-thaw, whereas WEY extender enhanced membrane integrity post-thaw, and mitochondrial activity, membrane integrity and motility parameters post-gradient. Hence, after undergoing density gradient and removing WEY bigger particles and viscosity, motility parameters were better for highly viable samples treated with WEY. Despite these differences, no significant effects of extender or cooling methods were observed on IVP rates. Both extenders supported blastocyst formation similarly, exceeding 19 %. These findings highlight the suitability of both protocols for feline sperm cryopreservation and underscore the need for further research into embryo quality outcomes