Short‐Term Cold Storage of Gray Ratsnake ( <i>Pantherophis spiloides</i> ) Sperm

The management of species in human care, hereafter ex situ management, has been identified as a key tool in the effort to restore global biodiversity. However, sustaining reproduction and genetic diversity in an ex situ environment can be challenging. Assisted reproductive technologies (assisted reproduction) can help. In this study, we begin development of an assisted reproduction program for an ex situ population of gray ratsnake (Pantherophis spiloides), a federally 'threatened' species in Canada. Assisted reproduction application in squamates has had limited attention, despite the disproportionate representation of the taxa among threatened and endangered species worldwide. Hence, we hoped to provide a model for the development of assisted reproduction in squamates in Canada more broadly. As such, we focused on the essential first steps of gamete collection and short-term cold preservation. We collected sperm samples monthly from 17 individuals in the ex situ population from May to October. We assessed concentration, motility, rapid progressive motility, and membrane integrity (viability). We used generalized linear models to assess the impact of four common sperm extenders on refrigerated sperm quality over a period of 5 days. We found that TL HEPES with 10% fetal bovine sera maintained the highest quality sperm. Further, we discovered previously unknown seasonality in sperm viability, where the highest percentage of viable sperm was collected in October, rather than May-June, at the height of the species breeding season. Our work highlights that the timing of sperm collection and the type of sperm extender used both influence the preservation of sperm ex vivo.