Assessing species-specific reproductive variation to inform assisted reproductive technologies for Canadian snakes

Abstract

Assisted reproductive technologies (ART), frequently used to manage domestic livestock, are increasingly incorporated into conservation mating programs for species-at-risk. Species-specific insights on reproductive biology, including seasonal variation and endocrine function, improve ART outcomes. Despite representation in many zoological collections, snakes are underrepresented in the ART literature and research is needed before it can be broadly implemented in conservation. My research characterizes temporal variation in spermatozoa quality and sexual steroids for two endangered Canadian snakes to facilitate gamete cryo-preservation and artificial insemination. These focal species have different life history strategies: an oviviparous viperid, the eastern massasauga rattlesnake (Sistrurus catenatus) and oviparous colubrid, the gray ratsnake (Pantherophis spiloides). I collected spermatozoa samples monthly from May to October in 2023 and 2024 from individuals of both species held in captive population. Spermatozoa samples were quantified for concentration, motility, membrane integrity and rapid forward progression of spermatozoa. I predicted the highest concentration and quality spermatozoa would be produced during the period corresponding to the mating season in wild population; late July through August in rattlesnakes, and late May through June in ratsnakes. I found species-specific variation in quality and concentration of spermatozoa, but not during the intervals predicted. To characterize the reproductive hormone cycle, faeces from captive rattlesnakes was collected from March 2023 to September in 2024. Sexual steroid faecal metabolites of progesterone (FPM) and testosterone (FTM) were quantified using commercial enzyme-linked immunosorbent assay kits. I predicted FTM concentrations would be highest from July to August, corresponding to spermatogenesis and the peak in mating behaviour. I predicted that FPM would increase in May and June during vitellogenesis, be expressed in the highest concentrations annually from May to August and remain at high concentration through to parturition in gravid females. I found annual variation in FTM and FPM, but not in the patterns which I had predicted. My research provides novel insights into the reproductive physiology of two Canadian at-risk species and facilitates efforts to develop ART in managed population. The integration of this information into conservation programs will improve fecundity, ensuring that captive population can best contribute to conservation.