Sperm cryopreservation and reproductive characteristics in the beauty snake (Elaphe taeniura) as a model for endangered Colubridae

Sperm collection and cryopreservation are key technologies for assisted reproduction, with post-mortem sperm collection being the main tool to prevent the decline of endangered species. The present study describes post-mortem sperm collection and two cryopreservation protocols in beauty snakes (Elaphe taeniura), as a model for Colubridae. The vasa deferentia of 18 snakes were collected post-mortem and incubated for 30 min at room temperature to retrieve sperm by float up. Afterwards, fresh sperm was analysed, and 11 samples were diluted into either a single or a double-step TEST-egg yolk-based extender to reach a final concentration of 12 % glycerol. Anatomical and histological analyses of the reproductive organs revealed that those on the right side were larger, heavier, and positioned more cranially than the left ones (P = 0.007, P = 0.003, and P = 0.0002, respectively), but sperm parameters did not differ. Successful sperm retrieval was achieved in 61.1 % of snakes, with positive correlations between seminiferous tubules length and body weight (P = 0.0004) and between seminiferous tubules length and body length (P = 0.001). Sperm-producing animals were heavier and longer compared to azoospermic ones (P = 0.04 and P = 0.04 respectively). Specifically, males longer than 130 cm and heavier than 177g have higher reproductive potential. Both cryopreservation protocols yielded comparable results, preserving sperm viability, motility, and morphology. Therefore, a single-step protocol should be preferred due to its technical simplicity. In conclusion, we described for the first time the reproductive features and characteristics of spermatozoa collected post-mortem in beauty snakes. Although the described protocols are effective, further research is warranted to optimize these techniques.