161 CRYOPRESERVATION OF CORN SNAKE, ELAPHE GUTATTA, SEMEN

Abstract

The purpose of this investigation was to develop a protocol for cryopreserving snake semen using the corn snake, Elaphe gutatta, as the model species. This experiment is part of a five year investigation where the influences of diluents, cryoprotectants, cooling and thawing rates on sperm survival were studied. This report presents one protocol found to be effective for cryopreserving corn snake semen as determined by post-thaw motility parameters in vitro. Semen was collected by applying pressure to the lower abdomen and continuing distally towards the cloaca to remove any feces or urates. The cloaca was washed using PBS, then a more local pressure was applied to each side of the vent to cause the hemipenes to evert and subsequently ejaculate. The semen (approximately 5 μL) was then collected using a sterile transfer pipette, placed in 120 μL Biladyl A containing 20% egg yolk (Minitube, 13502/0501), and analyzed for motility, rate of forward progression (RFP; 0–5), and concentration. The semen was further diluted at room temperature at 1:1 v/v with Biladyl A containing 20% egg yolk and 34% Glycerol (Sigma, G2025), yielding a final concentration of 17% Glycerol. The diluted semen was then loaded into 250-μL straws and slowly cooled for 1 hour. The straws were then placed 1 inch above a liquid nitrogen bath for ten minutes and finally plunged into the nitrogen where it remained frozen. The cryopreserved semen was thawed by placing the straws into a 50°C water bath for 8 s, then emptied into microcentrifuge tubes and the sperm were evaluated for motility and RFP. The mean motility of the fresh samples was 72.5% (66.4–77.7%). The mean post-thaw motility of sperm over six trials was 27.1% (17.8–50.2%). The mean RFP was 0.75 (0.5–1.0). The differences between fresh and post-thawed mean motilities were shown to be significant using a chi-square analysis (P < 0.0001). Density gradient centrifugation (DGC) was applied in one trial where the semen had an initial post-thaw motility of 50.2% with an RFP of 0.5. After the centrifugation treatment, the motility increased to 64.8% with an RFP of 3. The DGC media was composed of 400 μL 45% Percoll (Sigma, P4937) layered over 400 μL 90% Percoll. The density gradients were centrifuged at 700g for 30 min after which time the pellets were washed in 500 μL pre-warmed TL Hepes Solution (Lonza, 04-616F) and centrifuged at 300g for 10 min to remove the Percoll. The resulting sperm pellets were then resuspended in a small volume of the pre-warmed Hepes. Thus far, the protocol using 17% Glycerol in Biladyl A with 20% egg yolk has proven to be the most successful for cryopreserving corn snake semen. The use of DGC enhanced the number of usable sperm leaving sperm of higher motility and RFP possibly due to the absence of seminal plasma or cryoprotective agents that may detrimentally affect sperm quality. There are no known reports of the use of DCG with snake semen. Further studies are underway to improve these results and successfully use cryopreserved snake semen for artificial insemination and cryobanking for the long-term genetic management of endangered snake species.