Evaluation of commercial semen extenders and crystalloids for short term cooled extension of epididymal spermatozoa in squamates and chelonians

Abstract

Our working knowledge of assisted reproduction in male reptiles is limited to our ability to collect semen from less than 0.2% reptile species. To date, cooled storage using egg yolk based semen extender has had some short term benefits for corn snakes (Elaphe guttata), green iguanas (Iguana iguana), and leopard tortoises (Stigmochelys pardalis).1-3 Our objective was to evaluate commercial semen extenders and crystalloid solutions utility to provide short-term sperm storage in squamates and chelonians. The hypothesis was that commercial semen extenders maintain motility of squamate and chelonian sperm, specifically the green anole (Anolis carolinensis), the diamondback water snake (Nerodia fasciata), and the red eared slider (Trachemys scripta elegans). Epididymal sperm from A. carolinensis (n = 6), N. fasciata (n = 6), and T. scripta elegans (n = 6) were collected post mortem. A. carolinensis and N. fasciata samples were aliquoted into 6 commercial extender treatments (Ham’s F 10 without albumin, TEST yolk buffer, INRA 96, sperm washing media, Andro pro chill LT and Hank’s balanced salt solution) and 3 crystalloid treatments (0.9% sodium chloride injection, phosphate buffered saline, and Lactated Ringer’s solution), whereas T. scripta elegans samples were aliquoted into 6 extender treatments (Ham’s F 10 without albumin, TEST yolk buffer, INRA 96, sperm washing media, electrolyte free media, and Hank’s balanced salt solution). Samples were stored at 4°C for 72 and 96 hours respectively. Motility analysis was performed using a Computer Assisted Sperm Analysis system at time 0, 12, 24, 48, and 72 hours for A. carolinensis and N. fasciata and at 0, 6, 12, 24, 48, 72, and 96 hours for T. scripta elegans. Linear mixed models were used for statistical analyses. Epididymal sperm motility in A. carolinensis declined over time for each treatment over the first 24 hours (p < 0.001), with no significant difference in motility between commercial semen extenders and crystalloid solutions. Epididymal sperm motility in N. fasciata remained high in commercial semen extenders compared to crystalloid solutions (p < 0.001) for more than 72 hours. Sperm wash media, F 10, and INRA 96 maintained highest sperm motility for N. fasciata. Epididymal sperm motility in T. scripta elegans remained high over 96 hours in the sperm wash media, Test yolk buffer, and INRA 96 (p < 0.001). However, motility declined over time in all treatments except INRA 96 (p < 0.001). We concluded that INRA 96, Sperm wash media, or F 10 were the best choices for handling and cool storing reptile sperm.