117 CRYOPRESERVATION OF SNAKE SEMEN: ARE WE FROZEN IN TIME?

Abstract

The increasing number of endangered snake species, isolation of small fragmented populations with associated inbreeding and mating or conception problems in captivity underscore the need to develop assisted reproductive techniques such as semen cryopreservation and artificial insemination to enhance conservation efforts. However, no efficient protocols for semen evaluation, cooling, or freezing are described in the 4 known publications on snake semen cryopreservation. In this initial study, semen was collected noninvasively from 4 live adult red diamond rattlesnakes (Crotalus ruber) by ventral massage. An aliquot of each ejaculate was diluted in Ham's F10 and evaluated for motility and speed of progression (SOP, scale of 1–5, 5 being fastest) before and after freezing. Because ejaculate volumes were very small, each one was subjected to 2 to 4 treatments only. Samples were diluted in Lake's extender with 2 or 4% dimethyl sulfoxide or Test-Yolk extender with 4%, 8%, or 10% glycerol before cooling to 4°C. Semen extended with Lake's (a common avian semen extender) was then frozen by one of two protocols (FP): FP1, pelleted in liquid nitrogen vapor; FP2, placed in cryovials in an alcohol bath for 4 h in a –80°C freezer (cooling rate of 1°C min^–1). Semen extended with Test-Yolk (a widely used mammalian semen extender) was frozen by one of two protocols: FP3, placed in cryovials in a controlled-rate freezer and cooled at 1°C min^–1 to –20°C, then 50°C min^–1 to –80°C; FP4, placed in cryovials in a controlled-rate freezer and cooled at 1°C min^–1 to –8°C, then 3°C min^–1 to –16°C, then 5°C min^–1 to –80°C. Interestingly, semen frozen in Lake's extender exhibited no post-thaw motility, regardless of dimethyl sulfoxide concentration or FP (Table 1). In contrast, all semen frozen in Test-Yolk exhibited motility post-thaw. Semen from male 42 frozen by FP3 with 8% glycerol resulted in the greatest post-thaw motility of all treatments for both males. However, the same treatment for male 122 was not as successful, suggesting a possible male effect on semen cryosurvival (Table 1). The single sample frozen with 4% glycerol exhibited the lowest post-thaw motility, indicating that higher concentrations of the cryoprotectant may be beneficial to snake sperm. The exciting results of this preliminary study may guide future research in the long-neglected field of snake semen cryopreservation.

Table 1.  Motility/speed of progression for fresh and cryopreserved semen of red diamond rattlesnakes frozen with Test-Yolk extender and glycerol