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(179) EFFECTS OF CRYOPRESERVATION PROTOCOLS ON EXOTIC SHEEP SEMEN. Anderson, Autumn1, Korenstein, Michelle1, Amodeo, Dianne1, Wetsman, Irene1, Ravida, Nicole1, Durrant, Barbara1, 1 Center for Reproduction of Endangered Species, San Diego, CA ABSTRACT- In the past century a number of exotic sheep species have become endangered or threatened. Zoos can contribute to the conservation of these species by cryopreserving sperm of valuable males. For this study, sperm was collected post-mortem from eight rams representing 6 species. Epididymides were minced into TEST-Yolk buffer (T-Y) and filtered to separate sperm from tissue. Initial motility (MOT) and speed of progression (SOP) were expressed as initial motility score (IMS, MOT x SOP2). Initial viability was recorded as % intact plasma membrane (%IL) by eosin-nigrosin stain. Diluted sperm was aliquotted into cryovials and further extended with glycerolated T-Y for a final glycerol concentration of 4%. Four pre-freeze cool methods were used: 1) no cool (NC); 2) cooled to 4°C over 30 m (30" cl); 3) cooled to 4°C over 30 m, then equilibrated at 4°C for 2 h (2.5 FAST); 4) cooled to 4°C over 2.5 h (2.5 SLOW). Vials were frozen at 0.5 ml over LN2 vapor at 7.3°C/m. These were thawed for 1 m in a 37°C water bath, maintained at 37°C and assessed for %IMS and %IL at T5, T30, and T60 minutes. Sperm was then divided into five 0.1 ml aliquots, each subjected to one of the following post-thaw treatments: 1) no treatment (NT, placed directly into 37°C incubation); 2) diluted in 0.1 ml BWW with BSA before incubation (1:1 BWW); 3) diluted with 0.1 ml T-Y (1:1 T-Y); 4) washed by centrifugation, sperm pellet resuspended in 0.1 ml BWW (Wash BWW); 5) washed and resuspended in T-Y (Wash T-Y). Pre-freeze cool did not affect %IMS at T30, however, T5 and T60 %IMS was significantly higher (p<.05) in the 2.5 FAST treatment group. This cool method also preserved the greatest %IL (p<.05) at all time periods. Post-thaw treatment did not affect %IMS at any time period. Although NT and 1:1 BWW treatments did not differ significantly from each other, NT resulted in greater %IL at T30 and T60 compared to the three other treatments (p<.05). At T30, 1:1 BWW significantly improved %IL (p<.05) compared to both wash treatments, however, by T60 these differences were not significant. It may be concluded that the best cryosurvival of epididymal sheep sperm is achieved with a fast cool to 4°C followed by a 2 h equilibration prior to freezing. After thawing, maximum viability is maintained when sperm are not subjected to washing by centrifugation. KEY WORDS: cryopreservation, epididymal, sperm, sheep |
