283 DNA FRAGMENTATION DYNAMICS AND POST-THAW MOTILITY OF WHITE-TAILED DEER SPERM

The main objective herein was to study the level of DNA damage and post-thaw motility of White-tailed deer sperm before (neat sample) and after sex-sorting and conventional-sorting using a MoFlo® SX flow cytometer (SX, Dako, Fort Collins, CO, USA). For assessing DNA damage, a comparison of frozen–thawed (F-T) neat sperm (control) was made with F-T sex-sorted, F-T conventional-sorted, and F-T conventional sperm samples. Sperm motility was assessed by bright field microscopy using a Nikon Eclipse 80i microscope and slide-coverslips (25.4 × 76.2 mm slides, 22 × 22 #1.5 coverslips). A direct comparison of all 4 aforementioned sperm groups could not be made for some bucks. Live/dead sorting of the sperm (i.e. conventional-sorted sperm) can remove membrane compromised sperm and nonaligned live sperm, which may result, in part, from abnormal morphologies. White-tailed deer (Odocoileus virginianus; n = 13) from 1 to 7 years old were used for the experiments. The White-tailed deer were selected based on a genetic predisposition for producing large antlers (i.e. Boone and Crockett antler scores ≥200 points). Sperm DNA fragmentation levels were assessed using the Sperm-Halomax® kit (Halotech DNA, Madrid, Spain), counting 300 sperm per sample. The level of baseline DNA fragmentation was similar for conventional F-T sperm samples (<5%), but even lower after sex-sorting and conventional-sorting (2.39 and 1.69%, respectively). The conventional sperm samples had lower post-thaw motilities compared with sex-sorted samples from the same individual bucks (n = 6), with average post-thaw motilities of 43 ± 26% and 56.5 ± 20%, respectively. The statistical comparison of the dynamic loss of DNA quality (i.e. DNA fragmentation of samples incubated in a 34°C water bath for 96 h) was assessed using the nonparametric maximum likelihood Kaplan-Meier estimator and a Breslow (Generalized Wilcoxon) test. When comparing sperm samples taken from the same bucks (n = 6), the conventional samples had significantly greater (P < 0.05) DNA fragmentation levels over time than the sex-sorted sperm. Conventional-sorted White-tailed deer (n = 8) sperm samples did not have significantly greater (P > 0.05) DNA fragmentation levels when compared with the sex–sorted sperm. When comparing X-chromosome sorted sperm to Y-chromosome sorted sperm, the DNA fragmentation levels were not significantly different (n = 10; P > 0.05), averaging 2.59 ± 3.61% and 2.18 ± 0.53% after 96 h. Based on the sperm DNA fragmentation and post-thaw motility results in the present study, the sex-sorting of White-tailed deer sperm may be a viable technique for the White-tailed deer industry and perhaps serve as a model for the conservation of endangered species such as the Eld’s deer (Cervus eldithamin). Future work should be implemented for examining the fertilizing potential of sex-sorted White-tailed deer sperm. The authors thank Maurice Rosenstein, Laura Belluzzo, Jared Templeton, Mike Bringans, Pat Cooper, Suzanne Menges, Miguel Ramirez, Altea Gosálbez, and Sexing Technologies staff for technical assistance. This research was funded by Sexing Technologies.