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

Abstract

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.