247 COMPARISON OF THE DNA FRAGMENTATION INDEX BETWEEN CRYOPRESERVED EJACULATED SPERM AND EPIDIDYMAL SPERM IN STALLIONS

Abstract

The development of a reliable technique for freezing epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The semen analysis method with the best ability to predict fertility is an examination of the sperm chromatin structure. This test evaluates the susceptibility of spermatozoa DNA to denaturation. The ability of spermatozoal DNA to maintain an intact double-stranded configuration is determined by exposure to an acid environment. The aim of this study was to compare the DNA fragmentation index of sperm obtained from ejaculate (G1) and sperm from the cauda epididymis (G2). For G1, two ejaculates from each of seven stallions were collected and then subjected to cryopreservation using BotuCrio^TM extender. One week after the last semen collection, the stallions underwent bilateral orchiectomy. Sperm from the cauda epididymis was harvested immediately after castration (G2) by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender (BotuSemen^TM). The recovered sperm was then cryopreserved using BotuCrio^TM extender. The sperm motility parameters were analysed by computer-assisted sperm analysis (HTM IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA), and the DNA fragmentation index was estimated using acridine orange test epifluorescence microscopy. The samples were evaluated immediately (0 h) and 8 h after thawing. The total motility, progressive motility, and percentage of rapid cells of the G1 v. G2 samples at 0 h were, respectively, 62.3 ± 12.9^a v. 72.6 ± 8.4^a, 31.6 ± 9.2^a v. 35.3 ± 10.32^a, and 49.3 ± 14.33^a v. 59.7 ± 13.59^a. At 8 h, the results were 26.0 ± 21.6^b v. 54.7 ± 12.2^a, 6.1 ± 6.4^b v. 17.4 ± 8.54^a, and 13.7 ± 14.85^b v. 37.6 ± 14.15^a. Evaluation of the DNA fragmentation in the G1 and G2 samples yielded 6.7 ± 1.41^a v. 5.7 ± 1.60^a at 0 h and 8.3 ± 1.78^b v. 7.2 ± 1.19^b at 8 h for percentage of DNA fragmentation after thawing. At 0 h, no differences in the sperm parameters were observed between groups, but statistical differences were observed in the sperm motility parameters between the treatment groups after 8 h. For the DNA fragmentation index, no difference was found at 0 and 8 h between the groups. However, after thawing, a higher percentage of DNA fragmentation was observed in the ejaculated sperm (8 h) as compared with the epididymal sperm (0 h). On the basis of these results, we can conclude that frozen–thawed cauda epididymal sperm had similar or higher motion parameters than ejaculated sperm after thawing. In addition, incubating the sperm at 20°C for 8 h after thawing resulted in higher motion parameters and less DNA fragmentation of the epididymal sperm. This finding suggests that epididymal sperm are more resistant to the cold shock caused by cryopreservation.

FAPESP for financial support and Botupharma for donation of BotuSemen^TM and BotuCrio^TM extender.