226 CRYOPRESERVED EJACULATED AND EPIDIDYMAL SPERM COLLECTED FROM THE SAME HOLSTEIN BULLS USED FOR IN VITRO FERTILIZATION

Abstract

Exposure to seminal plasma may modify the ability of sperm to survive cryopreservation, undergo capacitation, and fertilize oocytes. The present work was designed to compare embryo development after IVF of oocytes with ejaculated and epididymal bovine sperm from bulls previously tested and showing similar responses to freezing. However, we also found that this ejaculated and epididymal sperm differed in their in vitro culture dynamics (capacitation, viability, and auto-acrosome reaction) and ability to fertilize oocytes in vitro. Ejaculated and epididymal sperm were collected from the same fertile mature Holstein bulls (n = 4) by artificial vagina and post-castration retrograde caudal epididymal flush, respectively. Collection of epididymal sperm was conducted 2 weeks after the last collection of ejaculated sperm. After collection, ejaculated and epididymal sperm were cryopreserved and stored in LN until use. Before IVF, a viable sperm population was isolated by centrifugation through a Percoll density gradient. Ejaculated and epididymal sperm were then added to fertilization drops at a final concentration of 1 × 106 mL–1, and IVF was conducted with and without the capacitation agent heparin. Oocytes were washed and randomly assigned to one of four treatment groups (ejaculated ± heparin or epididymal ± heparin). Embryo development was determined at 72 and 186 h after IVF. Differences in the mean values among treatment groups were analysed by one-way ANOVA, followed by the Holm-Sidak pairwise multiple comparisons. Embryo cleavage after IVF using ejaculated sperm without heparin (45.2%) was significantly lower (P < 0.05) than in all other groups. Cleavage rates of ejaculated sperm with heparin (56.9%) and epididymal sperm with (58.1%) and without (57.5%) heparin were found to be similar. No difference was noted between ejaculated and epididymal sperm in blastocyst development, although the inclusion of heparin did significantly (P < 0.01) increase blastocyst development in both ejaculated (9.3 compared with 23.6%) and epididymal sperm (2.5 compared with 23.3%). In conclusion, cryopreserved ejaculated and epididymal sperm collected from the same bulls can be successfully used for the in vitro production of bovine blastocysts without changing the existing protocols. This may increase the efficiency when using epididymal sperm in assisted reproductive techniques.