154 Cryo-Acrosome Reaction/Capacitation of Ejaculated and Epididymal Bovine Sperm and Their in Vitro Fertilization Rates

Abstract

Seminal plasma has been shown to affect the composition and function of sperm. This exposure may alter the ability of sperm to endure cryopreservation, undergo capacitation and fertilize oocytes. Earlier studies demonstrated that the freezing response of epididymal and ejaculated sperm from the bulls utilised in these studies was similar (Alapati et al. 2009). Subsequent studies are designed to investigate the response of ejaculated and epididymal bovine sperm to cryopreservation and their ability to undergo capacitation. Ejaculated and epididymal sperm from the same bulls (n = 4) were collected by an artificial vagina and retrograde caudal epididymal flush, respectively and were cryopreserved in standard egg-yolk glycerol extender. Sperm was compared by level of cryo-acrosome reaction/capacitation and their fertilization rates in vitro with and without the capacitating agent heparin. Upon evaluation, a sample was thawed and washed and the viable sperm population was isolated through a discontinuous Percoll® gradient. Sperm viability and acrosome status were assessed by fluorescent staining with propidium iodine and fluorescein isothiocyanate-PNA followed by evaluation with flow cytometry. Capacitated sperm were then induced to undergo the acrosome reaction by exposure to lysophosphatidylcholine (10 μg mL–1). Cryo-capacitation was determined by the difference between cryo- and lysophosphatidylcholine-induced acrosome-reacted sperm levels. Differences in the mean values between groups were analysed by ANOVA. Ejaculated and epididymal sperm differed by level of cryo-acrosome reaction (15 vs 4%, respectively; P < 0.001), but did not differ by level of cryo-capacitation (6.2 vs 5.9%, respectively; P = 0.92). The ability of epididymal and ejaculated sperm to fertilize oocytes in vitro with and without heparin was also investigated. Sperm were thawed and washed and the viable population was isolated through a Percoll® gradient. Oocytes were washed and randomly assigned to a treatment group, either ejaculated ± heparin or epididymal ± heparin. Oocytes and sperm were added to a capacitating medium (TALP) with and without heparin. Following 18 h of incubation, fertilization rate was determined by pronuclear fluorescent staining with Hoechst 33342 followed by confirmation with aceto-orcein staining. Differences in the mean values among treatment groups were analysed by one-way ANOVA followed by Holm-Sidak pairwise multiple comparisons. Fertilization rates of epididymal sperm with and without heparin and ejaculated sperm with heparin were similar (76, 80 and 70%, respectively). Ejaculated sperm without heparin was lower than all other groups, with a fertilization rate of 42% (P < 0.001). In summary, epididymal sperm displayed lower levels of cryo-acrosome reaction but were similar by level of cryo-capacitation. In addition, epididymal and ejaculated sperm differed in the need for a capacitating agent such as heparin when used in vitro. This may be useful to increase efficiency when using epididymal sperm in assisted reproductive techniques.