264 IN VITRO FERTILIZATION OF MACACA NEMESTRINA OOCYTES WITH FRESH AND FROZEN-THAWED EPIDIDYMAL SPERM

Abstract

In an effort to develop methods to maximize the use of reproductive tissues in our own tissue distribution program (WaNPRC TDP) and to serve as platforms for application of ART to endangered non-human primate (NHP) species, we have examined the effects of collection and cryostorage on epididymal sperm (ES) collected from Macaca nemestrina (Mn). Fresh ES was collected by needle aspiration from the cauda epididymis and prepared by centrifugation in mHTF (Irvine Scientific, Santa Ana, CA, USA) + BSA (3 mg mL⁻¹) at 700g for 10 min. The resultant pellet was resuspended in fresh mHTF + BSA and held at RT until required. ES subjected to freeze-thaw was collected from the cauda epididymis of testes provided by the WaNPRC TDP by slicing and swim-out into mHTF + BSA. ES was frozen in mTTE medium + 5% glycerol (Sankai T et al., 1994 J. Reprod. Fertil. 101, 273). Frozen ES straws were removed from liquid nitrogen and thawed in air at RT for 10 min. Thawed ES was centrifuged through an 80% PureSperm gradient (Spectrum Technologies, Healdsburg, CA, USA) at 700 g for 15 min. The resultant pellet was retrieved and washed in mHTF + BSA at 600 g for 5 min. For capacitation sperm were incubated in pre-equilibrated HTF + BSA containing 1.0 mM caffeine and 0.1 mM dbcAMP (Sigma, St. Louis, MO, USA) for 5–10 min in a humidified atmosphere of 6% CO₂ in air. In vitro-matured Mn oocytes collected from unstimulated ovaries were exposed to fresh or frozen-thawed ES whereas in vivo-matured Mn oocytes collected from superovulated ovaries were exposed to fresh ES in 50-μL Fertilized oocytes were cultured for 24–48 h and assessed for cleavage on Day 3 post-insemination. Data were analyzed by single-factor ANOVA or t-test where appropriate and are expressed as mean ± SD. Fresh Mn ES (n = 4) exhibited very low motility at collection (38 ± 10%) but recovered motility following processing (58 ± 20%). Frozen-thawed ES (n = 4) also exhibited low motility post-thaw (53 ± 17%) and recovered after processing (66 ± 28%). The percentage of acrosome-intact sperm was not significantly different (P > 0.05) for fresh ES (58.9 ± 8.6%) compared to frozen-thawed ES (70.6 ± 21.9%). Fresh and frozen-thawed ES fertilized in vitro-matured Mn oocytes at similar rates (fresh 68 ± 10.3%, n = 170 v. frozen-thawed 71.2 ± 6.7%, n = 90; P > 0.05). Cleavage rates of fertilized IVM oocytes were not significantly different (fresh 79 ± 7.2% v. frozen-thawed 79.8 ± 5.0%; P > 0.05). Fresh ES was also able to fertilize in vivo-matured Mn oocytes collected from superovulated ovaries (95.1 ± 2.5%, n = 98) and fertilized oocytes went on to cleave at a high rate (96.1 ± 2.6%). These results suggest that fresh and frozen-thawed ES may be useful for applied ART in endangered species. This work was supported by NIH grant #RR00166 and the WaNPRC TDP.