219 IN VITRO FERTILIZATION RATE OF MATURED PIG OOCYTES BY FROZEN–THAWED KOLBROEK PIG SPERM CELLS

Abstract

No studies have investigated the IVF rate of South African indigenous Kolbroek sperm cells following cryopreservation. The objective of this study was to test if frozen–thawed Kolbroek pig sperm cells could penetrate pig oocytes matured in vitro. Pig ovaries were collected from a local abattoir and cumulus–oocytes complexes were obtained by aspiration and were then in vitro matured in TCM-199 supplemented with 10% pig follicular fluid, 10% fetal bovine serum, and 1 μg mL^–1 of FSH and LH. Following 44 h of incubation, 200 matured pig oocytes were randomly assigned to 2 treatments with frozen–thawed and fresh (control) Kolbroek pig sperm cells. For IVF, Kolbroek sperm cells were in vitro capacitated using Brackett and Oliphant’s sperm wash medium. Matured pig oocytes and sperm cells were co-incubated for 24 h in Brackett and Oliphant’s IVF medium. Following fertilization, presumptive zygotes were in vitro cultured at 39°C in 5% CO₂, 5% O₂, and 90% N₂. Rate of fertilization was identified by the number of cleaved zygotes. Data were analysed by ANOVA. The total motility of Kolboek pig sperm cells used for IVF was 40% for frozen–thawed sperm cells and 80% for fresh sperm cells. The results showed that Kolbroek pig sperm cells were able to penetrate pig oocytes in vitro. However, no significant (P < 0.05) difference was observed in the percentage of cleavage of pig oocytes fertilized with either frozen–thawed (13.25%) or fresh (13.0%) Kolbroek pig sperm cells. The percentage of embryos that developed to the morulae stage was 2% in frozen–thawed sperm cells and was 0% in fresh Kolbroek sperm cells. Furthermore, oocytes fertilized with Kolboek sperm cells did not develop to the blastocyst stage in either treatment. In conclusion, this study demonstrated that frozen–thawed Kolbroek sperm cells are able to fertilize matured pig oocytes in vitro.