Reproduction Abstracts

Introduction: We aimed to establish an efficient in vitro fertilization (IVF) system without optimization of sperm concentration of individual bulls, which can obtain stable fertilization and development. We examined the effects of PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine, and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentrations (1, 2, or 5×10⁶ spermatozoa/ml) on fertilization and development of bovine oocytes in vitro.

Materials and Methods: Firstly, matured cumulus-oocyte complexes (COCs) were co-incubated with 1 or 2×10⁶ spermatozoa/ml with or without PHE mixture and/or theophylline for 12 or 18 h, and fertilization was examined. Secondly, matured COCs were co-incubated with different sperm concentrations (1, 2, or 5×10⁶ spermatozoa/ml) from 3 bulls with PHE mixture and/or theophylline for 18 h, and fertilization and development were examined. Finally, matured COCs were co-incubated with 2×10⁶ spermatozoa/ml from 8 bulls with PHE mixture and theophylline for 18 h, and development was examined.

Results and Discussion: When PHE mixture and theophylline were added to IVF medium, normal fertilization (two pronuclei) for 12 h at 1×10⁶ spermatozoa/ml became higher, and we obtained stable normal fertilization (70–80%) from 3 bulls, cleavage (80–90%), and blastocyst rates (30–50%) from 8 bulls at 2×10⁶ spermatozoa/ml. However, 5×10⁶ spermatozoa/ml was necessary when only theophylline was added to fertilization media to obtain high fertilization. In conclusion, fertilization was synergistically enhanced by PHE mixture and theophylline, and the blastocyst development became stable even if 2×10⁶ spermatozoa/ml from any bulls used for IVF.