The objective of this experiment was to compare the effectiveness of two bulls and two different semen treatments on rates of in-vitro fertilization of in-vitro matured bovine oocytes, cleavage and development of embryos to the stage of morula and blastocyst by using a 2 × 2 factorial design. Oocytes (n=946) aspirated from follicles 2-6 mm in diameter were incubated for 20-22 h in TCM-199 supplemented with 5% calf serum, and then inseminated with frozen-thawed semen from two single ejaculates of two Holstein bulls of proven fertility. For the conventional washing method (C), semen was washed twice by centrifugation at 500 g for 5 min with modified BO medium (BO: without glucose or bovine serum albumin (BSA)) supplemented with 10 mM caffeine and 2.5 IU/ml heparin (BO-wash), and then diluted with the same amount of BO containing 20 mg/ml BSA (BO-dilute). For the Percoll washing method (P) semen was layered on a 45 to 90% percoll gradient in a 15 ml centrifugation tube. After 30 min of centrifugation at 700 g, the sperm pellet was recovered from the bottom of the tube, and then resuspended in BO-wash and double diluted with BO-dilute. With this sperm suspension insemination drops (100 μl) were prepared and the sperm was cultured with oocytes for 5 h. Then the oocytes were transferred to CR1aa supplemented with 5% calf serum for 7 days. The rates of cleavage and morula or blastocyst development of oocytes fertilized in vitro were similar in P (14.2% and 7.9%, respectively) and C (17.7% and 7.7%, respectively) methods for bull A, but these rates were significantly higher (P<0.005) with C (72.0% and 46.9%, respectively) method than with P (58.7% and 28.2%, respectively) method for bull B. In total rates of fertilization, cleavage and morula or blastocyst (combined data for C and P methods) were significantly higher (P<0.05) for bull B (83.8%, 65.8% and 33.9%, respectively) than those for bull A (42.4%, 16.0% and 7.9%, respectively). These results indicate that the Percoll washing method is not superior to the conventional washing method for in-vitro production of bovine embryos independ-ently of the significant differences between bulls.

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