The Effects of Sperm Pre-Incubation Times on the Cleavage, Development and Sexing of Bovine Embryos

Abstract

We assessed the effect of various sperm pre-incubation times (0, 5, 10, 15 and 20 h) on the cleavage, blastocyst rate and sexing of bovine embryos. Cumulus oocyte complexs (COCs) were cultured in maturation medium containing TCM-199 supplemented with 5% superovulated cow serum (SCS), 0.002 AU/ml FSH and 50 μg/ml gentamicin for 22 h at 38.5°C with 5% CO₂. Frozen-thawed spermatozoa were pre-incubated in Brackett and Oliphant's medium containing 2.5 mM caffeine and 3.6 IU/ml heparin at various times. After 5 h of fertilization, the COCs were cultured in culture medium containing TCM-199 supplemented with 5% SCS, 5 μg/ml insulin and 50 μg/ml gentamicin. The cleavage and blastocyst rates were observed on days 2 and 8. Frozen-thawed blastocysts were bisected and analyzed for sexing by using PCR and XY selector. The cleavage rate was higher (P<0.01) in the groups of sperm which were cultured for 0 (80.5%) and 5 h (77.5%) than in the other groups (10 h; 61.5, 15 h; 65.7 and 20 h; 24.7%). The blastocyst rate was higher (P<0.01) in the group of sperm which was cultured for 0 h (32.1%) than in the other groups (5 h; 22.8, 10 h; 18.4, 15 h; 20.1 and 20 h; 8.4%). In addition, the sexing rates of blastocysts were not directly related to the in vivo sperm culture period.