266 IN VITRO-DERIVED EMBRYO PRODUCTION WITH SEXED AND UNSEXED SEMEN FROM DIFFERENT BULLS

Abstract

The production of pre-sex-selected calves by in vitro fertilization (IVF), using sexed semen, does show some benefits due to the small quantity of sperms needed for the process as compared to other reproductive technologies. The objective of this study was to determine differences among bulls and sperm concentrations in embryo development with sexed and unsexed semen. Follicles ranging from 2 to 6 mm in diameter were aspirated from slaughterhouse ovaries. COC were selected and matured in groups of maximum of 30 in 1.8 mL of TCM-199, supplemented with 10% fetal calf serum, 0.01 U mL⁻¹ bFSH, 0.01 U mL⁻¹ bLH and 10 μL mL⁻¹ penicillin-streptomycin for 24 h at 38.5°C. Fertilization (Day 0) was carried out in micro-drops (50 μL) with TALP-FERT medium containing PHE (3 μg mL⁻¹ penicillamine, 11 μg mL⁻¹ hypotaurine and 0.18 μg mL⁻¹ epinephrine), 10 μL mL⁻¹ non-essential amino acid and 2 μg mL⁻¹ heparin. Frozen/thawed sexed (female) and non-sexed sperms from five bulls were selected in a discontinuous percoll gradient. Sperm concentration was 1 × 10⁶ for non-sexed semen and 1 × 10⁶ or 2 × 10⁶ for sexed semen. After 18–20 h, presumptive zygotes were denuded and cultured in groups of 10 in 50-μL micro-drops of SOF citrate with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700) under paraffin oil in a 5% O₂, 5% CO₂, 90% N₂ atmosphere with high humidity. On Day 7, blastocysts (BL) were morphologically evaluated and recorded. Results are shown in Table 1. Data was compared by chi-square analysis. Sexed frozen bovine sperm can be used successfully in IVF systems. More research needs to be done to optimize and standardize bovine in vitro fertilization with sexed semen.

Table 1 
Results of comparisons between bulls, sperm concentrations, cleavage and embryo development