Mammalian pre-implantation embryos are sensitive to their environment and numerous publications suggest that post-fertilization culture is the most critical period affecting embryo quality and subsequent embryo development. This study was conducted to examine the viability, twining, sex ratio and birthweight (BW) after transfer of fresh and vitrified in vitro-produced sheep embryos derived from oocytes cultured in a semi- or defined medium supplemented with fatty-acid–free bovine serum albumin (BSA) with or without hyaluronan (HA) versus fetal bovine serum (FBS) as a control. A total of 649 oocytes were matured in TCM-199 containing 2 mM glutamine, 4 mg mL–1 BSA, 100 μM cysteamine, 0.3 mM sodium pyruvate, 1 μg mL–1 oestradiol-17β and fertilized with fresh semen prepared with SOF, enriched with 20% oestrus sheep serum. Presumptive zygotes were cultured for 48 h in SOF supplemented with 1% BME-essential and 1% MEM-nonessential amino acids, 1 mM glutamine and 8 mg mL–1 BSA. On the 3rd day of culture, presumptive zygotes were divided randomly into 3 groups: (1) BSA+HA, medium supplemented with 8 mg mL–1 BSA and 6 mg mL–1 HA, (2) BSA, medium supplemented with 8 mg mL–1 BSA only, or (3) control, medium supplemented with 5% charcoal-stripped fetal bovine serum (FBS). On the 5th day of culture, media were replaced accordingly in all groups. Embryos that reached the expanded blastocyst stage by 6 or 7 days/group were recorded and transferred as fresh or vitrified. The cleavage rate, blastocyst formation, pregnancies (PR) at 40, 60 and 80 days, lambing, twinning, sex ratio and BW were all recorded. Among all groups, no significant differences were observed in cleavage and blastocyst rates. The proportion of zygotes that developed to blastocysts at 6 days was significantly higher after culture with BSA+HA vs BSA and control (67/92, 72.8%), (46/118, 39.0%) and (29/72, 40.3%), respectively. Compared with the control group, vitrified BSA blastocysts were associated with increased (P < 0.05) PRs after 60 and 80 days and lambing rates. In comparison, there were no significant differences in pregnancy and lambing rate after transfer of fresh blastocysts. There was a positive correlation between age of embryo at transfer and higher BW. The mean BWs were higher after transfer of 7-day vs 6-day embryos (3.95 and 3.37 kg), respectively. However, there were no significant differences in twining and sex ratio after transfer fresh/or vitrified treated embryos versus control group. In conclusion, the results indicate that sheep zygotes cultured with BSA+HA had an increased development to blastocysts at 6 days, but the rate of blastocyst formation was not increased, PR and lambing rate were comparable with the BSA and control groups. In addition, transfer of vitrified embryos produced in the presence of BSA significantly increased the PR at 60, 80 days and lambing rate versus control group.
Elucidation of the Interaction between Flavan-3-ols and Bovine Serum Albumin and Its Effect on Their In-Vitro Cytotoxicity
