156 INFLUENCE OF SERUM AND BSA ON BLASTOCYST DEVELOPMENT AND HATCHING IN IVF AND NUCLEAR TRANSFER BOVINE EMBRYOS

Abstract

Important differences exist between in vivo- and in vitro-produced bovine embryos. Studies have shown that various components in culture media affect embryo development, with serum producing some of the more detrimental effects. Efforts to develop a serum-free culture system have included looking at the effects of BSA, polyvinyl pyrrolidone and polyvinyl alcohol on embryo development. In this study, we compare serum and BSA during oocyte maturation and embryo culture of IVF and nuclear transfer (NT) embryos. Experiment A: Oocytes were aspirated from follicles and matured in either collection medium (Hams F-10 + 2% steer serum (SS); F-10) or in follicular fluid alone (FF). They were subjected to IVM-IVF-IVC as follows: 20–22 h maturation in synthetic oviductal fluid +8 mg mL⁻¹ fatty acid-free BSA (SOF + BSA-FAF) supplemented with hormones, 18 h co-incubation with sperm in IVF-TALP, and culture for 9 days in SOF + BSA-FAF. Experiment B: Oocytes were randomly distributed for IVM-IVF-IVC into the following treatment groups: (i) IVM and IVC in SOF + 2% SS (SER), (ii) IVM in SOF + 2% SS and IVC in SOF + BSA-FAF (SER-FAF), (iii) IVM and IVC in SOF + BSA-FAF (FAF), and (iv) IVM and IVC in SOF + BSA-FrV (FrV). Experiment C: Oocytes were matured for 18 h in either SOF + 2% SS (SER) or SOF + BSA-FAF (FAF). Couplets were constructed with adult skin fibroblasts, exposed to a single pulse of 1.5 kV cm⁻¹ for 40 s and activated using ionomycin and cycloheximide. Embryos were cultured in SOF + BSA-FAF. Three replicates with 100–120 oocytes per treatment group were carried out. Results: Cleavage rates were similar among all treatments in experiments A and B. No differences were observed between oocytes collected in F-10 or FF indicating that short-term exposure to serum does not have long-term effects on embryo development. Although a higher number of blastocysts was observed in the SER group on Day 6 (3.2% v. <0.5%; P < 0.05), no differences were seen in blastocyst development among the IVF treatment groups from Day 8 onwards (SER: 29.7%, SER-FAF: 21.1%, FAF: 20.4%, FrV: 19.9%). However, hatching rates on Days 8 and 9 were significantly greater (P < 0.05) in groups with serum, with the exception of FAF on Day 9 (SER: 31.1%, 57.2%; SER-FAF: 29.4%, 50.6%; FAF: 23.1%, 46.4%; FrV: 18.5%, 34.2%). In the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes at 18 h, better oocyte quality for manipulation, and increased blastocyst development and hatching rates (SER: 31.4%, 18.2%; FAF: 21.7%, 4.8%). These results indicate that both serum and fatty acid-free BSA provide comparable embryo development during IVF. However, development in serum occurs at an accelerated rate as indicated by the shorter nuclear maturation times and blastocyst development on Day 6, which has been associated with adverse outcomes. Despite this, serum may provide the oocyte with factors that are important for membrane flexibility and repair, enabling greater survival after manipulation. Funding from NSERC and OMAFRA. Sperm provided by Gencor.