Fatty acid and antioxidant effects on development in vitro of bovine and ovine embryos
This thesis provides evidence that in vitro produced ruminant embryos accumulate lipid during culture in the presence of serum; the increase due to serum in the area occupied by lipid droplets was 30 to 40% (P<0.001). Innovative HPLC techniques revealed similar increases in fatty acid content of bovine (from 57 to 74 ng embryo-1, P<0.05) and ovine (from 73 to 84 ng embryo-1, P<0.001) embryos when foetal calf serum (FCS) was used as a media supplement. For ovine embryos, their total fatty acid content reached 130 ng embryo-1 in a polyunsaturated fatty acids (PUFA)-enriched medium (P<0.001). Serum-exposed embryos benefited from supplementation of their culture medium with Vitamin E, an inhibitor of lipid peroxidation. Despite a >20% increase in the total fatty acid content of embryos cultured in serum-supplemented medium, Vitamin E significantly improved blastocyst yields and cell proliferation rates. For bovine embryos cultured with FCS-supplemented medium, yields increased from 20 to 33% (P<0.001) and, for ovine embryos in a PUFA-enriched medium, from 47 to 64% (P<0.001). It was only when PUFA-enriched serum was used in the culture of ovine embryos, that Vitamin E prevented a decline in pyruvate oxidation (P<0.05), indicating maintenance of mitochondrial activity. It also precluded increased generation of H2O2 and lipid peroxides (P<0.05). Lipid accumulation increased from 111 to 130 ng embryo-1 when ovine embryos were cultured in the presence of albumin supplemented with docosahexaenoic acid (DHA, C22:6n-3, P<0.05). DHA increased the production of peroxides (P<0.001). It also inhibited embryo development, completely, beyond the 9- to 16-cell stage (DHA concentration = 1.7 mg ml-1 medium) and partially when its concentration was lowered (0.5 mg ml-1 medium, P<0.001). Partial inhibition involved a reduction in cell proliferation (P<0.05) and pyruvate oxidation (P<0.001) and increased apoptosis (P<0.05), features avoided by inclusion of Vitamin E or Trolex, a water-soluble analogue, in the culture media.