Title:
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Fatty acid and antioxidant effects on development in vitro of bovine and ovine embryos
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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.
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