Improving biosecurity of bovine in vitro embryo production and cryopreservation

The experiments reported in this thesis were conducted to overcome limitations in conventional processes of in vitro-production of embryos and to develop novel procedures for cryopreservation of bovine oocytes and embryos. The experiments, concentrating initially on unfertilized oocytes and subsequently on embryos, used methods relevant to livestock reproductive biotechnology. Key aims were to overcome reliance on media using animal-derived constituents such as serum and albumin. Bovine oocytes matured in vitro in a novel biosafe formulation (MM1mat) excluding serum and albumin reached blastocyst stages (after in vitro fertilization) at rates equivalent to those achieved with conventional protocols (P>0.05). Experiments that investigated development of bovine zygotes in an original series of protein-free culture media (V1 for embryos to Day 4 post-fertilization; V2a and similar formulations for Day 4 onwards) showed that sequential culture in V1/V2a did not compromise development to the blastocyst stage when compared to ‘monoculture’ in an albumin-supplemented medium (P>0.05). Blastocysts produced in V1/V2a survived better after vitrification than counterparts produced in presence of albumin. Vitrification solutions (also often reliant on harmful serum-supplemented media) could be superseded by novel alternatives using V2a as a biohazard-free base medium. It was concluded that bovine blastocysts can be produced and cryopreserved safely in the novel media. A further important aspect of the investigations was that they developed a new biosecure ‘closed system’ for cryopreservation of oocytes and embryos. That system employed the CryoTip –which could be sealed before contact with liquid nitrogen – and the subsequent metabolic and biochemical studies on V1/V2a-produced blastocysts, either fresh or vitrified, indicated pyruvate metabolism, amino acids depletion and peroxide status parameters were equivalent to controls.EThOS - Electronic Theses Online ServiceGBUnited Kingdo