Currently, the in vitro production (IVP) of porcine embryos suffers from low efficiency and reduced blastocyst quality. Poor outcomes from in vitro matured oocytes and in vitro fertilised embryos have limited the use of assisted reproductive technologies (ARTs) within commercial porcine herds, reducing the potential for global genetic improvement programs. It is believed that this reduced developmental competency compared to in vivo embryos is attributable to altered metabolism resulting from in vitro culture. Improper or incomplete metabolic support from the culture media leads to production of inferior embryos. Much of the prior research centres on metabolism of carbohydrates by oocytes and embryos, with the formulation of media based on this knowledge. However, oocytes and embryos also contain endogenous lipid substrates, and there is a lack of understanding as to how and when these stores are utilised. Lipids are a dense form of energy storage, and there is evidence of their metabolism by oocytes and embryos for energy generation. Porcine oocytes and embryos have higher intracellular lipid content than other domestic livestock species, and this makes them an excellent model for studying aspects of lipid metabolism in vitro. The aim of this study was to examine the impact of lipid metabolism on the acquisition of developmental competence during porcine IVP, and how this is affected by the presence or absence of exogenous carbohydrates. Stimulation or inhibition of the β-oxidation pathway was used to discern the importance of fatty acid oxidation to oocyte maturation and embryo development during in vitro maturation (IVM), in vitro fertilisation (IVF) and in vitro embryo culture (IVC). During IVM, it was identified that porcine oocytes are capable of using different substrates to compensate for deficiencies in others. While pyruvate and glucose are preferentially utilised to support maturation, upregulation of β-oxidation can compensate for a low glucose concentration and an absence of pyruvate to support nuclear maturation. Although there was no discernible decrease in lipid content associated with this, lipids provide such a dense energy reserve that any usage may have been beyond the limit of detection. Inhibition of β-oxidation in the absence of carbohydrates had a greater effect on nuclear maturation compared to inhibition in complete media. This indicates that lipid metabolism plays a minor role during oocyte maturation in the presence of carbohydrates and is likely to be more important when other substrates are deficient. Energy generation prior to fertilisation is an important factor in the developmental outcomes of subsequent embryos. Upregulation of β-oxidation for the duration of IVF increased cleavage rates, but doses above 6mM L-carnitine led to decreased blastocyst development. This effect may be attributable to the antioxidant activity of L-carnitine, with low levels of reactive oxygen species (ROS) being required at fertilisation for normal sperm function and sperm-oocyte interactions. Oocyte incubation in media supplemented with 3mM L-carnitine for an hour prior to insemination increased cleavage and improved cryosurvival of Day 7 embryos after vitrification. While ATP content of oocytes did not increase over this period, it is unclear if lipid content was reduced. Previous studies have shown that L-carnitine treatment of oocytes and embryos decreased lipid content, thereby increasing cryotolerance. It would therefore appear that there is a limited role for β-oxidation during the IVF period itself, although upregulation immediately prior to fertilisation may have beneficial effects on metabolic processes and may provide antioxidant protection leading to improved development in early cleavage stage embryos. During embryo culture, there was a greater effect of upregulating lipid metabolism seen in the absence of carbohydrate substrates than in complete media. However, this could not support embryo development to the same extent as carbohydrate substrates. Changing nutrient requirements of embryos has led to the development of sequential media, leading to the production of better quality IVP embryos. Upregulation of β-oxidation for the first three days of culture in a single media system increased embryo quality to the same extent as a sequential carbohydrate media system, implying there is some level of plasticity to embryo metabolism allowing for adaptability to different substrates. Inclusion of L-carnitine for either a three day period or the duration of culture increased cryosurvival, suggesting decreased lipid content due to increased β-oxidation activity. Similarly for oocyte maturation, β-oxidation appears to be able to compensate for carbohydrate deficiencies during embryo culture to some extent, and oxidation of lipids has a greater role in promoting embryo quality over increasing production rates. The findings reported in this thesis represent a contribution to the understanding of lipid metabolism during the in vitro production of porcine embryos. These results provide evidence to support a level of adaptability of porcine oocytes and embryos to different substrates available during maturation and culture. There is a preference shown for carbohydrates substrates, with the ability to utilise lipids to compensate for certain deficiencies. This would justify the inclusion of co-factors of lipid metabolism such as L-carnitine in culture media, to ensure that any deficiencies in other substrates might be corrected for and to promote higher embryo quality. Upregulation of β-oxidation also increased the cryosurvival of porcine embryos following vitrification, with this being a major development in the global movement of superior genetics for herd improvement programs. These findings will also have implications for improving in vitro culture of oocytes and embryos of other species, most notably advancements in human ARTs where research is predominantly limited to work in animal models. The understanding of how lipids are metabolised alongside exogenous carbohydrates will contribute to improving media formulations for better metabolic support in vitro, further to improving embryo production and quality
