Production of bovine embryos in vitro through in vitro fertilisation (ivf) and intracytoplasmic sperm injection (icsi) techniques / Nor Fadillah Binti Awang

The objective of this study was to develop an optimal in vitro produced embryos (IVP) protocol using two methods of insemination, namely in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) protocols by using different qualities of oocytes in Malaysian cattle. The ovaries were collected from slaughtered cattle from the local abattoir. The oocyte morphology was observed and grouped into three groups: Groups A, B and C, based on cumulus cell compactness. Then the oocytes were subdivided based on the cytoplasm quality, namely homogenous and heterogenous (Experiment 1). The oocytes were matured in humidified 5% CO2 incubator at 38.5°C at different maturation durations of 20, 24 and 28 hours (Experiment 2). The matured oocytes were fertilised with frozen-thawed sperm before being cultured. The cleavage rate and embryo development were evaluated. The serum was added at every embryo development stage to develop up to blastocyst stage (Experiment 3). In ICSI study, the different oocytes and sperm groups were used (Experiments 4 and 5) to produce ICSI embryos. Finally, comparison was made between IVF- and ICSI-derived embryos (Experiment 6). The oocyte quality (the presence of cumulus cells and different cytoplasmic group), IVM duration and culture medium supplement were considered among the important factors affecting the maturation, fertilisation and culture competence of bovine embryos during IVMFC procedures to produce IVF-derived embryos. In Experiment 1, the percentage of matured-fertilised oocytes differed between the three groups of oocytes. Homogenous cytoplasm, Grade A oocytes gave the highest 2-cell cleavage rate (78.30%) followed by Grade B (65.92%) and Grade C (24.02%) iii compared to heterogenous cytoplasm with 50.90%, 30.48% and 11.66%, respectively. It was consistently shown that homogenous cytoplasm gave higher cleavage rates for all the grades of oocyte. It was also observed that blastocysts were obtained for Grade A – Homogenous (28.55%), Grade A – Heterogenous (2.66%) and Grade B – Homogenous (14.40%) oocytes. In Experiment 2, 24 hours IVM duration gave better results (P