Studies on in vitro maturation of dog oocytes to improve maturation rate and development potentials
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AbstractIn vitro maturation of dog oocytes has always been the main obstacle preventing reproductive biologist from producing canine in vitro cultured embryos. The unsuccessful oocyte maturation in canine species originates from their unique physiological and biological specifications. Ovulation of dominant follicles in bitch (6-12 in each oestrous cycle) occurs at prophase I stage of oocyte nucleus and meiotic resumption develops during 3-5 days of oviductal transition. During this PhD thesis, studies were designed in order to speculate characteristics of canine oocyte maturation in vitro in terms of maturation media components, gas composition of the incubator and hormonal requirements. Level of oxidative stress during 72h (culture period) of in vitro maturation showed that 5%O2, 5% CO2 and 90% N2 composition improves meiotic resumption and reduces degeneration rate significantly compared to 5% CO2 in air. Utilization of caffeine as a non specific phosphodiesterase inhibitor at 10mM for 12h at the beginning of the 72h culture (12+60) also improved MII maturation rate (16.9% ± 2.4; P < 0.05). Among several hormonal treatments recombinant porcine Growth Hormone (PGH) at 100ng/ml and Melatonin (MTN) at 100nM concentrations had outstanding improvement over meiotic resumption (28.9% ±10.0 and 56.2% ±8.6 respectively; P < 0.05). Attempts were made to study developmental potentials of optimally matured oocytes by parthenogenetic activation (PA) and in vitro fertilization (IVF) using chilled semen. Partial digestion of the zona pellucida prior to IVF improved the cleavage rate at 48h 6.4% ± 0.3 and resulted in production of a single 8 cell embryo. Moreover; canine follicular cells were culture in order to characterize their primary culture morphology and steroidogenic responsiveness to physiological and pharmaceutical substances. Immunolocalization of aromatase (CYP19) positive cell clumps, presumptive oestrogen producing colonies, was identified. This primary culture also maintained its steroidogenic machinery up to 96h (measured by radioimmunoassay) with a significant increase in production of estradiol and progesterone after 72h compare to the start of the culture.
CitationSalavati, M. (2013) 'Studies on in vitro maturation of dog oocytes to improve maturation rate and development potentials'. PhD thesis. University of Bedfordshire.
PublisherUniversity of Bedfordshire
TypeThesis or dissertation
DescriptionA thesis submitted to the University of Bedfordshire, in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Institute of Biomedical and Environmental Science and Technology (iBEST)
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