A 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)In 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