Effect of cumulus cells and duration of culture on nuclear maturation of oocytes of pubertal and pre-pubertal individuals from Sus scrofa domesticus

Abstract

Objective: to evaluate the nuclear maturation ability of pig oocytes in relation to the morphology of the surrounding cumulus cells, the duration of in vitro maturation (IVM) and the physiological status of cumulus-oocyte complexes (COCs) donor animals.Materials and methods. СOCs collected from cycling (pubertal) and non-cycling (prepubertal) pigs ovaries were categorized according to the number of cumulus cell (CC) layers: COC I – more then 4, COC II – 3-4, COC III – 1-2 and СOC with granulosa cells (GCOC). Oocytes within COCs were matured during the first 22 h of culture in the TC-199 medium with 25 mM HEPES, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.4% BSA, 10% follicular fluid, 0.5 μg/mL FSH, 0.5 μg/mL LH and 50 μg/mL gentamicin sulfate and thereafter in medium of the same composition but without hormones. After 42, 44, 46, 48 hours of culture the maturation of oocytes of all categories and age groups was assessed by the sign of their release of the first polar body. Results. It was shown that at least 42 hours of in vitro culture of pubertal pig oocytes are required to ensure a high level of nuclear maturation. The duration of the in vitro maturation was found to be 44-48 hours for pre-pubertal pigs. Oocytes surrounded by 1-2 layers of CC, when cultured for less than 48 hours, are inferior in nuclear maturation to oocytes with a more multilayered cumulus. The presence of GC (granulosa cells) around COC retards retards the progression of nuclear maturation when cultured for less than 42 hours in prepubertal and 44 hours in pubertal gilts. Oocytes from cycling pigs reach peak maturation earlier than those from non-cycling gilts and can be used in assisted reproductive technologies (ART). Conclusions. It was concluded that both morphology of the surrounding cumulus cells and physiologic status of COC donor animals should be considered when choosing the duration of pig oocytes culture for the purpose of their in vitro maturation.