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Cytoplasmic maturation as determined by male pronuclear formation following fertilization in vitro was examined in pig oocytes cultured under different hormonal conditions during either the first or second 20 h period of in vitro maturation. Exposure to several combinations of pregnant mares' serum gonadotrophin (PMSG) (10 iu ml–1), hCG (10 iu ml–1) and oestradiol (1 µg ml–1) for a second 20 h period following culture in a medium supplemented with these hormones for 20 h did not result in differences among treatment groups in maturation rates, penetration rates or polyspermy rates. However, supplementation with PMSG and oestradiol for the last 20 h of culture reduced male pronuclear formation rates significantly. When oocyte–cumulus complexes were cultured in hormone-free media for 20 h after culture in several combinations of supplemental hormones for the first 20 h period, germinal vesicle breakdown rates and maturation rates were lower in oocytes previously exposed to oestradiol alone or no hormonal supplements (68–70% and 45–49%, respectively) than in oocytes previously exposed to PMSG or hCG (89–99% and 71–89%, respectively). Exposure of oocytes to oestradiol alone also reduced the penetration rate (61%) compared with PMSG or hCG (86–99%). Supplementation of media with PMSG alone or together with other hormones increased the male pronuclear formation rate (63–72%) compared with supplementation with oestradiol (33%) or no hormonal supplements (32%). The concentration of oestradiol in maturation medium decreased at filtration (to 216.5 ± 72.6 ng ml–1) before culture. Under paraffin oil, the concentration further decreased during equilibration (to 128.0 ± 13.3 ng ml–1), during the first 20 h of culture (42.8 ± 19.4 ng ml–1) and also during the second 20 h period of culture without hormonal supplements (0.925 ± 0.544 ng ml–1). The concentrations of progesterone in maturation media under paraffin oil were lower throughout maturation (1.0 ± 0.2 ng ml–1 at 0 h to 6.9±1.5 ng ml–1 after 40 h). These results demonstrate that at least two different hormonal conditions during maturation, which are the presence of PMSG during the first 20 h of culture and the absence of PMSG and oestradiol during the second 20 h of culture, are beneficial to meiotic and cytoplasmic maturation of pig oocytes. Furthermore, it was demonstrated that oocyte–cumulus complexes under paraffin oil were exposed to extremely low concentrations of steroids during maturation.
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