| The technology of immature porcine oocyte's vitrification and maturation has become the basic technology of modern agriculture as well as biomedical study, however, the many problems have not be resolved, especially vitrification of immature porcine oocyte. For resolving these difficulties, in present study, we studied the effect of some factors on maturation rates after vitrified-thawed immature porcine oocytes, and these factors as follow: membrane permeating, membrane non-permeating, the processes of vitrified-thawed, different pretreatments. It is the first time for Lycium barbarum polysaccharide (LBP) to be studied as membrane non-permeating cryoprotectant. In addition, the effect of the volume of culture medium, growth factor, porcine follicular fluid and co-culture medium on the maturation in vitro have been studied. The results showed:1. The maturation rate of the group adding ethylene glycol (EG, 12.9%) was significantly higher these adding 1, 2-propanediol (PROH, 3.7%), glycerol (GLY, 1.9%) and control (0.0%, P<0.05), but was not significantly different from that adding dimethyl sulfoxide (DMSO, 8.6%). When using mixture of two cryoprotectants, the highest maturation rates was obtained in EG +DMSO group (9.6%), was significantly higher than PROH+GLY (0.0%) group and control (0.0%, P<0.05).2. In glucose group, adding 1.5 M obtained the highest maturation rate (8.7%), but was not significantly different from other concentrations (P>0.05). In sucrose group, the highest maturation rate (19.9%) when adding 0.75 M was significantly higher than that adding 0.25 M (8.1%, P<0.05), but was not significantly different from those adding 1.0 M (16.9%) and 0.5 M (18.6%, P>0.05). The highest maturation rate (13.4%) was obtained in the group adding 3.0 g/ml ficoll, was not significantly different from those adding other concentrations ficoll. In addition, the highest maturation rate was obtained in 0.10 g/ml LBP (21.9%), and it was significantly higher than those in 0.05 M (10.5%, P<0.05) and 0.025 M (6.9%, P<0.05), but it was not significantly different from that vitrified in 0.075 g/ml LBP (18.7%, P>0.05).3. The maturation rates of six-step (12.3%) and three-step treatment (11.1%) were significantly higher than that using one-step treatment (1.3%, P<0.05). The maturation rates of four-step (12.7%) and three-step thaw (10.5%) was significantly higher than that using one-step thaw (6.7%, P<0.05), but was not significantly different from that using two-step thaw (7.3%, P>0.05). When using cytochalasin B, the maturation rate of oocyte exposed to vitrification solution long time (17.3%) were significantly higher than that short time(4.8%,P<0.05), and without cytochalasin B, the maturation rate of oocyte exposed to vitrification solution long time (6.9%)were significantly lower than that short time(16.4%, P<0.05).4. In vitro maturation, the maturation rates using 500 ul (73.6%),750 ul (73.3%) and 1000 ul (69.1%) culture medium were significantly higher than that culture in 50 ul drop (52.4%, P<0.05), and were not significantly different from that using 250 ul medium(66.0%, P>0.05).5. In vitro maturation, the maturation rate using 10 ng/ml EGF (75.7%) was significantly higher than that using 10 ng/ml FGF (60.5%) and control (57.9%, P<0.05), but was not significantly different from that using 10 ng/ml IGF-1(68.0%, P>0.05).6. In vitro maturation, the maturation rates using porcine follicular fluid (PFF) in follicular term (69.8%) , in ovulation(65.5%) and luteal term (68.4%) were not significantly different from that control (69.0%, P>0.05). But the maturation rate using PFF in lean type term (54.4%, P<0.05) was significantly lower than that control (69.0%, P<0.05).7. In vitro co-culture medium, the maturation rate co-cultured with granulose cells (75.8%) was significantly higher than that control group (66.7%, P<0.05). Co-culturing with oviduct epithelial or pancreas cells could improved the maturation rates, but it was not significantly different from that control (P>0.05). |
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