| As a main technology in embryo engineering, in vitro maturation (IVM) can providethe highly qualified mature oocytes (metaphase II, MII) for the other biotechnologies, suchas in vitro fertilization (IVF), nucleus transfer and transgene. In order to optimize thebovine in vitro maturation system, the factors influencing in vitro maturation of bovineoocytes were investigated in this study. The cumulus oocyte complexes (COCs) werecollected by aspiration from the follicles on the ovaries which were picked off from thelocal slaughter house, and the COCs were cultured in different conditions. (1). The COCswere incubated in 0, 5, 10, 20 μg/m FSH, 0, 5, 10, 20 μg/m LH, 0, 0.5, 1.0, 1.5 μg/m 17β-estrogen (E2), and 0, 0.1, 0.3, 0.5 mM sodium pyruvate respectively. It was found that FSH,LH, E2 and sodium pyruvate increased the maturation rate of bovine oocytes. The optimalconcentrations were FSH 10 μg/ml (80.18%), LH 10 μg/ml (80%), E2 1 μg/ml (76.67%),and sodium pyruvate 0.3 mM (77.5%), respectively. (2). The COCs were cultured in 10%fetal bovine serum (FBS) treated with 56℃ waterbath 30 min or not. It was found thatthere was no significant difference of maturation rate between them (P>0.05). (3). Themedium was equilibrated 0 h, 5 h, 10 h, 20 h respectively before used for COCs culture.The results showed that the 20 h equilibration had significantly higher maturation rate(80%) than 0 h, 5 h and 10 h equilibration (P<0.05). (4). The COCs were cultured in 5, 10,15 and 20/50 μl medium droplet. It was found that the culture density of 10/50 μl mediumdroplet had significantly higher maturation rate (76.67%) than 5 COCs/50 μl mediumdroplet (33.33%, P<0.05), but had no significant difference with 15 and 20/50 μl mediumdroplet (P>0.05). (5). The COCs were cultured 16 h, 20 h, 24 h, and 28 h respectively. Thepreferable in vitro maturation time was 24 h, and the maturation rate (80%) wassignificantly higher than 16 h (P<0.05), but had no significant difference with that of 20 hand 28 h (P>0.05). Specifically, the rates of type I polar body 1 (PB1) extrusion reducedgradually from 16 h to 28 h (80%, 50%, 48.78%, 31.28%). (6). The COCs were collectedfrom the follicles 2-6 mm, <2 mm, >6 mm in diameter and congestion follicles respectively.The collection rate of COCs at Grade A, B, C, D, E, F, and then the maturation rate werecompared. It was found that the morphological quality of COCs from the 2-6 mm follicleswas better than the others. The percentage of A&B oocytes from the 2-6 mm follicles(87.73%) was significantly higher than that from other size follicles (66.67~13.10%), andthe maturation rate of 2-6 mm follicle was also higher than the others (P<0.05). (7). COCswere cultured and the relation between cumulus expansion (0, +1, +2, +3, +4) andmaturation rate was investigated. The results showed that the maturation rates of +4expansion (76.43%) and +3 expansion (76.12%) were higher than that of +2, +1 and 0expansion (P<0.05). (8). The ovaries were taken back to laboratory in 2 h after slaughter,and then the ovaries were stored 0.5 h at 4℃, 15℃, 25℃, 37℃, and 39℃ respectively. Itwas found that the maturation rates at 25℃, 37℃ and 39℃ (63%, 73%, 60%) weresignificantly higher than those of 4℃ and 15℃ (P<0.05). The ovaries were stored at 25℃and 37℃ for 6 h respectively. The maturation rates were 53.33% and 26.67% respectively(P<0.05). While the ovaries were stored at 25℃ and 37℃ for 9 h respectively, thematuration rates were 6.67% and 0% respectively (P<0.05). The results show that thesupplementation of 10% FBS, 10 μg/ml FSH,10 μg/ml LH,1 μg/ml 17-β E2 and 0.3 mMsodium pyruvate is the optimal conditions fitting for local bovine oocytes in vitromaturation; the 20 h equilibration of medium before incubation is optimal for bovineoocyte in vitro maturation; the oocytes aspired from 2-6 mm follicles with more than twolayers of cumulus cells has better maturation effect after 24 h IVM; the culture density of10 COCs/50 μl medium droplet covered with mineral oil. When the ovaries are stored inshort time, the optimal storag... |
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