| This study was performed to optimize the system for producing matured porcine oocytes in vitro, which by comparing different medium used for maturing porcine oocytes in vitro,and optimizing the system by adding some materials. The two parts of this thesis is as follows:1 The optimization on maturtion of porcine oocytes in vitroThis part was to investigate the effects between a medium 1600 (ART-1600), which is used for maturation of human oocytes in vitro, and the other two commonly used media (mM199 and NCSU-23) on the maturation and development of porcine oocytes and parthenogenetic embryos in vitro. We also compared the effects of three embryo culture medium, including PZM-3, NCSU-23 and G1/G2, on porcine parthenogenetic embryo development. The results showed that: (1) In porcine oocyte maturation in vitro experiments, the maturing rate of the ART-1600 group was significantly higher than that of NCSU-23 and mM199 groups, respectively (P <0.05, 81.6±4.9 vs 71.7±3.2 and 69.6±3.9), the rate of parthenogenetic blastocyst in the ART-1600 group was significantly higher than NCSU-23 and mM199 group (P <0.05, 22.6±3.8 vs 18.7±1.2, 17.7±1.9); (2) Adding 10% PFF in medium compared with the control group was no significant difference in maturated rate (P>0.05), but it can significantly improve cleavage rate of parthenogenetic embryos (P <0.05); (3) It was find that adding 3mg/mL BSA can significantly improve the Parthenogenetic embryos cleavage rate(P <0.05) Also found that≥6mg/mL BSA in oocy maturation medium decreased the matured rate significantly(P <0.01), and a large number of oocytes degenerated and died; (4) Compared with oocytes maturing in four well plate, that maturing in droplets and covering with mineral oil did not shrink.(5)When the PZM-3 was used to culture porcine parthenogenetic embryos, the both cleavage rate and blastocyst rate of embryo were better than NCSU23 group and the G1/G2 group, and the differences were significantly (P<0.01, 77.0±3.9 vs 63.9±4.2, 63.6±8.3, 19.0±0.3 vs 13.2±1.1,10.9±1.2). Our results showed that ART-1600 as a medium for porcine oocytes maturation in vitro(IVM) is better than mM199 and NCSU-23. As culture medium for the porcine parthenogenetic embryos in vitro, PZM-3 is better than NCSU-23 and G1/G2.2 Effects of ButyrolactoneⅠon maturation and embryo development of Porcine oocytes in vitroThis part was to investigate the effects of specific cdc2 kinase inhibitor, butyrolactone I (BL-I) on the prevention of the porcine oocytes maturation in vitro. Porcine oocytes were cultured (44 h) in medium containing different concentrations that 0,10,20,40 and 80μmol/L of BL-I. As the concentration increased, with an increasing degree of inhibition on the maturation of porcine oocytes.The percentages of oocytes remaining at GV stage were 25 %,47% and 66% and the MⅡstage rates were 55 %,38% and 9% for oocytes treated with 20, 40,and 80μmol/L of BL-I, the rates were significantly different with control ,respectively. Showed that BL inhibited the porcine oocytes maturation significantly; When oocytes were released from BL I incubation and cultured for an additional 44 h, 51%,47% and 5% reached MⅡstage in the groups treated with 20, 40 and 80μmol/L BL-I, the rates were significantly different with control, respectively.This showed that BL inhibited the porcine oocytes maturation could be reversible;A duration of BL I incubation (20h) at 20μmol/L and culture for 24h without BL-I (20h+24 htreatment group),could reduced the percentage of oocytes at MⅡs tage compared to the control group (culture for 44h without BL-I ) (P < 0.05,65.6% vs 70.1%). When the matured oocytes from 20h+24h treatment group were parthenogenetic activated, the cleavage rate, blastocyst rate and blastocyst total cell number had no significant difference compared with the control group.When the matured oocytes were used for nuclear transfer, the cleavage rate,blastocyst rate and blastocyst total cell number were better than the control group, but only significant difference in cleavagerate (P <0.05, 69% vs 62 %); Indicated that matured oocytes treated with BL-I still maintain a certain capacity of parthenogenetic embryos, and the proper concentration of BL-I could slightly improved the development ability of NT embryos. |
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