Studies On Porcine Oocytes In Vitro Maturation And Parthenogenetic Activation

This study was conducted to establish optimum maturation conditions of porcine oocytes in vitro maturation(IVM) through different experiments to research the main factors which affect the porcine oocytes IVM.The thesis was also undertaken to determine various methods that influnence the parthenogenetic development at the same time.The results show:The maturation rates when it was cultivated for 44h and 46 h (57.56±2.80%vs61. 40±14.44%)were significantly higher than that cultivated for 40h (27.26±6.54%)in vitro(p<0.05).The optimum time of porcine matured in vitro is 44-46h. The results show that maturation rate were no difference between the condition in high O₂ tension (82.74±5.15%) and low O₂ tension (79.59±0.92%),(p>0.05).The low O₂ tension can not increase the maturation rate on porcine oocytes IVMThe porcine oocytes were cultivated in such five different maturated culture medium as TCM-199+FCS,TCM-199+ SP,NCSU23+FCS,NCSU23+SP and NCSU23(no serum) respectively.The rate of cleavage and blastocysts of porcine oocytes maturated in TCM-199+FCS(74.44±5.45%, 17.03 ± 8.55%) and TCM-199+ SP(79.22±2.45%,20.42±6.58%) were no difference(p>0.05),however the cell number of the blastocyst from TCM-199+ SP(38 ± 4.83) was significantly higher than that from others(p<0.05).The rate of cleavage in NCSU23+SP(62.81±4.44%) is significantly higher than that in NCSU23+FCS(47.25±2.85%) and NCSU23(no serum)( 47.80±3.08%),(p<0.05). The maturation rate and the rate of cleavage of porcine oocytes cultured in culture medium without growth factor added (62.61±4.97%, 58. 72±0.70%)was significantly lower than that in culture medium added with bFGF(72. 94 ±1. 33%, 72.07 ± 5.75%) or EGF(79. 54 ± 2.87%, 75. 87 ± 5. 69%),(p<0.05). The maturation rate ,the rate of cleavage and blastocyst and the blastocyst numbers were no difference when it was cultured in culture medium with bFGF or EGF.Different electrical activation methods were studied in order to choose the best one . The rate of cleavage and blastocyst of porcine oocytes after Electric/ Chemic activation(72.90±5.59%, 18.08±3.69%) was higher than other two methods(ethanol alone:8.61 ± 1.43%, 1.44±2.50% ;ionomycin: 17.58 ± 1.42%, 6.93 ±0.90%).The data show that the Electric/ Chemic activation is the first election .The ionomycin on the porcine oocytes activation took second place and the ethanol alone on the activation was the last.The different Ca²⁺ concentration(0.05mM Ca²⁺vs0.1mM Ca²⁺) can not influence the rate of cleavage, blastocyst and blastocyst number of porcine oocytes after activation.When the field strength was 0.8kv/cm (81.31±9.03%, 17.09 ± 2.53%)and 1.2v/cm(72.90±5.59%, 18. 08± 3.69%),the rate of cleavage and blastocyst of porcine oocytes after Electric/ Chemic activation was significantly higher than when the field strength was 1.6 kv/cm(50.96±7.29),(p<0.05). The optimum pulse duration is 40us with which got the highest cleavage rate and the higher blastocyst rate (88.86± 0.62%, 18.45±2.30%)among the pulse duration 30μs,40μs and 50μs.The rate of cleavage of electric combinating with CB+CHX (79.55±3.85%)was siganificantly higher than the treatment CB(72.90 ± 5.59%) and CHX(71.55 ±3.78%) on the parthenogenetic embryonic development in vitro.