| Porcine ovaries were obtained from Changsha Wulipai abattoir.Cumulus-oocyte complexes (COC) with more than three layer' s compact cumulu cells were mechanically isolate& from 3~-6 mm sized ovarian surface follicles. The COC were cultured in vitro to study the porcine ovarian oocyte' s in vitro maturation (IVM), in vitro fertilization (lyE) and in vitro culture (IVC) of the early embryos. The results were presented as follows:1.Oocyte' s in vitro maturation In the experiment, COCs were cultured in 300 microliters droplets (38. 8~C, 5%C02, 100% humidity). After cultured 42-44h, the cumulu cells expansion, some oocytes extrusion the first polar body (PBI). mTCM199-'-lO%NCS(v/v) and NCSU23-4-l0%PFF(v/v) were used to study their effects on oocyte' s IVM. The results show that, when supplemented with l5iu/ml PMSG and HCG the maturation rate were 41.2±3.%98 and 38.28±4.02% (P>0.03); When supplemented with 2Oiu/ml PMSG and HCG the maturation rate were 44. 8±5. 34% and 39. 12±l.25%(P>0. 05). When supplemented 0, 10%, 13%, 20%(v/v) NCS(New-born cattle serum) into mTCM199-15iu1"ml PMSG--lSiu ml HCG, the maturation rates were 0,37.78±1.92%, 43. 33±2.72% 43 33±3.85%, maturation rates were remarkbabiy different (P<0.0l) between the groups with lO%-----20%(vlv) serum and the group without supplemented serum, the difference of the maturation rate among 10% and 20% or 15% and 10% were significant (P<0. 05), the difference of the maturation rate among 15% and 20% was not significant (P>0. 05) . When supplemented 0, lOiu/ml, lSiu/ml, 2Oiu/ml PMSG and L-JCG into mTCM199~-l5%NCS, the maturation rates were 0, 35.56±1.92%, 41.2±3.98%, 46.27±7 36% the maturation rates between 0 (group) and l0iui'mb- 20iu11m1 (groups) were remarklablv different (P<0. 01); between l5iu/ml and 2Oiu ml or lSiu,"'mi and lOiu/ml were not significantly differen? (p>O.OS> between 2Oiu"ml (group) and lOiu/ml (group) were3significant different(p<0. 05). The results showed that add serum~ PMSG and HCG can improve oocyte' s maturation rate in vitro.2.In vitro fertilization Fresh sperm were capacitated in medium mTBM+100 ~ g/ml Heparin with the "swim-up" method. After capacitated 40- 60 mm. the sperm and the maturation oocytes were incubated in mTBM+50 ii g/ml Heparin (39. 40C, 5%C02, 100% humidity) for Gb. After 24'-28h some fertifized eggs extrusion the second polar body (PB II) and some fertilized eggs were clevaged. The clevage rate was 37. 95%(48-50h).3.Early embryo' s in vitro culture After in vitro fertilization, the fertilized eggs were cultured in three systems. I .TCM199+l5%NCS, II. Porcine oviduct epithelial cell monolayer (POEC)+TCM199-r15%NCS, III. granule/cumulus cell monolayer(G/C>TCM199+l5%NCS. The culture condition was 38. 80C, 5%C02, 100% humidity. Their development rates were 38. 49±6. 59% 37 19±5.19%,37.85±4.25%(48 -~ 50h), there were no significant different (P>0. 05) between them. In group I, the embryos were blocked at 4 cell stage, in group II and Ill some embryos can surmount 4 cell stage block and developed to 8-~l6 cell stage and morula. The morula rates were 0 12 84± 6. 04%, 4. 76 ±6. 73%. The difference of the morula rates among group II and I was remarkably (P0.05).In sumftiary, under the present culture conditions, the porcine ovarian oocvtes can matured in vitro, fertilized in vitro and developed to early embryos. When the early embryos co-cultured with monolayer porcine oviduct epithelial cells or granual/cumulus cells some of them can surmount the 4 cell stage block and developed to morula...
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