Studies Related To Somatic Cells Nuclear Transfer In Porcine

1.The in vitro meiotic process and developmental competence of porcine oocytes were mainly investigated in this study.(1)There was the difference in the meiotic progress and timing from different porcine oocytes with the different folliclal diameter.Most of oocytes from the diameter over 2mm follicles were taken place the GVBD(Germinal Vesicle Breakdown)after 16h in vitro culture.The rate of oocytes in GV stage was down from 68.00%at Oh to 15.38%at 16h.After 20h in vitro maturation,the rate of diakinesis stage was up to the peak value(27.06%).The peak value of Mâ… (69.69%)appeared after 28h in virto culture.In 32～44h in vitro culture,the rate of Ana-â… /Tel-â… stage was up to the peak value(53.33%).The rate of the porcine oocytes in Mâ…¡stage was up to the peak value(63.64%)after 48h in vitro maturation.(2)The developmental compentence of oocytes in different follicular diameter was different.Maturation rates of oocytes from the group of diameter＜2mm follicles,the group of diameter of 2-6mm and the group of diameter＞6mm were 5.12%,66.63%and 46.72%,respectively.Cleavage rates of three groups were 1.42%,72.19%and 58.54%,respectively.Blastocyst rates of three groups were 0,28.76%and 23.13%,respectively.Cleavage rates of oocytes from the diameter＜2mm follicles and ones from the diameter＞6mm follicles were markedly lower than the one of oocyte from the diameter 2～6mm follicles(P＜0.05).Oocytes from the 2～6mm follicles matured faster and owned the high developmental competence.2.The effects of hormones and factors on in vitro maturation of porcine oocytes were explored in this study.It was shown that the maturation medium supplemented with hormone concentration 0.1μg/mL FSH(Follicle Stimulating Hormone)and 0.01IU/mL hMG(human Menopausen Gonadotropin)were significant difference in the maturation rate(P＜0.05)and no significant difference was found in the cleavage rate and the blastocyst formation rate(P＞0.05).The maturation medium supplemented with 0,10, 30,50,70,90ng/mL EGF(Epithelium Growth Factor),the maturation rate,the cleavage rate and the blastocysts rate in group 50ng/mL EGF were up to 66.89%,84.90%and 30.20%,respectively.The maturation rate of the group of 50ng/mL EGF was markedly higher than other groups(P＜0.05).The cleavage rate and the blastocyst rate in group 50ng/mL EGF were higher than ones of the control group.The rate of oocytes extruded the first polar body(PB₁)could be significantly improved in the maturation medium supplemented with 10 ng/mL insulin like growth factor I(IGF-I).The maturation medium supplemented with 50ng/mL EGF and 10 ng/mL IGF-I could promote the developmental capacity of the porcine oocytes in vitro(P＜0.05).Compared with the medium supplemented with TCM-199 and NCSU-23 and PZM-3 in vitro maturation and in vitro culture in porcine oocytes,the effect of the maturation rate and the cleavage rate and the blastocysts rate in TCM-199 group,NCSU-23 group and PZM-3 group were not different (P＞0.05).TCM-199 medium,PZM-3 medium and NCSU-23 medium were available and beneficial to improve the developmental potential of porcine oocytes.3.The systematical analysis on the effect of parthenogenetic activation protocols was carried out in parthenogenetic development of porcine oocytes in vitro in the present study.It was shown that(1)The treatment of concentrations of 9%EH in the NCSU-23 medium and exposure times of 10min for the in vitro matured porcine oocytes was better than one of concentrations of 9%EH in the NCSU-23 medium and exposure times of 15min.(2) Oocytes were activated with the NCSU-23 medium containing 9%ethanol for 10 min. After that,Oocytes were incubated in the 2μmol/L 6-DMAP medium for 3～4h,the cleavage rate and blastocyst rate of porcine oocytes were 82.86%and 22.86%and were significant higher than ones of 10μg/mL CHX+10 mmol/L SrCL₂(Sr²⁺)group,10 mmol/L SrCL₂(Sr²⁺)+2μmol/L 6-DMAP group,10μg/mL CHX+2μmol/L 6-DMAP group and 10μg/mL CHX+2μmol/L 6-DMAP+10 mmol/L SrCL₂(Sr²⁺)group treated for 3～4h.(3)Compared the parthenogenetic activation effectiveness in porcine oocytes with using the different activation(5μmol/L Ion treated for 5min,9%EH activated for 10min and 50 v/mm,50μs,2 times pulse).The result was shown that the effectiveness of the parthenogenetic activation in porcine oocytes with using the Ion treatment was the best and was up to 37.50%.(4)There was the significant influence in the layer number of porcine cumulus oocyte surrounding the oocytes on parthenogenesis development after parthenogenetic activation.The cleavage rate in the group of 4～6 layers of cumulus cells and thegroup of＞6 layers of cumulus cells were(68.99%&75.36%)and blastocysts rates in two groups were(32.56%&37.68%),respectively and there were significantly higher than other groups(p＜0.05).4.The isolation and culture of porcine cumulus cells,porcine fetal fibroblasts and porcine leydig cell of testis were systematically carried out in this study.The results was showed that the monolayer formation of the porcine cumulus cells needed 5～6d.Monolayer of porcine fetal fibroblasts derived from tissue explant culture and enzymatic digestive culture needed 10～11d and 8～9d,respectively.The porcine leydig cell of testis could be obtained by enzymatic digestive culture methods and the filtration methods of the steel net. The testis ledyig cell formed the monolayer in 3～5d.After thawed the frozen porcine fetal fibroblasts,the survival rate of porcine fetal fibroblasts was 68.36%.Through the culture and observation,the porcine fetal fibroblasts had the regular growth curve.5.The influence of the somatic cells nuclear transfer in porcine was explored.(1)The developmental capacity of the reconstructed embryos from oocytes maturation of different time at 28h,32h,36h,40h,44h,48h,52h and 56h was discussed through the treatment of enucleation and injection.The result was shown that the fusion rate(58.99%&56.51%) and cleavage rate(67.52%&65.73%)and blastocyst rate(22.78%,&15.96%)of reconstructed embryos at 44h and 48h in vitro maturation were higher in experimental groups.The cleavage rate and blastocyst rate of oocytes at 44h in vitro maturation were significantly high than the ones of 40h,36h,32h,28h in vitro maturation(P＜0.05).The fusion rate of 48h oocytes in vitro maturation was higher than the one of 52h in vitro maturation.(2)Compared the enucleated methods using blindness sucking method, Hochest33342 staining method and Spindle-view system method,the result was shown that enucleated rates Using blindness sucking method,Hochest33342 staining method and Spindle-view method were 76.33%,100.00%and 98.40%,respectively.The enucleated rate of Hoechest 33342 staining method was higher than the blindness sucking method and was significant difference(P＜0.05).Compared with the enucleating method using the Spindle-view system,there was no significant difference(P＞0.05).The cleavage rate of Hoechest 33342 staining method was the lowest and was significant difference in three groups(P＜0.05).There was no significant difference in the fusion rate and blastocyst rate among three groups(P＞0.05).(3)The influence of developmental capacity was explored in reconstructed embryos by using different injection methods(Intracytoplasmin injection and Injection under zona pellucid)in porcine somatic cells nuclear transfer.The result was shown that cleavage rates of reconstructed embryos by using the method of inntracytoplasmin injection and the method of injection under zona pellucid were(68.13% & 60.37%)and blastocyst rates were(6.44%& 8.08%),so there were no significant diffenence(P＞0.05).6.In this research,influences aspects of the selection and treatments of donor cells were mainly discussed in SCNT in porcine.(1)The developmental capacity of reconstructed embryos using granulous cells,cumulus cells and fetal fibroblast was compared.The result was shown that fusion rates of fetal fibroblast,granulous cells and cumulus cells were 64.74%,51.05%and 56.89%,respectively and there were significant difference (P＜0.05).The fusion rate of fetal fibroblast was high and the efficiency was better.The cleavage rate and blastocyst rate of granulous cells,cumulus cells and fetal fibroblast were no significant difference(P＞0.05).Granulous cells,cumulus cells and fetal fibroblast can also be used as the material of donor cells in reconstructed embryos.(2)The efficiency of nuclear transfer of different culture passage of fetal fibroblast after serum starvation was viewed.The result was shown that the fusion rate of 6～9 culture passage was higher than ones of 3～5culture passage and over 10 culture passage.(P＜0.05).(3)The developmental capacity of reconstructed embryos of different sex(male donor cells and female donor cells)was discussed.The result was shown that the fusion rate and the cleavage rate of male fetal fibroblast were not significant difference compared the one of female fetal fibroblast(P＞0.05).The blastocyst rate of reconstructed embryos using the male fetal fibroblast was lower than the female fetal fibroblast and was significantly different(P＜0.05).(4)The fusion rate in group of 100%contact and confluence was higher than one of the 70～80%contact group(P＜0.05).The cleavage rate of fetal fibroblast in 100%contact and confluence group was high than one of serum starvation group(P＜0.05).Blastocyst rates in groups were not significant difference(P＞0.05).(5) The cleavage rate and the blastocyst rate of reconstructed embryos by using the fetal fibroblast in routine digestion group and 4℃frozen group were markedly higher than the frozen and thawed group(P＜0.05).Fusion rates among three groups were no significantly different(P＞0.05).The result was shown that the fetal fibroblast was no suitable as the donor cells after the freezing and thawing treatment.(6)The cleavage rate and blastocyst rate of reconstructed embryos using smooth granulous cells as donor cells were higher than one of the rough granulous cells as donor cells and were significant difference (P＜0.05).Fusion rates in two groups were no significantly different(P＞0.05).(7) The fusion rate of reconstructed embryos using diameter＜15μm donor cells was lower and was significant difference compared with the fusion rate of the diameter＞30μm group (P＜0.05).The cleavage rate and blastocyst rate(63.73%&21.54%)of reconstructed embryos using diameter of 20～30μm as donor cells were higher than other groups and were significantly different(P＜0.05).(8)By using tissue explant culture method and enzymatic digestive culture method,the developmental capacity of reconstructed embryos was discussed.The result was shown that the fusion rate,the cleavage rate and the blastocyst rate of the reconstructed embryos were not significantly different(P＞0.05).