In Vitro Production Of Embryos And Development Of Parthenogenetic Blastulas In Pigs

Establishment of a more mature technology of in vitro production porcine embryos, improving its operating procedures to improve the productivity of pig embryos, can lay the foundation for use of porcine embryos in vitro to isolate and culture embryonic stem cell. Presently porcine oocytes in vitro maturation, in vitro fertilization and parthenogenetic activation etc. has made a great progress, However, some aspects remain to be optimized; The development capacity of embryos produced in vitro, especially whether can make use of pig embryos in vitro production to isolate embryonic stem cell has not gain a breakthrough. In this study, the pig slaughterhouse ovaries and the pig farm fresh semen are used for raw materials, production of porcine embryo in vitro fertilization and parthenogenetic activation and the developmental capacity of parthenogenetic blastocysts on feeder layer were researched, aim to improve the production systems of porcine embryo laboratory and approach the post-development capacity of parthenogenetic blastocysts, This study can Provide a reference and basis on porcine embryos in vitro production and separation of embryonic stem cell research. Test results are as follows:(1) Number of effective oocytes obtained from each ovarian from slaughter house were statisted and summarized at different month locally: Effect of different time of PMSG and hCG added to the oocyte maturation medium on oocyte maturation was approached. It was concluded that: Months of August, September, October and April were the best time to collect ovarian for experiment,; Aspects in culture, the maturation rate of oocytes in group added gonadotropic hormone and group added gonadotropic hormone later 22h was 80.35±0.76%and 82.75±0.85%, which had no significant difference (P>0.05).(2) The relation between time of porcine semen preserved at 17℃constant temperature and sperm motility was discussed, and the effect of fertilization also be discussed. The results showed that, porcine sperm motility can maintain in 0.9 when semen preserved within 24h, sperm motility can maintain above 0.7 when semen preserved for 3 days, the motility decreased below 0.5 after semen preserved for 3days; The semen preserved within 1 days, compared with 4-5 days, used for fertilization, the cleavage rate, morula rate and blastocyst rate of zygotes had significant difference (P<0.05), The semen preserved within 3 days used for fertilization, the cleavage rate, morula rate and blastocyst rate of zygotes had no significant difference (P>0.05), The semen preserved within 4-5 days used for fertilization, the cleavage rate, morula rate and blastocyst rate of zygotes had no significant difference(P>0.05).(3) The effect of development of zygotes on the Incubation time and RA were discussed. The results showed that, oocytes co-incubated with sperm after 6h and 7h, the cleavage rate of zygotes was 55.90±9.77% and 54.51±13.25%, which had no significant difference(P>0.05), oocytes co-incubated with sperm after 5h and 8h, the cleavage rate of zygotes was 52.31±4.84%and 53.85±7.16%, which had no significant difference(P>0.05), but in the aspect of blastocyst rate, group of 6h was 11.89±3.64% ,which had significant difference(P<0.05) compare with group of 6h vs 8h(5.55±0.63% vs 3.85±2.12%); The concentration of RA (0nM,5nM,25nM and 50nM) were added to maturation medium, oocytes cultured in these mediums and then used for fertilization, the cleavage rate, morula rate and blastocyst rate of zygotes had no significant difference(P>0.05), but groups of 0nM and 5nM were higher than groups of 25M and 50nM in rate of blastocyst.(4)The effect of different electric parameters on the development of oocytes matured in vitro after parthenogenetic activation was discussed. The results showed that, the electric parameter of 1.5Kv/㎝, 1DC, 100μs can activate oocytes matured in vitro to develop to blastocyst, rate of blastocyst was 17.13±0.34%, had significant difference compared with the group of 1.5Kv/㎝/1DC/60μs and 1.5Kv/㎝/1DC/80μs (P<0.05); When the electric parameter was 2.0Kv/㎝,1DC,60μs, the rate of blastocyst was 18.70±0.93%, had significant difference compared with the group of 2.0Kv/㎝/1DC/100μs (P<0.05). It had no significant difference when the pulse frequencies were 1DC or 2DC.(5)The development capacities of Parthenogenetic blastocysts in vitro were discussed. The results showed that, when the small molecules CHIR99021 and PD0325901 were added to the medium which contained feeder layer cells to culture parthenogenetic blastocysts, the rate of blastocyst developed to attachment had no significant difference compared with the the control group (P>0.05); The CEF as feeder cells was used to culture parthenogenetic blastocysts, the rate of attachment of blastocysts were 33.67±9.84%,which had significant difference compared with the the group of MEF and PEF(19.08±0.52% vs 23.47±5.69%) (P<0.05), MEF had no significant difference compared with PEF on the aspect of supporting blastocyst cells to attachment (P>0.05); Using NCSU-23 to culture parthenogenetic blastocysts, the rate of attachment of blastocysts was 22.53±2.46%, significantly higher than groups of DMEM and DMEM/NCSU-23 (10.41±1.75%vs 12.05±2.23%)(P<0.05).To sum up, the best time that collecting ovarian for laboratory using were August, September, October and April in this region; The sperm motility can maintain above 0.7 when the porcine fresh semen preserved within 3 days at 17℃constant temperature,which all can be used for fertilization; It had best development capacity when sperm were co-incubated with oocytes for 6h; The electric parameters of 1.5Kv/㎝/100μs/1DC or 2DC and 2.0Kv/㎝/60μs/1DC or 2DC all can effectively activate oocytes in vitro maturation to develop to the blastocyst; Using CEF as feeder cells and NCSU-23 as medium can support blastocysts to develop to attachment.