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The Elementary Research in Pig Embryos in Vitro Production

Author: WuCaiFeng
Tutor: ZhangDeFu；XuYinXue
School: Nanjing Agricultural College
Course: Animal Genetic Breeding and Reproduction
Keywords: Pig parthenogenetic IVF Nuclear transfer
CLC: S828
Type: Master's thesis
Year: 2008
Downloads: 60
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Abstract


Pigs are the optimal animal donors of tissues and organs for xenograft of human beings. We should modify the gene of the early embryos of the pig in order to investigate xenograft. Compared with in vivo production, until now, the total efficiency of in vitro production of porcine embryos remains low. Therefore, it is of great importance to optimize the conditions for in vitro production of porcine embryos. The oocyte activation is one of the key procedures of nuclear transfer(NT) in mammals, and the efficiency of oocytes activation affects the efficiency of NT directly. Nowadays the efficiency of NT in mammals is very low due to the fact that the oocytes are not well activated. This experiment was conducted to establish optimum in vitro production conditions of porcine embryos through getting better understanding of the factors affecting the porcine oocytes maturation, fertilization, parthenogenetic and cloned embryos transfer.The effects of seasons on porcine oocytes maturation in vitro was analysed in this paper, Comparing the A class oocytes’ collection efficiency, the rate of nuclear maturation and nuclear transfer embryos development protential from ovaries in different seasons. It was found that both the pregnancy rate and birthrate in spring season were higher than in autumn and winter. This maybe due to too high or too low temperature during transportation and embryo transfer, especially to the 1-2 cells embryos. The effects of CHX-pretreatment on meiotic progression, the reversibility of this inhibition and the pronuclei formation after electrical activation of oocytes were investigated, After electrical activation, the activation rates inTCM199 +CHXgroup were lower than those of M199 group, but no significant difference was found(P>0.05).Repeated parthenogenetic activation experiments confirmed that in the different activation conditions, The best electrical activation parameter was 120v/mm, 40μs, IDC. The activation rate of by ethanol was significantly lower than associated with 7.5μg/mLCB +10μg/mL CHX, 7.5μg/mL CB +2mM 6-DMAP or 7.5μg/mL CB +10μg/mL CHX +2mM 6-DMAP(P<0.05). The highest blastocyst rate for 7.5μg/ml CB +10μg/mL CHX +2mM 6-DMAP was 22.22%. Before electrical activation, the oocytes cultured in the medium with 8% ethanol, After electrical activation, associated with CHX, 6-DMAP, CHX +6-DMAP. The blastocyst rate of Ethanol+ DC pulse group was significantly lower than Ethanol +DC pulse +CHX, Ethanol +DC pulse +6-DMAP, Ethanol +DC pulse +CHX +6-DMAP groups, Ethanol +DC pulse +CHX +6-DMAP group achieved the highest blastocyst rate. The object of this study was to investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos. Blastocyst rates of cumulus-free mature oocytes in the group that expose to NCSU-23 medium containing cycloheximide (10 mg/mL) for 10min was 25%, which was higher than 0 min, 30 min, 60 min treatment groups. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic.Taking IVF using sperm vitality 0.3 after defrost, the sperm density of 10~7 was found optimum. No significant difference was seen on the blastocyst rate between 0.25mL frozen sperm and fresh sperm groups, but the cleavage rates were significant difference (P <0.05). With hatching time and sperm density increasing, frozen sperm IVF and polyspermia rate increased. The effect of zygote altogether 6h is the best. There was a relationship between embryonic development speed at different stages and blastocyst development rates. The 4-cell and 8-cell embryos had significantly higher blastocyst development rates than those derived from 2-cell and > 8-cell embryos. The incidence of chromosomal abnormalities was higher in the blastocysts derived from 2-cell and > 8-cell stage embryos than in the blastocysts derived from the other stage embryos.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Pig
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