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Study on Parthenogenetic Activation of Porcine in Vitro Maturation Oocytes

Author: ZhangTingYu
Tutor: MaHengDong;ZhangDeFu
School: Sichuan Agricultural University
Course: Animal Genetic Breeding and Reproduction
Keywords: Pig Oocyte In Vitro Maturation Parthenogenetic Activation Ca2+ Electric-Activation Chemical-Activation
CLC: S828
Type: Master's thesis
Year: 2008
Downloads: 23
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Pigs are considered as ideal organ donor for human future xenotransplantation.Somatic cell nuclear transfer provides an opportunity for producing hyper-acute-rejection(HAR) and endoviruses free pigs.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 artificial activation conditions of pig though getting better understanding of factors affecting the in vitro maturation porcine oocytes parthenogenetic activation.Compared the parthenogenetic activation efficincy from ovaries in different seasons; and was evaluated the gourped of Ca2+ concentration in activation medium and electrical pulse strength on activation of porcine ooctyes;and the application of alternating current(AC) in sorbitol or sorbitol and mannitol mixture electroporation medium were first examined in this paper;and the effects of electrical pulses conbined with 6-dimethylaminopurine(6-DMAP),cycloheximid(CHX) and cytochalasin B(CB) treatment time on the rates of cleavage and blastocyst of porcine in vitro maturation oocytes were investigated,respectively;and with the CHX treatment alone 2h after united the chemical activator treatment 4h was also discussed.It was showed that:(1)The spring collection slaughtered by the ovary oocytes parthenogenetic activation after in vitro maturation,the cleavage and blastocyst rate was the highest,respectively 36.45%and 79.70%,significantly higher than in the summer and winter(63.90%,16.80%;69.48%,20.13%,P<0.01).In the fall,although the rate of cleavage was significantly lower than in the spring,but the blastocyst rate was no significant difference(P<0.05).(2)With low Ca2+ concentration(0.01,0.05 mM),the increase of electrical pulse strength had positive effect on the development of parthenogenetic embryos.The cleavage rates(73.75%,74.70%) and blastocyst rates(37.50%,36.83%) obtained under 0.05mM Ca2+ and 1.6kV/cm pulse strength,and 0.1mM Ca2+ and 1.2kV/cm,respectively,were high significantly higher than under other groups(P<0.01).Under excessive level of Ca2+ concentration(0.5mM),however,increase electrical pulse strength led to high degradation and low development rate;(3)Activated by the two 15μs consecutively pulses of 1.2kV/cm DC,the rate of cleavage and blastocyst alike to given 30μs single pulse of by the same strength,were significantly higher than that Activated by the two 30μs consecutively pulses of 1.2kV/cm DC.Two electrical pulses given 30 min apart of strength of 1.2kV/cm DC,the rate of cleavage and blastocyst are significantly lower than that activated by a single 30μs pulse of 1.2kV/cm DC.(4)Using sorbitol as electro-activation medium,the highest cleavage(77.23%) and blastocyst rates (34.15%) could be obtained under 1.6kV/cm DC;significantly higher than that activated by 1.0kV/cm DC and 1.2kV/cm DC(P<0.01);and no significant difference to 1.4kV/cm DC(P>0.05).(5)Given the AC pulse before DC pulse,the sorbitol electroporation medium could not promoting parthenogenetic development.(6)Using equally mixed sorbitol and mannitol as electro-activation medium,the cleavage rates were 72.33%and 70.03%under 1.2 and 1.6kV/cm electrical pulse,and Blastocysts rates were 31.03%and 29.60%, respectively.The development of mixture group was a little lower than mannitol group (77.07%,36.03%),and higher than sorbitol group(69.63%,26.93%),but no significantly difference(P>0.05).(7)Electrical puples followed by 6-DMAP treated 4 hours,CB treated 4 hours,CHX treated 6 hours,to achieve the best efficiency of activation,cleavage and blastocyst rates were 73.30%,32.68%,71.92%,30.14%and 59.58%,19.16%.(8)United 6-DMAP,CB and CHX treatment with both the 4h or 6h,or with the CHX treatment alone 2h after united the chemical avtivator treated 4h(4h+2h),the cleavage rate was significantly higher than that of the three chemical activated alone(P<0.01).Parthenogenetic activation efficincy of the 4h+2h group same with 4h group;although the cleavage rate was significantly lower than treated united 6h(P<0.05),but the blastocyst rate was significantly higher than that one,respectively,41.24%,35.60%(P<0.01);and nearly half of the cleavaged eggs development to the blastocyst stage.These results demonstrated that The best time to collection of slaughter ovarian oocyte periods Spring and autumn parts,the collection period,the in vitro maturated oocytes,has great capability parthenogenetic activation and development;summer and winter collection ovarian pig oocytes not suitable for use in vitro culture.The low efficiency parthenogenetic activation due to low level of Ca2+ concentration in electroporation medium and electrical pulse strength,the high level of Ca2+ concentration and electrical pulse strength,activated eggs development may be inhibited.Given consecutively pulse,resulting in one single Ca2+ rise in oocytes;one single Ca2+ rise is enough to activate the in vitro matured porcine oocytes;two electrical pulses given 30min apart was not necessary activate the in vitro matured porcine oocytes.The mannitol could be replaced by Sorbitol as fundamental component in electro-activation of porcine oocytes.Mixture of sorbitol and mannitol also got a satisfied activation results.Electrical pules followed by chemical activator to activated,6-DMAP activated the best activation efficincy was treatmented 4h;CB activated the best activation efficincy was treatmented 4h;and CHX activated the best activation efficincy was treatmented 6h.Activated by the CHX treatment alone 2h after united the 6-DMAP,CB and CHX treatmented 4h,has a better activation efficincy could be used as artificial activation in vitro matured porcine oocytes.On the whole,the optimal parthenogenetic development condition was that the porcine ooctyes collected in spring and autumn were manual matured and activated in mannitol electroporation medium,under DC pulse 1.2kV/cm and 30μs,and then treated using CHX treatment alone 2h after united the 6-DMAP,CB and CHX for 4h.sorbitol or sorbitol and mannitol mixture electroporation medium were useful in manual activation of the porcine ooctyes,however,the optimal conditions need further research.

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