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Study on the Techniques in Bovine Oocytes Freezing and Parthenogenesis in Vitro

Author: NiMing
Tutor: ZhangJuNong;LvZiLi
School: Shihezi University
Course: Animal Genetic Breeding and Reproduction
Keywords: Freezing Damage Bovine oocytes Parthenogenetic activation Holstein cattle
CLC: S823
Type: Master's thesis
Year: 2009
Downloads: 14
Quote: 0
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IVF - (In vitro fertilization embryo transplantation, IVF-ET) and the development of refrigeration technology, frozen able to promote the use of technology in the preservation of animal germplasm resources, oocyte freezing method in constant development, but due to damage by freezing its development will have some impact. Especially oocytes after freezing, then parthenogenetic culture is sufficient to use an important part of the oocyte. This will definitely help to take full advantage of Holstein cattle slaughterhouse oocytes. The the study frozen mature oocytes parthenogenetic activation, as well as the freezing of Parthenogenic. The study is divided into three experimental results are as follows. Test a different freezing methods of bovine oocytes in vitro maturation and parthenogenetic embryo development. Frozen oocytes were morphologically normal rate, cleavage rate, blastocyst rate with the control group difference is extremely significant (P lt; 0.01), cleavage rate and blastocyst rate is less than the control group, the freezing of oocytes damage, and the subsequent activation of oocytes and culture have a certain impact. Observed in this study, straws Act OPS method differences are highly significant (P lt; 0.01), the OPS Law frozen oocytes were morphologically normal rate, cleavage rate, blastocyst rate significantly higher than frozen straws law, indicating The the OPS method can effectively protect oocytes, subsequent parthenogenetic activation, the impact of the frozen program oocytes smaller (P gt; 0.05). Frozen oocytes were morphologically normal rate, cleavage rate, blastocyst rate with the control group compared to different significantly (P lt; 0.01). The difference between protective agent with protective agent is not significant, but the protective agent than protecting agent 1 To, the ability to protect the protective agent of bovine oocytes effect is better than the protective agent, protective agent oocyte cleavage rate to be slightly higher than the protective agent (P gt; 0.05). 3.OPS law equilibrium temperature of 38 ° C and 25 ℃, the difference was not significant. Oocytes were morphologically normal rate, cleavage rate, blastocyst rate slightly higher than 25 ° C in 38 ℃. The experimental results show that the equilibrium temperature between 38 ℃ and 25 ° C has little effect on the frozen oocytes parthenogenetic development. This experiment confirmed that 38 ° C at 25 ° C when compared with 4 ℃ difference was very significant equilibrium temperature should not be less than 25 ° C, when equilibrium temperature significantly reduced, be parthenogenetic development of oocytes caused significant impact. 4 ℃ frozen oocytes activated blastocyst, the experimental results show that the temperature of the low temperature level can have a greater impact on the development of parthenogenetic blastocysts stage. Test two different culture conditions in vitro maturation of bovine oocytes and parthenogenetic embryo development in the maturation medium with different concentrations in follicular fluid found: Add 0% -20% of follicular fluid, oocytes parthenogenetic activated cleavage rate did not significantly affect (P gt; 0.05); add 10%, 20% follicular fluid oocyte parthenogenetic activation, blastocyst rates (14.6%, 14.8%) was significantly higher than the addition of 5% and 0% group (10.9%, 11.4%, P lt; 0.05) The results show that, in the maturation medium supplemented with 10%, 20% follicular fluid role in promoting the maturation of the oocytes and subsequent blastocyst. The test were used triple-distilled water and Mill-Q ultrapure water preparation were cultured bovine parthenogenetic embryos, results showed that the oocyte maturation rate (PbI emission rates) and rates of cleavage and blastocyst rate None significantly (P gt; 0.05). The water is not the key factors that affect parthenogenetic activation of embryonic development, as long as clean water, and to cultivate water would not give greater impact subsequent culture. Test three different duration of action of bovine oocytes in vitro maturation and parthenogenetic embryo development test results showed that the 24 h-30 h in vitro maturation of oocytes activated after cleavage rate difference is not significant (P gt; 0.05 ), but with the mature time, cleavage rate upward trend. The oocyte maturation 28 h or 30h blastocyst development rate (16.4%, 15.3%) was significantly higher than the mature 24 h or 26 h blastocyst rates (10.5%, 9.8%, P lt; 0.05). Another factor oocytes With prolonged maturation time, the oocyte maturation process more fully, will increase the number of mature oocytes, oocyte activation may blastocyst rate. 2 the experimental findings confirmed bovine mature oocytes activated with ionomycin for 5 min, then 6-DMAP activation 2h cleavage rate was significantly lower than the activation 4h, 6h group (P lt; 0.05) ,6-DMAP role 4h or 6h blastocyst rates significantly higher blastocyst rates (P lt; 0.05) 2h or 8h. The results showed that the the oocyte maturation 28h, 5 min, and then in the culture in 6-DMAP culture medium containing 2mM 4h-6h appropriate culture medium with 5 um ionomycin activation. Activation 4h 6h compared with 6-DMAP activation time difference was not significant. The duration of action of 6-DMAP for 8h, the blastocyst rates downward trend. Showed that the duration of action of 6-DMAP activation efficiency of bovine oocytes matured influential. The test that the appropriate the ionomycin joint 6-DMAP of activated bovine oocyte ,6-DMAP role time to 4-6 h.

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