The Expression Of Adding Different Concentrations Of CLC And LDL On Pig Oocyte Cryopreservation

Oocytes cryopreserved technology has been improved, due to the uniqueness of its structure, the change of osmotic pressure and so on in the process of freezing and thawing will on the egg cell membranes and organelles caused a certain degree of damage, in order to explore the toxicity of pig oocytes at different periods, tin order to detect the CLC and LDL of porcine oocytes mitochondrial membrane potential and after freezing and thawing ZP ubiquitin level influence, this study applies vitrification freezing and slow freezing, two methods were compared to add two kinds of different concentrations of oocytes after freezing carrier LDL and CLC mitochondrial membrane potential difference, and analyze the M Ⅱ period after vitrification freezing oocytes transparent ubiquitin mineralization degree.Test mainly by sds-page and Western blot methods for different treatment group of pig oocytes with protein samples analysis, and mitochondrial membrane potential specific probe JC-1 mark each treatment group of mitochondrial membrane potential, the fluorescence intensity of fluorescence microscope to observe the red, green and distribution, for seeking better oocyte freezing and thawing method and make it better used in the production practice to provide theoretical support. The result is as follows:1. Vitrification freezing and slow freezing method after freeze-thaw GV stage oocytes, LDL and CLC treatment group survival rates were 74.84%,55.26% and 69.86%,44.53%, vitrification frozen pig M Ⅱstage oocytes thawed adding different concentration of CLC cell survival rate in treatment group were higher than LDL freezing treatment group;Add 10% CLC cell survival of freeze-thaw treatment group is significantly higher than other treatment group, and fresh group no significant difference (P> 0.05);2. Adding different concentrations of CLC and cryoprotectants LDL group (1%,10%,20%) treatment in the frozen thawed, M Ⅱ stage oocytes with protein immunoblot results show that in 63 kDa respectively,82 kDa,110 and 61 kDa kDa,80 kDa,106 kDa may cause different degrees of ubiquitin tag, and a significant difference was found in ubiquitin change with grey value, show that pig oocyte in vitro maturation culture in the process of transparent with proteins have been varying degrees of ubiquitin;3. The mitochondrial membrane potential specific probe JC-1 mark the vitrification freezing treatment group,10% of CLC treatment group △ bits of m were higher than in other frozen treatment group, compared with the fresh group had no significant difference (P> 0.05).