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Study on Vitrification of Pig Embryos and Their Production Protocol

Author: YangShanTing
Tutor: ZhangDeFu；XuYinXue
School: Nanjing Agricultural College
Course: Animal Genetic Breeding and Reproduction
Keywords: pig parthenogenetic IVF superovulation vitrification
CLC: S828
Type: Master's thesis
Year: 2009
Downloads: 8
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Abstract


Currently, cryopreservation of pig embryos had not yielded great breakthrough and the freezing protocol remained to optimize. Establishing and optimizing a freezing protocol of pig embryos was very useful in both theory and practice. The technology of vitrification have become very important, it could provide a new technical approach for preservation of domestic animal genetic resources and protection of endangered animals; On the other hand, speeding the application of new reproductive technologies, establish female animal genetic, and enrich developmental biology library. The aim of this study was to establish and optimize a simplified and efficient porcine embryo vitrification protocol, and to establish basis for it by IVF (in vitro fertilization), partheno-activation and superovulation.Blastocyst rate was higher, when the pig embryo production technique parthenogenetic activation at 0.10 M Ca2+and 1.2kV/cm pulse strength. Cleavage rate (80.70%) and blastocyst rate (11.94%) was significantly higher than the control. When Ca2+concentration was 0.50 mM, cleavage rate (61.40%) and blastocyst rate(7.84%) was lower than the control and degeneration rate was significant increase. Cleavage rate(92.80%) and blastocyst rates of oocytes was the highest(49.10%), which was chemokinesis by 7.5μg/mLCB+10μg/mLCHX+2mM6-DMAP treatmented (P<0.05), Sperm vitality was 0.3 after thawing, and 107/mL of sperm density was appropriate. Blastocyst by IVF using 0.25ml straw frozen sperm was significant lower fresh sperm groups. The cleavage rate was significantly increased, when 5mM Caffeine was added in mTBM.In the test of superovulation of pig, the effect of synchronization of estrus was the best by treating with PG+PMSG+hCG, rate of synchronization of estrus was 91.7%, and average ovum pick-up was 19.2, it was increased but had no significantly different with others. The superovulation treated with 1000IU PMSG+500IU hCG had the best effect, synchronization of estrus of nine pigs was 100%. The effect of superovulation was not increase with the increasing of PMSG. Superovulation techniques could obtain the best effect in the suitable periodicity, and it was 14d and 15d, the oestrus rate and blastula retrieve amount were higher than others, and it had no significant difference(P>0.05).The influence of different freezing methods, freezing pretreatment and freezing carrier for embryo were systematicaly compared. The results showed that CLV (cryoloop vitrification) was the best method in freezing oocytes and embryos. compared with freezing effect of OPS, Straw. The survival rate of freezing MⅡperiod oocyte of pigs reached (86.70%), which was significantly higher than that of the OPS(83.80%) and Straw(77.60%) methods (P<0.05). Cleavage rates after parthenogenetic activation by CLV method was significantly higher(7.22%) than the OPS(4.85%) and Straw(0%) methods(P<0.05). Oocyte normal morphology rate (84.35%) and FDA stain survival rate (80.00%) by CLV method was higher than the control (P<0.05). Different pretreatment influenced on freezing effect. The cleavage rates of MⅡperiod oocytes could be partly improved by Taxol (7.92%) or cytochalasin B (CB 7.69%), it had no significant differences with the control (6.93%). The normal morphology rate was no significant differences (P>0.05) treat with Taxol (87.94%) or CB (84.89%). Porcine oocytes treat with CB and centrifugation polarize didn’t behave significant decrease or increase in survival in comparison with the control. For CB+centrifugation group, the survival rate of vitrified MⅡoocytes could not be improved. The results of the effect of zona pellucida treatment to pig embryo survival rate showed that, although zona pellucida treatment(0.50%catenin enzyme 10s) had no significant influence to pig embryo survival rate, could significant improve blastocyst cell (60.1), had significant differences with the control (46.6).

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