Study On Vitrification Of Porcine Embryos Derived From Production In Vitro

The cryopreservation of mammalian embryos increasingly displayed its huge potential of application in animal resource protection.Long-term storage of porcine embryos would be worthwhile both for the preservation of genetic resources and for exportation of genotypes around the world.But it's very difficult for porcine embryo cryopreservation using the conventional slow freezing and vitrification due to lipids droplets contents,which making them extremely sensitive to chilling.Since Vajta successfully vitrified porcine morula and blastocysts with open pulled straw(OPS) in 1998,OPS technology has become the most widely employed to date.In the present study,some key fators that affected in the fertilization of porcine and sequential culture conditions were investigated.In addition,different stage embryos derived from in vitro maturation,in vitro fertilization and in vitro culture were vitrified using OPS,two cryoprotectants and pre-proceesing methods were compared.The results were shown as follows.1.This study investigated the effert of fresh boar spermatozoa of different quality and frozen-ejaculated spermatozoa on the developmental competence of porcine embryo fertilized in vitro.The result shows that,comparing to the group fertilized with high quality fresh spermatozoa(molitily＞0.8,morphologities abnormalities＜10%),the rate of cleavage and embryo developing to morula is not different(54.3 %vs51.1%,48.2%vs45.4%)fertilized with frozen-thawed ejaculated spermatozoa (molitily＞0.5),although cleavage rates were not different(51%vs53.6%) but the rate of embryo developing to morula is significantly reduced(28.9%vs47.4%)fertilized with fresh spermatozoa which have low motility(motility＜0.5)and poor morphology (morphologial abnormalities＞30%);Comparing to the group(frozen-thawed ejaculated spermatozoa,molitily＞0.5),there is not different for cleavage rates(56.4 %vs54.3%)but the rate of embryo developing to morula is significantly reduced fertilized with lower molitily(molitily≤0.4) frozen-thawed ejaculated spermatozoa (37.0%vs48.2%);Fertilized with different sperm-oocyte ratio(Group A:12000: 1,Group B:6000:1,Group C:3000:1),Comparing to the group B and Group C(54.3%vs 54.2%,48.5%vs 45.7%),the rate of cleavage and embryo developing to morula is significantly reduced In the Group A(37.5%,34.4%).The results indicate that boar spermazota quality can influence subsequent embryo developmental competence after fertilized in vitro;the sperm molity of fresh boar spermatozoa and frozen-ejaculated spermatozoa is not decisive fator for sperm fertilization ablity; using appropriate sperm-oocyte ratio in vitro fertilization,embryo sequential developmental ability can be improved.2.The cleavage rate and morula rate were 54.7%and 46.2%cultured in NCSU-23, which were not different from in PZM-3(55%,40.3%);Comparing the cultured in NCSU-23,while co-culturation with porcine oviductal epithelium shell cells there were more embryos cleaving(63.4%) and developed to morula(49.3%),but there were not significantly different from the control group(61.1%,47.2%,P＞0.05).3.Vitrified different stage porcine early embryos directly with EDS(15%EG, 15%DMSO,0.5M sucrose),we found that the survival rates of zoge-stage oocytes was significantly higher(62.3%,P＜0.05) than other stage early embryos,2-8 cell embryos,8-16 cells embryos and morulas(8.7%,4.4%and 5.6%,respectively).4.Directly vitrified porcine zygote-stage oocytes with EDS and ES(40%EG,0.5M sucrose),the frozen-thawing normal morphology rates(90%vs 53.3%) and survival rates(72.7%vs 31%) were significantly different between the two groups(P＜0.05); Treated the zygotes-stage oocytes before vitrification with Cytochalasin B can not improved the frozen-thawing normal morphology rate(60.6%vs 81.7%,P＜0.05) and survival rate(70.2%vs 61.2%,P＞0.05) comparing the directly vitrification group. The frozen-thawing zygote-stage oocytes form all vitrification groups can't develope during the sequential culturation in NCSU-23.