| The argali(Ovis ammon) is a conserved and endangered wild species.Because of being over-killed for a long time,the argali(Ovis ammon) have become endangered. Interspecies cloning has shown the great potential to conserve these endangered species.In the present study,we established argali(Ovis ammon) ear fibroblast cell lines by cell culture in vitro,and got higher development in vitro of argali(Ovis ammon)-sheep embryos by optimizing the procedures of nuclear transfer.The argali(Ovis ammon)-sheep embryos were transferred into sheep surrogate mothers,but two of these surrogate sheeps delayed estrus at 90th.day and finally no pregnancies were established.1 Establishment of donor cell linesWe established argali(Ovis ammon) and sheep fibroblast cell lines by tissue piece culture,which were from wild argali(Ovis ammon) car skin and sheep fetus skin in Xinjiang,and cultured and passaged cumulus cells from cumulus-oocyte-complexes by in vitro maturation.The growth curves of argali(Ovis ammon) fibroblasts,sheep fibroblasts and cumulus cells showed that latent phase of argali(Ovis ammon) fibroblasts was longer than that of sheep fibroblasts and cumulus cells.Argali(Ovis ammon) fibroblasts had no obvious plateau phase.The regular fluctuation of growth curve exhibited in vitro growth characteristics of fibroblasts.The fibroblasts subcultured like this could be used as nuclear transfer donors.In order to establish cryopreservation system for argali(Ovis ammon) fibroblasts,the effect of cryopreservation protocols and cryoprotectants on survival rate of argali(Ovis ammon) fibroblasts after being thawed were studied.Among three cryopreservation protocols,the survival rate of cells was 95.2%by protocol 1 and significantly higher than that by another two protocols.The adhesion rate reached 70%～80%at 18 h after being thawed.The protocol 1 was that cells could be equilibrated at 4℃for 30min,—20℃for 3h,—70℃overnight,and then finally plunged into liquid nitrogen.In protocol 1,cells could stay at—20℃for 3～5h which didn't affect survival rate and growth state after being thawed.The survival and adhesion rate of cells cryopreserved with cryoprotectant 1(50%FBS) were higher than those with cryoprotectant 2(20%FBS).The results imply that cells cryopreserved with 50%FBS cryoprotectant by protocol 1 have higher survival rate after being thawed.Chromosomal karyotypes of wild argali(Ovis ammon) and sheep in Xinjiang were studied by using the routine analysis method of chromosomal karyotype.The results showed that the number of the chromosomes in argali(Ovis ammon) was 2n= 56.There were 27 pairs of autosomes and 1 pair of sex chromosome.No.1 and No.2 of autosomes were metacentric chromosomes while No.3～No.27 of autosomes were telocentric chromosomes;X chromosome was the largest telocentric chromosome while Y was the smallest metacentric chromosome in all chromosomes.The number of the chromosomes in sheep was 2n=54.There were 26 pairs of autosomes and 1 pair of sex chromosome.Among 26 pairs of autosome,No.1,No.2 and No.3 chromosomes were metacentric chromosomes, while No.4～No.26 chromosomes were telocentric chromosomes;X chromosome was the largest telocentric chromosome while Y chromosome was the smallest metacentric chromosome.2 Reconstruction of argali(Ovis ammon)-sheep interspecies cloned embryosThe present study was conducted to investigate the effects of storage temperature,time and solution of sheep ovaries on the parthenogenetic developmental competence of oocytes obtained from stored ovaries.The results showed that storage of ovaries at 14～18℃for 15～17h did not affect oocyte maturation(80.9%vs 82.2%) and the potential of oocytes to develop into blastocysts after parthenogenetic activation(21.0%vs 22.0%).The type of storage solutions including modified phosphate-buffered saline(D-PBS) and physiological saline that the ovaries were immersed in had little effect on the results. However,the parthenogenetic oocytes from ovaries stored at 4℃or 30℃for 15～17h had a significantly decreased maturation rate(61.9%vs 19%) and cleavage rate(8.6%vs 9.1%), and did not develop into blastocysts.Thus,sheep ovaries can be stored at 14～18℃for 15～17h without decreasing oocyte maturation competence and the developmental potential of parthenogenetically activated oocytes.Enucleation of recipient oocytes is critical to nuclear transfer.Blind enucleation is a conventional enucleation method.Chemically assisted enucleation is that the location of sheep oocyte chromosome was displayed with colchicine and enucleated by micromanipulator.In this experiment,it focuses on the influence of the concertration of colchicine handling oocytes and the time of oocytes incubating in colchicine on displaying effect.The effects of blind enucleation and chemically assisted enucleation on enucleation rate were also studied.The results showed that(1) the rates of cytoplast protrusion and enueleation had no difference for ooeytes incubating in colehicine(0.4μg/mL) for 0.5h or 1h.(2) oocytes could be enucleated efficiently after being treated with medium containing 0.2μg/mL or 0.4μg/mL colchicine for 0.5h.(3) maturation for 18～23h didn't affect the rates of cytoplast protrusion and enucleation,and the enucleation rote of oocyes matured for 21～23h was significantly higher by chemically assisted enucleation than blind enucleation. (4) the development rate of reconstructed embryos hadn't been.influenced when oocytes matured for 18～20h were enucleated by blind enucleation and chemically assisted enucleation.It has demonstrated that matured oocytes incubating in colchicine(0.2μg/mL) for 0.5h could not only been enueleated fast and effectively,but didn't affect the early development of reconstituted embryos.The effects of electrofusion protocol,activation method and embryo culture media on the development of interspecies reconstructed embryos were examined.Results showed that the appropriate electrofusion parameters for interspecies cloned embryos were 121～125 V/mm of pulse intensity,40μs of pulse duration and 2 of pulse number or 126～130 V/mm of pulse intensity,20μs of pulse duration and 2 of pulse number.When those embryos which failed to fusion were refused with the same electrofusion parameters,the development of reconstituted embryos couldn't been influenced and more usable embryos were produced.Parthenogenetic oocytes matured for 25h or 26h resulted in the higher blastocyst rate than that matured for 24h;the higher activation rate was obtained when oocytes were treated with 6—DMAP for 2h;reconstituted embryos activated by Iono+6-DMAP had higher cleavage rate and blastocyst rate than those activated by EH+6-DMAP,but there was no significant difference.The development rate of blastocyst would tend to arise in new culture media though there was no significant difference. Embryo culture media with the addition of 10 ng/mL EGF enhanced the cleavage rate and blastocyst rate of interspecies reconstituted embryos(87.4%,19.3%,respectively).3 Effects of argali(Ovis ammon) donor cells on interspecies nuclear transferWe tested the cell cycle stages of argali(Ovis ammon) fibroblasts under different treatment conditions.Results showed that confluency,confluency duration and serum-starvation period had a positive impact on the percentage of G0/G1 cell.The aim of this study was to investigate the effect of individual,passage number, confluency,serum-starvation period,storage method and surface state of argali(Ovis ammon) fibroblasts on the development of embryos.The embryos were reconstructed by interspecies nuclear transfer using local ovine oocyte cytoplasm.Results showed that(1) there was no difference in the cleavage rate and blastocyst rate among three argali sheeps. (2) the passage number of fibroblast cells within 10 passages didn't affect the development rate of reconstructed embryos.(3) the blastocyst rates,which was produced by 70%～80% confluency,90%confluency and full-confluency for 1d fibroblast cells,were higher than those using full-confluency for 2～4d cells.(4) the serum-starvation period of donor cells didn't affect the development of embryos.(5) the cleavage rate of embryos reconstructed using donor cells stored for 2 days at 4℃was lower than that using donor cells from control group(P＜0.05),but no difference was found in the proportion of blastocysts in total embryos.(6) smooth cells led to a high fusion rate and blastocyst rate.Therefore,The embryos,which were reconstructed by interspecies nuclear transfer using more than 70%～80%confluent smooth fibroblasts within 10 passages from three argali sheeps in Xinjiang,had higher development competence.4 The in vitro development of ovine embryos produced by different manner and embryo transferWe conducted the present study to examine the developmental competence of preimplantation ovine embryos in vitro.Parthenogenetic activation(PA),somatic cell nuclear transfer(SCNT) and in vitro fertilization(IVF) were used for ovine PA embryos (PAEs),IVF embryos(IVFEs),ovine fetal fibroblast cell nuclear transfer embryos (FC-NTEs),ovine cumulus cell nuclear transfer embryos(CC-NTEs) and argali(Ovis ammon) fibroblast cell nuclear transfer embryos(AC-NTEs).These embryos were used to study the effects of genetic material differences on the developmental competence in vitro and the total cell number in the blastocyst,which caused by the different embryo-produced manner.Furthermore,effects of season,ovary storage,and embryonic culture media on the developmental potential of the ovine PA embryos(PAEs) and SCNT embryos(NTEs) were also investigated.The results showed:(1) there was no difference in the cleavage rates among PAEs,FC-NTEs,CC-NTEs,AC-NTEs and IVFEs.Blastocyst formation rate of PAEs was significantly higher than that of AC-NTEs(23.4%vs 15.9%),while no significant difference was obtained among blastocyst formation rate of FC-NTEs, CC-NTEs and IVFEs.The total cell number of FC-NT blastocyst was more than that of PA blastocyst(120 vs 96),but had no significant difference with that of IVF blastocyst(120 vs 107).(2) the developmental rates of PAEs and FC-NTEs were significantly higher during estrous seasons than those during anestrous seasons.(3) storaging ovaries at 14～18℃for 15～17 h did not affect the development in vitro of PAEs,but the cleavage rate of FC-NTEs was significantly decreased and no blastocyst was obtained.(4) there was no effect on the preimplantation development of PAEs and FC-NTEs that were cultured in SOFaa and CR1aa media.(5) we had maturated 57 oocytes and got 53 interspecies reconstructed embryos.The fusion rate was 84.9%and the cleavage rate was 77.8%.35 embryos were transferred to 3 surrogate mothers,but two of them delayed estrus at 90th day and finally no pregnancy had been achieved.
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