Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stern cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, ~lll-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neuregenesis related genes （Sox-1, Nestin, GABA, Pax6, Zic5 and Pitxl） in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

Effects of 5-aza-2’-deoxyctidine on the development of porcine parthenogenetic and nuclear transfer embryos

The current study was to investigate whether embryo or fetal fibroblast cells treated with 5-aza-2’-deoxyctidine (5-aza-dC) have a positive effect on the in vitro development of porcine parthenogenetic and cloned embryos. To this end, porcine fetal fibroblast cells were treated with different concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours), the results showed that DNA methylation in PRE-1 SINE region was gradually reduced over time in cells treated with 5-aza-dC. To determine the effect of 5-aza-dC on in vitro development of porcine activated oocytes, the parthenogenetic embryo was treated with 5-aza- dC. Notably, treatment with 5 nM 5-aza-dC for 1 hour led to a significant improvement in blastocyst development, compared with the control group. The effects of donor cell treatment with 5-aza-dC on porcine cloned embryos development were further examined by treating fetal fibroblast cells with various concentrations (5 nM, 50 nM and 500 nM) of 5-aza-dC for different exposure times (1, 6 and 20 hours). Exposure of cells in 5 nM 5-aza-dC for 1 - 20 hours led to a significant improvement in the percentage of developed blastocysts, while treatment with 500 nM 5-aza-dC did not affect blastocyst development, compared to untreated controls. These findings indicate that treatment of fetal fibro-blast cells with relatively low concentrations of 5-aza-dC for short exposure times improves subsequent blastocyst development of porcine cloned embryos.

Melatonin improves the first cleavage of parthenogenetic embryos from vitrified-warmed mouse oocytes potentially by promoting cell cycle progression

Background:This study investigated the effect of melatonin(MT)on cell cycle(G1/S/G2/M)of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II(MII)oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos.Results:After vitrification and warming,oocytes were parthenogenetically activated(PA)and in vitro cultured(IVC).Then the spindle morphology and chromosome segregation in oocytes,the maternal mRNA levels of genes including Miss,Doc1r,Setd2 and Ythdf2 in activated oocytes,pronuclear formation,the S phase duration in zygotes,mitochondrial function at G1 phase,reactive oxygen species(ROS)level at S phase,DNA damage at G2 phase,early apoptosis in 2-cell embryos,cleavage and blastocyst formation rates were evaluated.The results indicated that the vitrification/warming procedures led to following perturbations 1)spindle abnormalities and chromosome misalignment,alteration of maternal mRNAs and delay in pronucleus formation,2)decreased mitochondrial membrane potential(MMP)and lower adenosine triphosphate(ATP)levels,increased ROS production and DNA damage,G1/S and S/G2 phase transition delay,and delayed first cleavage,and 3)increased early apoptosis and lower levels of cleavage and blastocyst formation.Our results further revealed that such negative impacts of oocyte cryopreservation could be alleviated by supplementation of warming,recovery,PA and IVC media with 10^(−9) mol/L MT before the embryos moved into the 2-cell stage of development.Conclusions:MT might promote cell cycle progression via regulation of MMP,ATP,ROS and maternal mRNA levels,potentially increasing the first cleavage of parthenogenetic zygotes developed from vitrified-warmed mouse oocytes and their subsequent development.

Effects of Chemical Activation on the Parthenogenetic Development of Porcine in vitro Maturation Oocytes

The objective of this study was to determine the effects of ionomycin combined with cytochalasin B （CB）, cycloheximide （CHX）, or 6-dimethylaminopurine （6-DMAP） on the activation of porcine oocytes. In Experiment 1, in vitro matured oocytes were activated with 15, 20, 25 or 30 mmol L -1 ionomycin separately. Activation rates of 20, 25 mmol L-1 and 30 mmol L treatments were higher （P〈0.05） than that of 15 mmol L-1 treatment. In Experiment 2, in vitro matured oocytes were activated with 20 mmol L-1 ionomycin for 10, 20, 30, 40 or 50 rain and then incubated with 2 mmol L-1 6-DMAP for 6 h. Cleavage and blastocyst rates [（72.40 ± 13.02）%, （25.37 ± 11.43）%] after treatments for 40 min were higher （P〉0.05） than those of the other treatments. In Experiment 3, matured oocytes were activated with ionomycin and then incubated with 7.5 mg mL-1 CB, 10 mg mL-1 CHX, 2 mmol L-1 6-DMAP, 7.5 mg mL-1 CB ＋ 10 mg mL-1 CHX or 7.5 mg mL-1 CB ＋ 2 mmol L-1 6-DMAP for 6 h. The rates of activation, cleavage and blastocyst of 2 mmol L -1 6-DMAP treatment [（86.05 ± 4.29）%, （61.77 ±8.10）% and （21.62± 3.31）%] were higher （P〈0.05） than those of 7.5 mg mL-1 CB treatment. In Experiment 4, matured oocytes were activated with ionomycin and then incubated with 2 mmol L-1 6-DMAP for 3.5, 5.5 or 7.5 h. Cleavage rates and blastocyst rates of 5.5 h treatment [（66.59 ± 14.36）% and （25.40 ± 10.16）%] were higher （P〉0.05） than those of other treatments. In conclusion, activation of porcine oocytes appears to be most successful using the combination of ionomycin （20 mmol L-1, 40 min） followed by 6-DMAP （2 mmol L-1, 5.5 h）.

Effects of Ages of Donors and Conditions of Preserving Ovaries on Porcine Oocytes Maturation in vitro and Efficiency of Parthenogenetic Activation

Effects of different ages of donors and different conditions of preserving ovaries onporcine oocytes maturation in vitro and efficiency of parthenogenetic activation werestudied. The experiments included: 1) effects of different temperatures (22, 30, 37, 38.5and 40℃) of preserving ovaries on porcine oocytes maturation in vitro and developmentalpotential; 2) effects of periods of preserving ovaries on porcine oocytes maturation invitro and development in vitro; 3) effects of different ages of donors on porcine oocytesmaturation in vitro and developmental potential. The results of the experiment showed:1) There were no statistical differences (p>0.05) of the parthenogenetic cleavage rate(79.64% vs 76.18%) and blastocyst rate (18.11% vs 33.82%) between oocytes from ovariespreserved at 38.5℃ and those preserved at 37℃. When the preserving temperature wasincreased to 40℃, the cleavage rate (21.68%) and the blastocyst rate (0) were greatsignificantly lower than those at 37℃(p<0.01). The cleavage rate (80.79% vs 76.18%) andblastocyst rate (29.61% vs 33.82%) were not different between 30 and 37℃(p>0.05). Whenthe preserving temperature was decreased to 22℃, the rate of cleavage was not different,but the rate of blastocyst was significantly lower, compared with that at 37℃; 2) Thecleavage and blastocyst rates of the porcine oocytes collected after slaughter 2 or 6hwere not different (p>0.05); 3) The cleavage rate of oocytes from gilts and sows aftermaturation was not different, but the blastocyst rate of the sow group was significantlyhigher than that of gilt group (p<0.05). The blastocyst cell number of sows and giltshowed no difference (p>0.05).

Imprinting at the KBTBD6 locus involves species-specific m ternal methylation and monoallelic expression in livestock animals

Background The primary differentially methylated regions(DMRs) which are maternally hypermethylated serve as imprinting control regions(ICRs) that drive monoallelic gene expression, and these ICRs have been investigated due to their implications in mammalian development. Although a subset of genes has been identified as imprinted, in-depth comparative approach needs to be developed for identification of species-specific imprinted genes. Here, we examined DNA methylation status and allelic expression at the KBTBD6 locus across species and tissues and explored potential mechanisms of imprinting.Results Using whole-genome bisulfite sequencing and RNA-sequencing on parthenogenetic and normal porcine embryos, we identified a maternally hypermethylated DMR between the embryos at the KBTBD6 promoter Cp G island and paternal monoallelic expression of KBTBD6. Also, in analyzed domesticated mammals but not in humans, non-human primates and mice, the KBTBD6 promoter Cp G islands were methylated in oocytes and/or allelically methyl-ated in tissues, and monoallelic KBTBD6 expression was observed, indicating livestock-specific imprinting. Further analysis revealed that these Cp G islands were embedded within transcripts in porcine and bovine oocytes which coexisted with an active transcription mark and DNA methylation, implying the presence of transcription-dependent imprinting.Conclusions In this study, our comparative approach revealed an imprinted expression of the KBTBD6 gene in domesticated mammals, but not in humans, non-human primates, and mice which implicates species-specific evolution of genomic imprinting.

Human parthenogenetic embryonic stem cells:one potential resource for cell therapy

Pluripotent stem cells derived from somatic cells through such processes as nuclear transfer or induced pluripotent stem(iPS) cells present an important model for biomedical research and provide potential resources for cell replacement therapies.However,the overall efficiency of the conversional nuclear transfer is very low and the safety issue remains a major concern for iPS cells.Embryonic stem cells(ESCs) generated from parthenogenetic embryos are one attractive alternative as a source of histocompatible cells and tissues for cell therapy.Recent studies on human parthenogenetic embryonic stem cells(hPG ESCs) have revealed that these ESCs are very similar to the hESCs derived from IVF or in vivo produced blastocysts in gene expression and other characteristics,but full differentiation and development potential of these hPG ESCs have to be further investigated before clinical research and therapeutic interventions.To generate various pluripotent stem cells,diverse reprogramming techniques and approaches will be developed and integrated.This may help elucidate the fundamental mechanisms underlying reprogramming and stem cell biology,and ultimately benefit cell therapy and regenerative medicine.

Generation of developmentally competent oocytes and fertile mice from parthenogenetic embryonic stem cells

Parthenogenetic embryos,created by activation and diploidization of oocytes,arrest at mid-gestation for defective paternal imprints,which impair placental development.Also,viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells(pESCs)derived from parthenogenetic embryos,presumably attributable to their aberrant imprinting.We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring.Moreover,normal expression of imprinted genes is found in the germ cells and the mice.pESCs exhibited imprinting consistent with exclusively maternal lineage,and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background.pESCs differentiated into primordial germ cell-like cells(PGCLCs)and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function.The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs,consistent with efficient reprogramming of methylation and genomic imprinting.These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting,offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function.

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic,Androgenetic and Fertilized Blastocysts

MicroRNAs（miRNAs） are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions.In this study,miRNA and messenger RNA（mRNA） profiles were examined by Illumina microarray in mouse embryonic stem cells（ESCs） derived from parthenogenetic,androgenetic,and fertilized blastocysts.The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression.Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs（aESCs） and fertilized ESCs（fESCs）,a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs（pESCs） and fESCs,and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs.In addition,a total of 575,5 and 376 miRNA-mRNA target pairs were observed in aESCs vs.fESCs,pESCs vs.fESCs,and aESCs vs.pESCs,respectively.Furthermore,15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR.Finally,transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3,Dyrklb,and Prrg2 mRNA,inhibition of miR-363 increased the expression of Nfat5 and Soatl mRNA,and inhibition of miR-883b-5p increased Nfat5,Tacstd2,and Ppapdc1 mRNA.These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.

Advantages and limitations of the parthenogenetic embryonic stem cells in cell therapy

Parthenogenetic embryonic stem cells（pE SCs）, as ＂seed cells＂ for regenerative medicine, are an effective way to build patient-specific pluripotent stem cells, due to the fact that characteristics of self-renewal and pluripotent are similar to embryonic stem cells（ESCs）. Parthenogenetic activation can be performed at meiosis I or meiosis II describing the embryos with distinct patterns of homozygosity and heterozygosity.Heterozygous pE SCs are expected to be used for autologous transplantation, while homozygous pE SCs enable to be used for allogeneic gene therapy in theory but is hampered by immunological barriers defined by the recognition of natural killer（NK）cells. In this review, we describe the mechanism of deriving heterozygous and homozygous pE SCs, and summarize the advantages and limitations of pE SCs in the area of cell therapy.

Quantitative proteomics analysis of parthenogenetically induced pluripotent stem cells

Parthenogenetic embryonic stem(pES)cells isolated from parthenogenetic activation of oocytes and embryos,also called parthenogenetically induced pluripotent stem cells,exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential.Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated(IVO)oocytes or from the in vitro-matured(IVM)oocytes with that of fertilized embryonic stem(fES)cells derived from fertilized embryos.A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells,whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value.No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes.Notably,no differences were found in the protein expression of imprinted genes between the pES and fES cells,suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages.The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.

Insights into wing dimorphism in worldwide agricultural pest and hostalternating aphid Aphis gossypii

Background:The worldwide pest Aphis gossypii has three-winged morphs in its life cycle,namely,winged parthenogenetic female(WPF),winged gynopara(GP),and winged male,which are all produced by a wingless parthenogenetic female(WLPF).Most studies on A.gossypii have focused on WPF,while few have investigated GP and male.The shared molecular mechanism underlying the wing differentiation in the three wing morphs of A.gossypii remains unknown.The wing differentiation of WPF was explored in a previous study.Herein,GP and male were induced indoors.The characters of the body,internal genitals,wing veins,and fecundity of GP and male were compared with those of WPF or WLPF.Compared with WLPF,the shared and separate differentially expressed genes(DEGs)were identified in these three-wing morphs.Results:Newly-born nymphs reared in short photoperiod condition(8 L:16D,18°C)exclusively produced gynoparae(GPe)and males in adulthood successively,in which the sex ratio was GP biased.A total of 14 GPe and 9 males were produced by one mother aphid.Compared with WLPF,the three-wing morphs exhibited similar morphology and wing vein patterns but were obviously discriminated in the length of fore-and underwings,reproductive system,and fecundity.A total of 37090 annotated unigenes were obtained from libraries constructed using the four morphs via RNA sequencing(RNA-Seq).In addition,10867 and 19334 DEGs were identified in the pairwise comparison of GP versus WLPF and male versus WLPF,respectively.Compared with WLPF,the winged morphs demonstrated 2335 shared DEGs(1658 upregulated and 677 downregulated).The 1658 shared upregulated DEGs were enriched in multiple signaling pathways,including insulin,FoxO,MAPK,starch and sucrose metabolism,fatty acid biosynthesis,and degradation,suggesting their key roles in the regulation of wing plasticity in the cotton aphid.Forty-four genes that spanned the range of differential expression were chosen to validate statistical analysis based on RNA-Seq through the reverse transcription quantitative real time polymerase chain reaction(RT-qPCR).The comparison concurred with the expression pattern(either up-or downregulated)and supported the accuracy and reliability of RNA-Seq.Finally,the potential roles of DEGs related to the insulin signaling pathway in wing dimorphism were discussed in the cotton aphid.Conclusions:The present study established an efficiently standardized protocol for GP and male induction in cotton aphid by transferring newly-born nymphs to short photoperiod conditions(8 L:16D,18°C).The external morphological characters,especially wing vein patterns,were similar among WPFs,GPe,and males.However,their reproductive organs were strikingly different.Compared with WLPF,shared(2335)and exclusively(1470 in WLPF,2419 in GP,10774 male)expressed genes were identified in the three-wing morphs through RNA-Seq,and several signaling pathways that are potentially involved in their wing differentiation were obtained,including insulin signaling,starch and sucrose metabolism.

Mechanism of origin in two cases of chimerism

Chimerism is defined as the presence in a subject of more than one stable and genetically distinct cell line;cases reported so far include both patients with ambiguous genitalia and healthy subjects. The biological mechanisms, which may give origin to chimeras, are complex, and can be understood by analyzing DNA samples of the patients and their parents using molecular techniques. The objective of this study is to identify the mechanism of origin for the 2 cases we report. The first patient is a phenotipically normal girl with normal (external and internal) genitalia;the second patient had ambiguous genitalia and underwent surgery. DNA was purified from blood samples and, limited to Patient 1, from a sample of biliary cyst. Short tandem repeat polymorphisms were analyzed in order to identify the relative parental contribution to the patients. Molecular analyses carried out on the first patient are not fully informative because of two possible explanations (i.e. parthenogenetic and andrognetic chimera), while in the second case the presence of four alleles at some markers allowed us to identify a tetragametic chimera originnated from the fusion of two distinct embryos. Studies carried on one single tissue may not always be conclusive as they do not allow the precise identification of the mechanism of origin. In these cases, studies on more tissues are strongly suggested.

Human embryonic stem cell lines with ccr5-del32 allele conferring resistance to HIV

A 32bp deletion in the chemokine receptor 5 (CCR5) gene (CMKBR5) was shown to be linked to HIV resistance. Bone marrow transplantation from the homozygous CCR5-del32 donor to a CDC Stage 2 HIV-positive recipient was demonstrated to confer a HIV resistance, resulting in discontinuation of antiretroviral therapy. In search for an unlimited source of CCR5-del32 cells for transplantation purposes, we tested 137 human embryonic stem cell (hESC) lines from the Reproductive Genetics Institute’s hESC lines collection, and report here the finding of 12 hESC lines with the CCR5-del32 allele, one of which represents a unique partenogenetic ESC line containing two copies of this deletion and may be studied for utility in stem cell transplantation treatment of HIV.

No effect of exogenous melatonin on development of cryopreserved metaphase Ⅱ oocytes in mouse

Background： This study was conducted to investigate effect of exogenous melatonin on the development of mouse mature oocytes after cryopreservation. Results： First, mouse metaphase II （MII） oocytes were vitrified in the open-pulled straws （OPS）. After warming, they were cultured for 1 h in M2 medium containing melatonin at different concentrations （0, 10-9, 10-7, 10-s, 10 3 mol/L）. Then the oocytes were used to detect reactive oxygen species （ROS） and glutathione （GSH） levels （fluorescence microscopy）, and the developmental potential after parthenogenetic activation. The experimental results showed that the ROS level and cleavage rate in 10-3 mol/L melatonin group was significantly lower than that in melatonin-free group （control）. The GSH levels and blastocyst rates in all melatonin-treated groups were similar to that in control. Based on the above results, we detected the expression of gene Hsp9Oaal, Hsfl, Hspalb, Nrf2 and Bcl-xl with qRT-PCR in oocytes treated with 10-7, or 10-3 mol/L melatonin and untreated control. After warming and culture for 1 h, the oocytes showed higher Hsp90oal expression in 10-7 mol/L melatonin-treated group than in the control （P 〈 0.05）; the Hsfl, Hsp9Oaal and Bcl-xl expression were significantly decreased in 10-3 mol/L melatonin-treated group when compared to the control. Based on the above results and previous research, we detected the development of vitrified-warmed oocytes treated with either 10-7 or 0 mol/L melatonin by in vitro fertilization. No difference was observed between them. Conclusions： Our results indicate that the supplementation of melatonin （10-9 to 10-3 mol/L） in culture medium and incubation for 1 h did not improve the subsequent developmental potential of vitrified-warmed mouse MII oocytes, even if there were alteration in gene expression.

Haploid embryonic stem cells:an ideal tool for mammalian genetic analyses

Identification of the function of all genes in the mammalian genome is critical in understanding basic mechanisms of biology.However,the diploidy of mammalian somatic cells has greatly hindered efforts to elucidate the gene function in numerous biological processes by mutagenesis-based genetic approaches.Recently,mouse haploid embryonic stem(haES)cells have been successfully isolated from parthenogenetic and androgenetic embryos,providing an ideal tool for genetic analyses.In these studies,mouse haES cells have already shown that they could be used in cell-based forward or reverse genetic screenings and in generating gene-targeting via homologous recombination.In particular,haES cells from androgenetic embryos can be employed as novel,renewable form of fertilization agent for yielding live-born mice via injection into oocytes,thus showing the possibility that genetic analysis can be extended from cellular level to organism level.

Current advances in haploid stem cells

Diploidy is the typical genomic mode in all mammals.Haploid stem cells are artificial cell lines experimentally derived in vitro in the form of different types of stem cells,which combine the characteristics of haploidy with a broad developmental potential and open the possibility to uncover biological mysteries at a genomic scale.To date,a multitude of haploid stem cell types from mouse,rat,monkey and humans have been derived,as more are in development.They have been applied in high-throughput genetic screens and mammalian assisted reproduction.Here,we review the generation,unique properties and broad applications of these remarkable cells.

Effects of chemical activation and season on birth efficiency of cloned pigs

The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned pigs.Three different activation methods were used:(i) Electroporation(Ele);(ii) Ele followed by incubation with 6-dimethylaminopurine(6-DMAP);and(iii) Ele followed by a treatment with cycloheximide(CHX).In experiment 1,the rates of cleavage,developmental rates and cell number of porcine parthenogenetic(PA) embryos were investigated in the three treatment groups.In experiment 2,NT embryos produced by the three different activation treatments were compared for the rates of cleavage,development and cell number.Finally,the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were compared.The activated oocytes treated by combination activation generally showed a higher(P<0.05) blastocyst rate and produced more expanded blastocysts than oocytes activated with Ele.The rates of cleavage and total cell number of parthenotes were not significantly different.Parthenogenetic embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly(P<0.05) higher rate than those treated with Ele,but the developmental capability was dramatically decreased in NT embryos.With the CHX activation method,the NT embryo blastocyst rate was substantially(P<0.05) increased although the production of expanded blastocysts was not significantly different from that by the other two methods.The birth rate of cloned pigs increased in the CHX group,though the rate was not significantly different from Ele.The effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our study.Porcine oocytes collected in the spring had higher developmental capabilities than those collected in the winter.However,no difference in birth rate of the cloned pigs was found between the oocytes collected in the two seasons.The results obtained from PA and NT embryos,following different activation methods,were inconsistent,suggesting that activation mechanisms are dissimilar in PA and NT embryos.Although the chemical activation in our study leads to an elevation of the blastocyst rate,it does not improve the oocyte's molecular programming and so does not significantly improve the efficiency of producing cloned pig births.