RESUMO
In this issue of Cell, Liu et al. (2018) report the birth of two healthy cloned macaque monkeys using fetal fibroblasts. By artificially enhancing the arsenal of epigenetic modifiers in the oocyte, the authors overcome the earliest roadblocks that take place during somatic cell nuclear transfer (SCNT).
Assuntos
Haplorrinos , Macaca , Animais , Clonagem de Organismos , Fibroblastos , Técnicas de Transferência Nuclear , Oócitos , PrimatasRESUMO
Generation of genetically uniform non-human primates may help to establish animal models for primate biology and biomedical research. In this study, we have successfully cloned cynomolgus monkeys (Macaca fascicularis) by somatic cell nuclear transfer (SCNT). We found that injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at one-cell stage following SCNT greatly improved blastocyst development and pregnancy rate of transplanted SCNT embryos in surrogate monkeys. For SCNT using fetal monkey fibroblasts, 6 pregnancies were confirmed in 21 surrogates and yielded 2 healthy babies. For SCNT using adult monkey cumulus cells, 22 pregnancies were confirmed in 42 surrogates and yielded 2 babies that were short-lived. In both cases, genetic analyses confirmed that the nuclear DNA and mitochondria DNA of the monkey offspring originated from the nucleus donor cell and the oocyte donor monkey, respectively. Thus, cloning macaque monkeys by SCNT is feasible using fetal fibroblasts.
Assuntos
Clonagem de Organismos , Técnicas de Transferência Nuclear , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Feminino , Ácidos Hidroxâmicos/farmacologia , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/metabolismo , Macaca fascicularis , GravidezRESUMO
Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by in vitro fertilization (IVF) but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells and its removal by ectopically expressed H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.
Assuntos
Desenvolvimento Embrionário , Código das Histonas , Histonas/metabolismo , Técnicas de Transferência Nuclear , Animais , Clonagem de Organismos/métodos , Embrião de Mamíferos/metabolismo , Feminino , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Metilação , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas Repressoras/metabolismo , ZigotoRESUMO
A critical turning point was reached in research with the recent success in cloning rhesus monkeys (Macaca mulatta), a major advancement in primatology. This breakthrough marks the beginning of a new age in biomedical research, ushered by improved somatic cell nuclear transfer techniques and creative trophoblast replacement strategies. The successful cloning of rhesus monkeys presents the possibility of producing genetically homogeneous models that are highly advantageous for studying complex biological processes, testing drugs, and researching diseases. However, this achievement raises important ethical questions, particularly regarding animal welfare and the broader ramifications of primate cloning. Approaching the future of primate research with balance is critical, as the scientific world stands on the brink of these revolutionary breakthroughs. This paper aims to summarise the consequences, ethical challenges and possible paths forward in primatology arising from rhesus monkey cloning.
Assuntos
Clonagem de Organismos , Macaca mulatta , Animais , Clonagem de Organismos/ética , Bem-Estar do Animal/ética , Técnicas de Transferência Nuclear/ética , Técnicas de Transferência Nuclear/veterinária , Pesquisa Biomédica/éticaAssuntos
Sistemas CRISPR-Cas , Camelus , Bovinos , Clonagem de Organismos , Edição de Genes , Ovinos , Animais , Bovinos/genética , Camelus/genética , Clonagem de Organismos/tendências , Clonagem de Organismos/veterinária , Sistemas CRISPR-Cas/genética , Edição de Genes/tendências , Edição de Genes/veterinária , Ovinos/genéticaRESUMO
The reason for the poor development of cloned embryos is not yet clear. Several reports have suggested that some nuclear remodeling/reprogramming factors (RRFs) are removed from oocytes at the time of enucleation, which might cause the low success rate of animal cloning. However, there is currently no method to manipulate the amount of RRFs in oocytes. Here, we describe techniques we have developed to gradually reduce RRFs in mouse oocytes by injecting somatic cell nuclei into oocytes. These injected nuclei were remodeled and reprogrammed using RRFs, and then RRFs were removed by subsequent deletion of somatic nuclei from oocytes. The size of the metaphase II spindle reduced immediately, but did recover when transferred into fresh oocytes. Though affected, the full-term developmental potential of these RRF-reduced oocytes with MII-spindle shrinkage was not lost after fertilization. When somatic cell nuclear transfer was performed, the successful generation of cloned mice was somewhat improved and abnormalities were reduced when oocytes with slightly reduced RRF levels were used. These results suggest that a change in RRFs in oocytes, as achieved by the method described in this paper or by enucleation, is important but not the main reason for the incomplete reprogramming of somatic cell nuclei.
Assuntos
Núcleo Celular/metabolismo , Reprogramação Celular , Clonagem de Organismos , Metáfase , Técnicas de Transferência Nuclear , Oócitos/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos ICRRESUMO
In brief: Early embryonic development in goats is a complex and an important process. This study identified a novel long non-coding RNA (lncRNA), lncRNA3720, that appears to affect early embryonic development in goats through histone variants. Abstract: Although abundant lncRNAs have been found to be highly expressed in early embryos, the functions and mechanisms of most lncRNAs in regulating embryonic development remain unclear. This study was conducted to identify the key lncRNAs during embryonic genome activation (EGA) for promoting embryonic development after somatic cell nuclear transfer (SCNT) in goats. We screened and characterized lncRNAs from transcriptome data of in vitro-fertilized, two-cell (IVF-2c) and eight-cell embryos (IVF-8c) and eight-cell SCNT embryos (SCNT-8c). We obtained 12 differentially expressed lncRNAs that were highly expressed in IVF-8c embryos compared to IVF-2c and less expressed in SCNT-8c embryos. After target gene prediction, expression verification, and functional deletion experiments, we found that the expression level of lncRNA3720 affected the early embryonic development in goats. We cloned full-length lncRNA3720 and over-expressed it in goat fetal fibroblasts (GFFs). We identified histone variants by analyzing the transcriptome data from both GFFs and embryos. Gene annotation of the gene library and the literature search revealed that histone variants may have important roles in early embryo development, so we selected them as the potential target genes for lncRNA3720. Lastly, we compensated for the low expression of lncRNA3720 in SCNT embryos by microinjection and showed that the development rate and quality of SCNT embryos were significantly improved. We speculate that lncRNA3720 is a key promoter of embryonic development in goats by interacting with histone variants.
Assuntos
RNA Longo não Codificante , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Histonas/metabolismo , Cabras/genética , Embrião de Mamíferos , Técnicas de Transferência Nuclear/veterinária , Desenvolvimento Embrionário/genética , Fertilização in vitro , Clonagem de OrganismosRESUMO
A number of reviews have been written recently celebrating the 25th anniversary of the birth of Dolly the cloned sheep and the effect this breakthrough has had on various fields of research. However, arguably the biggest impact Dolly has had is on the field of xenotransplantation, described here based on our own experience and that of others.
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Clonagem de Organismos , Técnicas de Transferência Nuclear , Animais , Ovinos , Transplante HeterólogoRESUMO
CONTEXT: In vitro maturation is an important process in the production of embryos. It has been shown that three cytokines, fibroblast growth factor 2, leukemia inhibitory factor and insulin-like growth factor 1 (FLI), increased efficiency of in vitro maturation, somatic cell nuclear transfer (SCNT) blastocyst production, and in vivo development of genetically engineered piglets. AIMS: Assess effects of FLI on oocyte maturation, quality of oocytes, and embryo development in bovine in vitro fertilisation (IVF) and SCNT. KEY RESULTS: Cytokine supplementation resulted in significant increases in maturation rates and decreased levels of reactive oxygen species. Oocytes matured in FLI had increased blastocyst rates when used in IVF (35.6%vs 27.3%, P <0.05) and SCNT (40.6%vs 25.7%, P <0.05). SCNT blastocysts contained significantly more inner cell mass and trophectodermal cells when compared to the control group. Importantly, SCNT embryos derived from oocytes matured in FLI medium resulted in a four-fold increase in full-term development compared to control medium (23.3%vs 5.3%, P <0.05). Relative mRNA expression analysis of 37 genes associated with embryonic and fetal development revealed one gene had differential transcript abundance in metaphase II oocytes, nine genes at the 8-cell stage, 10 genes at the blastocyst stage in IVF embryos and four genes at the blastocyst stage in SCNT embryos. CONCLUSIONS: Cytokine supplementation increased efficiency of in vitro production of IVF and SCNT embryos and in vivo development of SCNT embryos to term. IMPLICATIONS: Cytokine supplementation is beneficial to embryo culture systems, which may shed light on requirements of early embryo development.
Assuntos
Citocinas , Técnicas de Transferência Nuclear , Animais , Bovinos , Suínos , Citocinas/genética , Citocinas/metabolismo , Técnicas de Transferência Nuclear/veterinária , Desenvolvimento Embrionário , Fertilização in vitro/veterinária , Blastocisto/metabolismo , Oócitos/metabolismo , Suplementos Nutricionais , Clonagem de OrganismosRESUMO
Somatic cell nuclear transfer (SCNT) is the only reproductive technology used to produce individuals from somatic cells by transferring them to enucleated oocytes. Although more than 25 years have passed since the first mammalian SCNT was reported in sheep, problems such as low birth rates and morphological abnormalities have persisted and limited its practical applications. The mouse is the ideal laboratory animal to unveil these questions due to its established reproductive technologies and extensive knowledge base of its genome and various strains. We investigated the causes of incomplete reprogramming after nuclear transfer of donor somatic cells and found that the loss of imprint in some placenta-specific imprinted genes could induce non-random SCNT abnormalities. By ameliorating aberrantly expressed imprinted genes, we succeeded in increasing the low birth rate and improving morphological abnormalities observed in SCNT fetuses. Furthermore, we sought appropriate mouse strains and cell types as nuclear donors to increase their developmental efficiencies and expand their applications in various fields. Peripheral blood cells are useful as ethical and economical cell species because they can be collected easily, even though SCNT embryos derived from hematopoietic cells show poor developmental abilities after reconstruction. Additionally, it is possible to obtain mice that are reactive to specific antigens of interest by using lymphocytes. Although there are still many limitations to the practical use of SCNT, its utilization is steadily expanding.
Assuntos
Metilação de DNA , Técnicas de Transferência Nuclear , Animais , Camundongos , Ovinos , Técnicas de Transferência Nuclear/veterinária , Mamíferos , Núcleo Celular/metabolismo , Embrião de Mamíferos/metabolismo , Clonagem de Organismos/veterináriaRESUMO
Interspecies somatic cell nuclear transfer (iSCNT) has an immense potential to rescue endangered animals and extinct species like mammoths. In this study, we successfully established an Asian elephant's fibroblast cell lines from ear tissues, performed iSCNT with porcine oocytes and evaluated the in vitro and in vivo development of reconstructed embryos. A total of 7780 elephant-pig iSCNT embryos were successfully reconstructed and showed in vitro development with cleavage rate, 4-cell, 8-cell and blastocyst rate of 73.01, 30.48, 5.64, and 4.73%, respectively. The total number of elephant-pig blastocyte cells and diameter of hatched blastocyte was 38.67 and 252.75 µm, respectively. Next, we designed species-specific markers targeting EDNRB, AGRP and TYR genes to verify the genome of reconstructed embryos with donor nucleus/species. The results indicated that 53.2, 60.8, and 60.8% of reconstructed embryos (n = 235) contained elephant genome at 1-cell, 2-cell and 4-cell stages, respectively. However, the percentages decreased to 32.3 and 32.7% at 8-cell and blastocyst stages, respectively. Furthermore, we also evaluated the in vivo development of elephant-pig iSCNT cloned embryos and transferred 2260 reconstructed embryos into two surrogate gilts that successfully became pregnant and a total of 11 (1 and 10) fetuses were surgically recovered after 17 and 19 days of gestation, respectively. The crown-rump length and width of elephant-pig cloned fetuses were smaller than the control group. Unfortunately, none of these fetuses contained elephant genomes, which suggested that elephant embryos failed to develop in vivo. In conclusion, we successfully obtained elephant-pig reconstructed embryos for the first time and these embryos are able to develop to blastocyst, but the in vivo developmental failure needs further investigated.
Assuntos
Clonagem de Organismos , Elefantes , Gravidez , Animais , Suínos , Feminino , Clonagem de Organismos/métodos , Elefantes/genética , Técnicas de Transferência Nuclear/veterinária , Oócitos/metabolismo , Blastocisto , Sus scrofa , Desenvolvimento Embrionário , Embrião de MamíferosRESUMO
The purpose of this study was to examine STC-1's structure, function, and differential expression in large and miniature pigs. We cloned the Hezuo pig's coding sequence, compared its homology, and used bioinformatics to assess the structure. RT-qPCR and Western blot were used to detect the expression in ten tissues of Hezuo pig and Landrace pig. The results showed that Hezuo pig was most closely related to Capra hircus and most distantly related to Danio rerio. The protein STC-1 has a signal peptide and its secondary structure is dominated by the alpha helix. The mRNA expression in the spleen, duodenum, jejunum, and stomach of Hezuo pigs was higher than that of Landrace pigs. And except for heart and duodenum, expression of the protein in Hezuo pig was higher than in another. In conclusion, STC-1 is highly conserved among different breeds of pigs, and the expression and distribution of its mRNA and protein are different in large and miniature pigs. This work can lay the foundation for future study into the mechanism of action of STC-1 in Hezuo pigs and the enhancement of breeding in miniature pigs.
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Clonagem de Organismos , Suínos/genética , Animais , Porco Miniatura/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Clonagem MolecularRESUMO
Adult male and female Murrah buffalo fibroblast cells were used as donors for the production of embryos using handmade cloning. Both donor cells and reconstructed embryos were treated with 50 nM trichostatin-A (TSA) and 7.5 nM 5-aza-2'-deoxycytidine (5-aza-dC). The blastocyst rate of both treated male (40.1% ± 2.05) and female (37.0% ± 0.83) embryos was significantly lower than in untreated control males (49.7% ± 3.80) and females (47.2% ± 2.44) but their apoptotic index was lower (male, control: 5.90 ± 0.48; treated: 4.96 ± 0.31): (female, control: 8.11 ± 0.67; treated: 6.65 ± 0.43) and epigenetic status in terms of global acetylation and methylation of histone was significantly improved. The expression level of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was higher (P < 0.05) and that of PGK, G6PD, OCT 4, IFN-tau and CASPASE3 was significantly lower (P < 0.05) in treated male blastocyst than control and the expression levels of DNMT1, IGF1R and BCL-XL were not significantly different between the two groups. In the female embryos, the relative mRNA abundance of OCT4 was significantly higher (P < 0.05), and that of XIST and CASPASE3 was significantly lower (P < 0.05) in the epigenetic modifier-treated group compared with that of the control group, whereas the expression levels of HPRT, PGK, G6PD, DNMT1, IFN-tau, IGF1R and BCL-XL were not significantly different between the two groups. In both embryos, a similar effect of treatment was observed on genes related to growth and development, but the effect on the expression of X-linked genes varied. These results indicate that not all X-linked genes respond to TSA and 5-aza-dC treatment in the same manner.
Assuntos
Búfalos , Epigênese Genética , Animais , Feminino , Masculino , Búfalos/genética , Búfalos/metabolismo , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Hipoxantina Fosforribosiltransferase/farmacologia , Blastocisto/metabolismo , Clonagem de Organismos/métodos , Azacitidina/farmacologia , Desenvolvimento Embrionário/genética , Técnicas de Transferência NuclearRESUMO
Since the announcement of the birth of Dolly, the world's first mammal produced by cloning, it was demonstrated for the first time that somatic cells could be reprogrammed to produce a whole individual. This represented a considerable change in paradigm in the field of embryo technologies both in humans and animals which led to an intense burst of research on nuclear transfer but also on the establishment of pluripotency and the directed edition of the genome. As such, induced pluripotent cells and gene editing tools, the best known being CRISPR-Cas9, are now available to the scientific community. Nevertheless, cloning was associated with important developmental abnormalities in a variable proportion of pregnancies, raising concern about the long-term effects of embryo technologies at a time when the concept of the developmental origins of health and disease had emerged, together with a better understanding of the underlying epigenetic modifications. The focus of this article is to review current knowledge on long-term effects of artificial reproduction technologies in mammals, leading to globally reassuring information although differences are present and caution remains necessary taking the current increasing number of in vitro-produced ruminant and equine embryos into account and their potential intergenerational consequences.
Assuntos
Clonagem de Organismos , Técnicas de Transferência Nuclear , Gravidez , Feminino , Cavalos , Animais , Humanos , Técnicas de Transferência Nuclear/veterinária , Clonagem de Organismos/veterinária , Epigênese Genética , Mamíferos , BiotecnologiaRESUMO
This study was focused on the effects of ovary acquisition season, embryo transfer season, and conditions of surrogate sows on cloning efficiency, with the objective of improving the production of cloned pigs. The statistical analysis documented that cloning efficiency was highest when ovary extraction and embryo transfer occurred in the spring, and lowest when such operations occurred in the autumn. This was evidenced by the higher number of recovered oocytes (3.2 ± 0.47 vs. 2.5 ± 0.51), rate of mature oocytes (57.4 ± 0.07% vs. 48.9 ± 0.06%), rate of developed cloned blastocysts (35.7 ± 0.12% vs. 34.4 ± 0.07%), pregnancy rate of surrogate sows (73.5% vs. 33.3%), delivery rate (67.6% vs. 16.7%), litter size (6.9 ± 2.3 vs. 2.3 ± 2.5), and the number of alive newborns (5.7 ± 2.2 vs. 1.3 ± 1.2). Cloning efficiency was little affected by the ovulatory status of the surrogate sow prior to embryo transfer. The length of pregnancy, the parity, and the length of labor of the surrogate sow significantly affected the efficiency of generating pigs cloned from somatic cells. Specifically, when length of pregnancy ranged from 111 to 117 days, surrogate sows with shorter gestation period had larger litter size (8.9 ± 2.8) and a higher number of stillbirths per litter (2.1 ± 2.0). Moreover, statistical analysis indicated that selecting sows with 2-4 parities as surrogates led to increased litter size (7.7 ± 3.0) and the number of alive newborns (6.4 ± 3.1). In comparison with naturally breeding sows, the surrogate sows spent more time giving birth and suffered higher rates of stillbirth. The data obtained in this study provide valuable insights for improving the production efficiency of somatic cell cloned pigs.
Assuntos
Clonagem de Organismos , Doenças dos Suínos , Suínos , Gravidez , Animais , Feminino , Estações do Ano , Paridade , Tamanho da Ninhada de Vivíparos , Clonagem de Organismos/veterinária , Natimorto/veterinária , Clonagem Molecular , LactaçãoRESUMO
In this study we treated the handmade cloned (HMC) buffalo embryos with the DNA methylation inhibitors; 5-aza-2'-deoxycytidine (AzadC) or Zebularine individually after post-fusion and during in vitro culture till eighth day. The blastocysts production rate significantly improved (p < .01) after treating embryos independently with 5 nM AzadC and 5 nM zebularine compared with 2 and 10 nM AzadC or zebularine groups, respectively. The highest cleavage rates were obtained for 5 nM treatment of AzadC and zebularine compared with other treatments and untreated control group. Quality of blastocysts were evaluated using total cell number (TCN) and the ratio of number of inner cell mass (ICM) cells/total cell number (ICM/TCN). Zebularine treatments (2/5/10 nM) significantly improved both TCN and ICM/TCN ratio compared with AzadC treatments (2/5/10 nM); however, control group TCN and ICM/TCN ratio was found lower. The methylation percentage of pDS4.1 and B. bubalis satellite DNA were comparatively more attenuated with 5 nM zebularine than 5 nM AzadC treatment. The increased in vitro development rates of the treated embryos were correlated with the decreased level of DNA methylation and the improved blastocyst quality. Following transfer of 5 nM zebularine treated embryos to 6 recipients, 4 were found to be pregnant, though the pregnancies were not carried to full term.
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Búfalos , Clonagem de Organismos , Gravidez , Feminino , Animais , Decitabina/farmacologia , Búfalos/genética , Clonagem de Organismos/veterinária , Técnicas de Transferência Nuclear/veterinária , Blastocisto , Metilação de DNA , Desenvolvimento EmbrionárioRESUMO
The abnormal modification of histone is an important factor restricting development of porcine cloned embryos. Overexpression of histone H3K9me3 demethylase KDM4 family can effectively improve the developmental efficiency of cloned embryos. In order to explore the effects of overexpression of H3K9me3 demethylase on the development of porcine cloned embryos, KDM4A mRNA and KDM4D mRNA were injected respectively into porcine cloned embryos at the 1-cell stage and 2-cell stage to detect the blastocyst rate; 2-cell stage cloned embryos injected with KDM4A mRNA and embryo injection water (the control group) at the 1-cell stage were collected to detect the expression level of H3K9me3, and 4-cell stage cloned embryos were collected for single cell transcriptome sequencing, then the sequencing data was analyzed with KEGG and GO. The results showed that the blastocyst rate of porcine cloned embryos injected with KDM4A mRNA at 1-cell stage was significantly higher than that of the control group (25.32 ± 0.74% vs 14.78 ± 0.87%), while cloned embryos injected with KDM4D mRNA had a similar blastocyst rate with cloned embryos in control group (16.27 ± 0.77% vs 14.78 ± 0.87%). Porcine cloned embryos injected with KDM4A mRNA and KDM4D mRNA at 2-cell stage had a similar blastocyst rate with cloned embryos in control group (32.18 ± 1.67%, 30.04 ± 0.91% vs 31.22 ± 1.40%). The expression level of H3K9me3 in cloned embryos injected with KDM4A mRNA at 1-cell stage was lower than that in control group. There were 133 differentially expressed genes detected by transcriptome sequencing, including 52 up-regulated genes and 81 down-regulated genes. Pathways enriched by GO analyses were mainly related to protein localization. Pathways enriched by KEGG analyses were related to cellular senescence and acute myeloid leukemia. These results suggest that overexpression of histone H3K9me3 demethylase KDM4A can significantly improve the developmental efficiency of porcine cloned embryos.
Assuntos
Histona Desmetilases , Histonas , Suínos/genética , Animais , Histona Desmetilases/metabolismo , Histona Desmetilases/farmacologia , Histonas/genética , Histonas/metabolismo , Técnicas de Transferência Nuclear , Desenvolvimento Embrionário/genética , Blastocisto/metabolismo , RNA Mensageiro/metabolismo , Clonagem de OrganismosRESUMO
Somatic cell nuclear transfer (SCNT) shows great application value in the generation of transgenic animals, protection of endangered species, and therapeutic cloning. However, the cloning efficiency is still very low, which greatly restricts its application. Compared to fertilized embryos, cloned embryos lack the sperm proteins, which are considered to play an important role in embryonic development. Here, we compared the sperm proteome, with that of donor fibroblasts and oocytes, and identified 342 proteins unique to sperm, with 42 being highly expressed. The 384 proteins were mainly enriched in the categories of post-translational modification and cytoskeletal arrangement. Extracts of soluble sperm or fibroblast proteins were injected into cloned embryos, and the result showed that injection of sperm protein significantly inhibited abnormal embryonic cleavage, significantly decreased the level of trimethylated histone H3 Lys9 (H3K9me3) and the apoptotic index, and increased the inner cell mass (ICM)-to-trophectoderm (TE) ratio. More importantly, the sperm proteins also significantly enhanced the birthrate. The results of in vitro and in vivo experiments demonstrate that sperm-derived proteins improve embryo cloning efficiency. Our findings not only provide new insights into ways to overcome low cloning efficiency, but also add to the understanding of sperm protein function.