Paternal high-fat diet altered H3K36me3 pattern of pre-implantation embryos.

The global transition towards diets high in calories has contributed to 2.1 billion people becoming overweight, or obese, which damages male reproduction and harms offspring. Recently, more and more studies have shown that paternal exposure to stress closely affects the health of offspring in an intergenerational and transgenerational way. SET Domain Containing 2 (SETD2), a key epigenetic gene, is highly conserved among species, is a crucial methyltransferase for converting histone 3 lysine 36 dimethylation (H3K36me2) into histone 3 lysine 36 trimethylation (H3K36me3), and plays an important regulator in the response to stress. In this study, we compared patterns of SETD2 expression and the H3K36me3 pattern in pre-implantation embryos derived from normal or obese mice induced by high diet. The results showed that SETD2 mRNA was significantly higher in the high-fat diet (HFD) group than the control diet (CD) group at the 2-cell, 4-cell, 8-cell, and 16-cell stages, and at the morula and blastocyst stages. The relative levels of H3K36me3 in the HFD group at the 2-cell, 4-cell, 8-cell, 16-cell, morula stage, and blastocyst stage were significantly higher than in the CD group. These results indicated that dietary changes in parental generation (F0) male mice fed a HFD were traceable in SETD2/H3K36me3 in embryos, and that a paternal high-fat diet brings about adverse effects for offspring that might be related to SETD2/H3K36me3, which throws new light on the effect of paternal obesity on offspring from an epigenetic perspective.

Significant alteration of protein profiles in a mouse model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disorder, affecting women of child-bearing age, and the incidence rate is growing and assuming epidemic proportions. The etiology of PCOS remains unknown and there is no cure. Some animal models for PCOS have been established which have enhanced our understanding of the underlying mechanisms, but omics data for revealing PCOS pathogenesis and for drug discovery are still lacking. In the present study, proteomics analysis was used to construct a protein profile of the ovaries in a PCOS mouse model. The result showed a clear difference in protein profile between the PCOS and control group, with 495 upregulated proteins and 404 downregulated proteins in the PCOS group. The GO term and KEGG pathway analyses of differentially expressed proteins mainly showed involvement in lipid metabolism, oxidative stress, and immune response, which are consistent with pathological characteristics of PCOS in terms of abnormal metabolism, endocrine disorders, chronic inflammation and imbalance between oxidant and antioxidant levels. Also, we found that inflammatory responses were activated in the PCOS ovarium, while lipid biosynthetic process peroxisome, and bile secretion were inhibited. In addition, we found some alteration in unexpected pathways, such as glyoxylate and dicarboxylate metabolism, which should be investigated. The present study makes an important contribution to the current lack of PCOS ovarian proteomic data and provides an important reference for research and development of effective drugs and treatments for PCOS.

DT-109 ameliorates nonalcoholic steatohepatitis in nonhuman primates.

Nonalcoholic steatohepatitis (NASH) prevalence is rising with no pharmacotherapy approved. A major hurdle in NASH drug development is the poor translatability of preclinical studies to safe/effective clinical outcomes, and recent failures highlight a need to identify new targetable pathways. Dysregulated glycine metabolism has emerged as a causative factor and therapeutic target in NASH. Here, we report that the tripeptide DT-109 (Gly-Gly-Leu) dose-dependently attenuates steatohepatitis and fibrosis in mice. To enhance the probability of successful translation, we developed a nonhuman primate model that histologically and transcriptionally mimics human NASH. Applying a multiomics approach combining transcriptomics, proteomics, metabolomics, and metagenomics, we found that DT-109 reverses hepatic steatosis and prevents fibrosis progression in nonhuman primates, not only by stimulating fatty acid degradation and glutathione formation, as found in mice, but also by modulating microbial bile acid metabolism. Our studies describe a highly translatable NASH model and highlight the need for clinical evaluation of DT-109.

Somatic Cell Nuclear Transfer in Rabbits.

Somatic cell nuclear transfer (SCNT) is a technology that enables differentiated somatic cells to acquire a totipotent state, thus making it of great value in developmental biology, biomedical research, and agricultural applications. Rabbit cloning associated with transgenesis has the potential to improve the applicability of this species for disease modeling, drug testing, and production of human recombinant proteins. In this chapter, we introduce our SCNT protocol for the production of live cloned rabbits.

Effect of Dietetic Obesity on Testicular Transcriptome in Cynomolgus Monkeys.

Obesity is a metabolic disorder resulting from behavioral, environmental and heritable causes, and can have a negative impact on male reproduction. There have been few experiments in mice, rats, and rabbits on the effects of obesity on reproduction, which has inhibited the development of better treatments for male subfertility caused by obesity. Nonhuman primates are most similar to human beings in anatomy, physiology, metabolism, and biochemistry and are appropriate subjects for obesity studies. In this investigation, we conducted a transcriptome analysis of the testes of cynomolgus monkeys on high-fat, high-fructose, and cholesterol-rich diets to determine the effect of obesity on gene expression in testes. The results showed that the testes of obese monkeys had abnormal morphology, and their testes transcriptome was significantly different from that of non-obese animals. We identified 507 differentially abundant genes (adjusted p value < 0.01, log2 [FC] > 2) including 163 up-regulated and 344 down-regulated genes. Among the differentially abundant genes were ten regulatory genes, including IRF1, IRF6, HERC5, HERC6, IFIH1, IFIT2, IFIT5, IFI35, RSAD2, and UBQLNL. Gene ontology (GO) and KEGG pathway analysis was conducted, and we found that processes and pathways associated with the blood testes barrier (BTB), immunity, inflammation, and DNA methylation in gametes were preferentially enriched. We also found abnormal expression of genes related to infertility (TDRD5, CLCN2, MORC1, RFX8, SOHLH1, IL2RB, MCIDAS, ZPBP, NFIA, PTPN11, TSC22D3, MAPK6, PLCB1, DCUN1D1, LPIN1, and GATM) and down-regulation of testosterone in monkeys with dietetic obesity. This work not only provides an important reference for research and treatment on male infertility caused by obesity, but also valuable insights into the effects of diet on gene expression in testes.

Sperm-borne proteins improve rabbit cloning efficiency via regulating embryonic cleavage and epigenetics.

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.

Loss of Renewal of Extracellular Vesicles: Harmful Effects on Embryo Development in vitro.

Background: Extracellular vesicles (EVs), as a promising platform for drug delivery, have attracted much attention. Degradation and regeneration of EVs maintain their homeostasis in vivo, but this regeneration is missing in the in vitro culture (IVC) system, which is likely to lead to negative effects. It is particularly concerning that most studies involving the addition of EVs in IVC seem to overlook this point. Methods: We used rabbit embryos and oviduct fluid EVs as a model of embryo development to examine the effect of loss or gain of EV functionality in an IVC system. Embryonic development ratios were determined in each group. Malondialdehyde and ammonium ions in the culture medium were measured. RNA-seq, reactive oxygen species (ROS) staining, immunofluorescence of LC3 and H3K36me3, and qPCR of oxidative stress-related genes and autophagy-related genes of blastocysts in the in vivo group, non-EVs group, con-EVs group, and R-EVsM group was implemented. Results: Incubation of embryos with 9.1×1011 EV particles/mL had a positive effect at 48 h and 72 h, which disappeared by 96 h, however. EVs at a concentration of 9.1×1012 particles/mL even showed a negative effect at 96 h. As culture time in the IVC system was increased, the amount of malondialdehyde and ammonium ions in the culture medium was increased, and there was a decrease in embryonic development activity of EVs. Lack of EV renewal in the IVC system impaired embryonic development competence, while replacement of EVs and medium during IVC could sustain embryonic development. Loss or gain of renewal in the IVC system affected EVs' influence on embryo transcriptome, embryonic ROS, autophagy, epigenetic state and apoptosis. Conclusion: Loss of renewal in the IVC system affected EVs' role in embryonic development by causing an imbalance in ROS, autophagy, abnormal H3K36me3 levels and apoptosis, while gain of renewal in the IVC system reduced these adverse effects and ensured the beneficial function of EVs.

Current Progress and Prospects in Rabbit Cloning.

Somatic cell nuclear transfer (SCNT) shows great value in the generation of transgenic animals, protection of endangered animals, and stem cell therapy. The combination of SCNT and gene editing has produced a variety of genetically modified animals for life science and medical research. Rabbits have unique advantages as transgenic bioreactors and human disease models; however, the low SCNT efficiency severely impedes the application of this technology. The difficulty in SCNT may be attributable to the abnormal reprogramming of somatic cells in rabbits. This review focuses on the abnormal reprogramming of cloned mammalian embryos and evaluates the progress and prospects of rabbit somatic cell cloning.

Effects of changing culture medium on preimplantation embryo development in rabbit.

Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

Sperm-borne small RNAs improve the developmental competence of pre-implantation cloned embryos in rabbit.

The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.

Sperm-borne miR-202 targets SEPT7 and regulates first cleavage of bovine embryos via cytoskeletal remodeling.

In mammals, sperm-borne regulators can be transferred to oocytes during fertilization and have different effects on the formation of pronuclei, the first cleavage of zygotes, the development of preimplantation embryos and even the metabolism of individuals after birth. The regulatory role of sperm microRNAs (miRNAs) in the development of bovine preimplantation embryos has not been reported in detail. By constructing and screening miRNA expression libraries, we found that miR-202 was highly enriched in bovine sperm. As a target gene of miR-202, co-injection of SEPT7 siRNA can partially reverse the accelerated first cleavage of bovine embryos caused by miR-202 inhibitor. In addition, both a miR-202 mimic and SEPT7 siRNA delayed the first cleavage of somatic cell nuclear transfer (SCNT) embryos, suggesting that miR-202-SEPT7 mediates the delay of first cleavage of bovine embryos. By further exploring the relationship between miR-202/SEPT7, HDAC6 and acetylated α-tubulin during embryonic development, we investigated how sperm-borne miR-202 regulates the first cleavage process of bovine embryos by SEPT7 and demonstrate the potential of sperm-borne miRNAs to improve the efficiency of SCNT.

Insights into the roles of sperm in animal cloning.

Somatic cell nuclear transfer (SCNT) has shown a wide application in the generation of transgenic animals, protection of endangered animals, and therapeutic cloning. However, the efficiency of SCNT remains very low due to some poorly characterized key factors. Compared with fertilized embryos, somatic donor cells lack some important components of sperm, such as sperm small noncoding RNA (sncRNA) and proteins. Loss of these factors is considered an important reason for the abnormal development of SCNT embryo. This study focused on recent advances of SCNT and the roles of sperm in development. Sperm-derived factors play an important role in nucleus reprogramming and cytoskeleton remodeling during SCNT embryo development. Hence, considering the role of sperm may provide a new strategy for improving cloning efficiency.

Melatonin Protects Rabbit Somatic Cell Nuclear Transfer (SCNT) Embryos from Electrofusion Damage.

The study's objectives were to examine the effects of electrofusion on rabbit somatic cell nuclear transfer (SCNT) embryos, and to test melatonin as a protective agent against electrofusion damage to SCNT embryos. The levels of reactive oxygen species (ROS), the epigenetic state (H3K9me3), and the content of endoplasmic reticulum (ER) stress-associated transcripts (IRE-1 and CHOP) were measured. Melatonin was added during the preimplantation development period. The total blastocyst cell numbers were counted, and the fragmentation rate and apoptotic index were determined and used to assess embryonic development. Electrofusion increased (1) ROS levels at the 1-, 2-, 4-, and 8-cell stages; (2) H3K9me3 levels at the 2-, 4-, and 8-cell stage; and (3) the expression of IRE-1 and CHOP at the 8-cell, 16-cell, morula, and blastocyst stages. The treatment of SCNT embryos with melatonin significantly reduced the level of ROS and H3K9me3, and the expression levels of IRE-1 and CHOP. This treatment also significantly reduced the fragmentation rate and apoptotic index of blastocysts and increased their total cell number. In conclusion, the electrofusion of rabbit SCNT embryos induced oxidative stress, disturbed the epigenetic state, and caused ER stress, while melatonin reduced this damage. Our findings are of signal importance for improving the efficiency of SCNT and for optimizing the application of electrical stimulation in other biomedical areas.

Extracellular vesicles and melatonin benefit embryonic develop by regulating reactive oxygen species and 5-methylcytosine.

Embryo culture conditions are crucial as they can affect embryo quality and even offspring. Oviductal extracellular vesicles (EVs) long been considered a major factor influencing interactions between the oviduct and embryos, and thus its absence is associated with inferior embryonic development in in vitro culture. Herein, we demonstrated that melatonin is present in oviduct fluids and oviduct fluid-derived EVs. Addition of either EVs (1.87 × 1011 particles/mL) or melatonin (340 ng/mL) led to a significant downregulation of reactive oxygen species (ROS) and 5-methylcytosine (5-mC), as well as an increase in the blastocyst rate of embryos, which was inhibited by the addition of luzindole-a melatonin receptor agonist. A combination of EVs (1.87 × 1010 particles/mL) and melatonin (at 34.3 pg/mL) led to the same results as well as a significant decrease in the apoptosis index and increase in the inner cell mass (ICM)/trophectoderm (TE) index. These results suggest that an EV-melatonin treatment benefits embryonic development. Our findings provide insights into the role of EVs and melatonin during cell communication and provide new evidence of the communication between embryos and maternal oviduct.

The effects of glycine-glutamine dipeptide replaced l-glutamine on bovine parthenogenetic and IVF embryo development.

Relative to alanine and serine amino acid levels, glutamine is highly abundant in follicular fluid, and is an important source of energy required for oocyte maturation and embryo development. Thus, glutamine is an essential component of in vitro embryo culture media. However, glutamine has poor stability and degrades spontaneously in solution to form ammonia and pyrrolidonecarboxylic acid. In the present study, we aimed to explore the effect of substituting l-glutamine with glycine-glutamine, a more stable glutamine, on development of early parthenogenetic embryos and in vitro fertilization (IVF) embryos in bovine. Results revealed that glycine-glutamine can significantly increase cleavage rate (parthenogenetic embryos:87.24% vs. 72.61%, IVF embryos:89.33% vs. 83.79%, P < 0.01), blastocyst number (parthenogenetic embryos:24.98% vs. 18.07%, IVF embryos:33.53% vs. 27.29%, P < 0.01), and blastocyst number (parthenogenetic embryos:96 vs. 76, IVF embryos:114 vs. 109, P < 0.01), reduce blastocyst apoptosis (parthenogenetic embryos:3.72% vs. 6.65%, IVF embryos:2.53% vs.6.23%, P < 0.01), alleviate embryo ammonia toxicity, and reduce the content of reactive oxygen species (ROS) compared with the l-glutamine. In addition, glycine-glutamine can alter epigenetic reprogramming by increasing the expression of HDAC1 (Histone Deacetylase 1) and decreasing the relative expression levels of H3K9 acetylation in early parthenogenetic embryos and IVF embryos. From our present study, we concluded that glycine-glutamine is an effective substitute of glutamine in modified synthetic oviduct fluid with amino acids (mSOFaa).

Use of oocytes selected by brilliant cresyl blue staining enhances rabbit cloned embryo development in vitro.

SummaryRabbits play an important role in people's lives due to their high nutritional value and high-quality hair that can be used as raw material for textiles. Furthermore, rabbits are an important animal model for human disease, as genome-edited animals are particularly valuable for studying gene functions and pathogenesis. Somatic cell nuclear transfer (SCNT) is an important technique for producing genome-edited animals and it has great value in saving endangered species and in clone stem cell therapy. However, the low efficiency of SCNT limits its application, with the selection of suitable rabbit oocytes being crucial to its success. In the present study, we collected oocytes from ovarian follicles and stained them with 26 µM brilliant cresyl blue (BCB). We then matured the oocytes in vitro and used them for SCNT. Comparison of the BCB-positive oocytes with BCB-negative oocytes and the control group showed that the BCB-positive group had a significantly higher maturation rate (81.4% vs. 48.9% and 65.3% for the negative and control groups, respectively), cleavage rate (86.6% vs. 67.9% and 77.9%), blastocyst rate (30.5% vs. 12.8% and 19.6%), total number of blastocysts (90±7.5 vs. 65.3±6.3 and 67.5±5.7), and inner cell mass (ICM)/ trophectoderm (TE) index (42.3±4.2 vs. 30.2±2.1 and 33.9±5.1) (P<0.05). The BCB-positive group had a significantly lower apoptosis index (2.1±0.6 vs. 8.2±0.9 and 6.7±1.1 for the negative and control groups, respectively) (P<0.05). These findings demonstrate that BCB-positive oocytes have a higher maturation ability and developmental competence in vitro, indicating that BCB staining is a reliable method for selecting oocytes to enhance the efficiency of SCNT.

Sperm-borne small RNAs regulate α-tubulin acetylation and epigenetic modification of early bovine somatic cell nuclear transfer embryos.

Accumulated evidence indicates that sperm-borne small RNA plays a crucial role in embryonic development, especially the absence of the sperm-borne small RNA might be a major cause of the abnormal development of cloned embryos. In this study, we found that sperm-borne small RNA can affect abnormal pronuclear-like structures, postpone the timing of first embryo cleavage and enhance developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos. In addition, the supplementation of sperm-borne small RNA can significantly increase live birth rates and decrease the birth weights of cloned offspring. To investigate the underlying mechanisms, the levels of α-tubulin K40 acetylation (Ac α-tubulin K40) and histone H3 lysine 9 trimethylation (H3K9me3) during early embryo development were investigated in SCNT embryos with sperm-borne small RNA supplementation (termed as T-NT), compared to those normal SCNT embryos and embryos obtained from standard IVF. The results showed that sperm-borne small RNA can significantly decrease the H3K9me3 levels at the pronuclear and two-cell stages, while significantly increase Ac α-tubulin K40 levels at anaphase and telophase of bovine SCNT embryos during the first cleavage. Collectively, our study for the first time demonstrates that sperm-borne small RNA plays a crucial role in the developmental competence of SCNT embryos by regulating H3K9me3 and Ac α-tubulin K40. Further studies will be required to determine how sperm small RNA regulate the H3K9me3 and Acα-tubulin K40. Our study suggests that the supplementation of sperm-borne small RNA is a potential application to improve the cloning efficiency.

Extracellular vesicles derived from donor oviduct fluid improved birth rates after embryo transfer in mice.

Embryo transfer (ET) is an important procedure for assisted reproduction. However, the relatively lower success rate of ET hampers its application potential. In this study we aimed to elucidate the effects of extracellular vesicles derived from donor oviduct fluid (EDOF) on embryo development after ET. Extracellular vesicles from the oviduct were isolated and purified using ultracentrifugation and identified using transmission electron microscopy, NanoSight, bicinchoninic acid (BCA) protein assay and western blotting. The results revealed that extracellular vesicles were present in donor oviduct fluid in higher concentrations (P<0.05) and contained more proteins (P<0.05) than extracellular vesicles derived from recipient oviduct fluid (EROF). EDOF or EROF were supplemented in an ET medium (ETM) and the results showed that EDOF significantly improved birth rate via resisting apoptosis and promoting differentiation. In conclusion, our study indicated that there are differences in EDOF and EROF and that supplementing EDOF to ETM can improve the efficiency of ET; improved ET efficiency promotes the use of gene editing and benefits assisted reproductive technology and animal welfare.

Tauroursodeoxycholic acid (TUDCA) alleviates endoplasmic reticulum stress of nuclear donor cells under serum starvation.

Serum starvation is a routine protocol for synchronizing nuclear donor cells to G0/G1 phase during somatic cell nuclear transfer (SCNT). However, abrupt serum deprivation can cause serious stress to the cells cultured in vitro, which might result in endoplasmic reticulum (ER) stress, chromosome damage, and finally reduce the success rate of SCNT. In the present study, the effects of tauroursodeoxycholic acid (TUDCA), an effective ER stress-relieving drug, on the nuclear donor cells under serum deprivation condition as well as following SCNT procedures were first assessed in the bovine. The results showed that TUDCA significantly reduced ER stress and cell apoptosis in those nuclear donor cells. Moreover, it significantly decreased the expression of Hdac1 and Dnmt1, and increased the level of H3K9 acetylation in nuclear donor cells compared with control group. SCNT reconstructed embryos cloned from TUDCA-treated donor cells showed significantly higher fusion, cleavage, blastocyst formation rate, total cell number in day 7 blastocysts, and lower apoptotic index than that from control group. In addition, the expression of Hdac1, Dnmt1 and Bax was significantly lower in blastocysts derived from TUDCA-treated donor cells than that from control group. In conclusion, TUDCA significantly reduced the ER stress of nuclear donor cells under serum starvation condition, and significantly improved the developmental competence of following SCNT reconstructed embryos when these TUDCA-treated cells were used as the nuclear donors.

Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine.

Accumulating evidence indicates the absence of paternally derived miRNAs, piwiRNAs, and proteins may be one important factor contributing to developmental failure in somatic cell cloned embryos. In the present study, we found microRNA-449b (miR-449b) was highly expressed in sperm. Target gene predictions and experimental verification indicate that several embryonic development-related genes, including CDK6, c-MYC, HDAC1 and BCL-2, are targets of miR-449b. We therefore investigated the role of miR-449b using somatic cell nuclear transfer (SCNT) embryo model. Bovine fetal fibroblasts, expressing miR-449b through a doxycycline (dox) induced expression system were used as nuclear donor cells for SCNT. The results showed that miR-449b expression in SCNT embryos significantly enhanced the cleavage rate at 48 h after activation and the levels of H3K9 acetylation at the 2-cell to 8-cell stages, meanwhile, significantly decreased the apoptosis index of blastocysts. In addition, we verified miR-449b could regulate the expression levels of CDK6, c-MYC, HDAC1 and BCL-2. In conclusion, the present study shows that miR-449b expression improves the first cleavage division, epigenetic reprogramming and apoptotic status of bovine preimplantation cloned embryos.