Sorbicillinoid HSL-2 inhibits the infection of influenza A virus via interaction with the PPAR-γ/NF-κB pathway.

BACKGROUND: Drug resistance is an important factor in the fight against influenza A virus (IAV). Natural products offer a rich source of lead compounds for the discovery of novel antiviral drugs. In a previous study, we isolated the sorbicillinoid polyketide HSL-2 from the mycelium of fungus Trichoderma sp. T-4-1. Here, we show that this compound exerts strong antiviral activity against a panel of IAVs. METHODS: The immunofluorescence and qRT-PCR assays were used to detect the inhibitory effect of HSL-2 toward the replication of influenza virus and IAV-induced expression of the pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1ß. RESULTS: The results indicated that HSL-2 inhibited influenza virus replication, and it significantly inhibited IAV-induced overexpression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß through modulating the PPAR-γ/NF-κB pathway. Notably, this effect was decreased when cells were transfected with PPAR-γ siRNA or treated with the PPAR-γ inhibitor T0070907. In addition, HSL-2 was able to attenuate lung inflammatory responses and to improve lung lesions in a mouse model of IAV infection. CONCLUSIONS: In this paper, we identified a microbial secondary metabolite, HSL-2, with anti-influenza virus activity. This report is the first to describe the antiviral activity and mechanism of action of HSL-2, and it provides a new strategy for the development of novel anti-influenza virus drugs from natural sources.

PTHrP promotes development of mouse preimplantation embryos through the AKT/cyclin D1 pathway and nuclear translocation of HDAC4.

Parathyroid hormone-related protein (PTHrP), the main cause of humoral hypercalcemia in malignancies, promotes cell proliferation and delays terminal cell maturation during embryonic development. Our previous study reported that PTHrP plays important roles in blastocyst formation, pluripotency gene expression, and histone acetylation during mouse preimplantation embryonic development. In this study, we further investigated the mechanism of preimplantation embryonic development regulated by PTHrP. Our results showed that Pthrp depletion decreased both the developmental rate of embryos at the cleavage stage and the cell number of morula-stage embryos. Pthrp-depleted embryos had significantly decreased levels of cyclin D1, phospho (p)-AKT (Thr308) and E2F1. However, Pthrp depletion did not cause significant changes in CDK4, ß-catenin or RUNX2 expression. In addition, our results indicated that Pthrp depletion promoted HDAC4 translocation from the cytoplasm to the nucleus in cleavage-stage embryos by stimulating the activity of protein phosphatase 2A (PP2A), which resulted in dephosphorylation of HDAC4. Taken together, these results suggest that PTHrP regulates cleavage division progression and blastocyst formation through the AKT/cyclin D1 pathway and that PTHrP modulates histone acetylation patterns through nuclear translocation of HDAC4 via PP2A-dependent HDAC4 dephosphorylation during preimplantation embryonic development in mice.

Antiviral Bafilomycins from a Feces-Inhabiting Streptomyces sp.

A new bafilomycin derivative (1) and another seven known bafilomycins (2-8) were isolated from feces-derived Streptomyces sp. HTL16. The structure of 1 was elucidated by 1D and 2D NMR spectroscopic analysis. Biological testing demonstrated that these bafilomycins exhibited potent antiviral activities against the influenza A and SARS-CoV-2 viruses, with IC50 values in the nanomolar range, by inhibiting the activity of endosomal ATP-driven proton pumps.

Broad-spectrum antivirals of protoporphyrins inhibit the entry of highly pathogenic emerging viruses.

Severe emerging and re-emerging viral infections such as Lassa fever, Avian influenza (AI), and COVID-19 caused by SARS-CoV-2 urgently call for new strategies for the development of broad-spectrum antivirals targeting conserved components in the virus life cycle. Viral lipids are essential components, and viral-cell membrane fusion is the required entry step for most unrelated enveloped viruses. In this paper, we identified a porphyrin derivative of protoporphyrin IX (PPIX) that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses including Lassa virus (LASV), Machupo virus (MACV), and SARS-CoV-2 as well as various subtypes of influenza A viral strains with IC50 values ranging from 0.91 ± 0.25 µM to 1.88 ± 0.34 µM. A mechanistic study using influenza A/Puerto Rico/8/34 (H1N1) as a testing strain showed that PPIX inhibits the infection in the early stage of virus entry through biophysically interacting with the hydrophobic lipids of enveloped virions, thereby inhibiting the entry of enveloped viruses into host cells. In addition, the preliminary antiviral activities of PPIX were further assessed by testing mice infected with the influenza A/Puerto Rico/8/34 (H1N1) virus. The results showed that compared with the control group without drug treatment, the survival rate and mean survival time of the mice treated with PPIX were apparently prolonged. These data encourage us to conduct further investigations using PPIX as a lead compound for the rational design of lipid-targeting antivirals for the treatment of infection with enveloped viruses.

Secondary Metabolites with Cytotoxic Activities from Streptomyces sp. BM-8 Isolated from the Feces of Equusquagga.

A new aliphatic acid, compound 1, together with six known metabolites, including nonactic acid (2), homononactic acid (3), ethyl homononactate (4), homononactylhomononactate (5), valinomycin (6), and cyclo-(Pro-Leu) (7), was isolated from the culture broth of Streptomyces sp. BM-8, an actinobacterial strain isolated from the feces of Equus quagga. The structures of these compounds were established by analyses of spectroscopic data, including 1D and 2D nuclear magnetic resonance spectra (NMR), as well as by HR-ESI-MS spectrometry and chemical derivative analyses. Additionally, a serial analogue of nonactic acid and homononacticacid (8-21) was synthesized. The cytotoxicity of 1-21 wastested against a panel of cancer cell lines, such as HT-29, MCF-7, A375 and K562, with MTT assay. In addition, the cytotoxicity tests revealed that 1 was less cytotoxic toward a panel of cancerous cells, as compared with valinomycin (6).

CRISPR/Cas9-mediated MSTN disruption accelerates the growth of Chinese Bama pigs.

CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed.

Melatonin alleviates the deterioration of oocytes from mice subjected to repeated superovulation.

Induction of repeated superovulation with exogenous hormones is widely used in assisted reproductive technology (ART). Though it is generally safe, emerging evidence has indicated that repeated superovulation may compromise oocyte quality. However, few studies have explored how to ameliorate such impairment. Because melatonin has beneficial influences on oocytes in various detrimental environments, we aimed to explore whether melatonin could protect mouse oocytes after repeated superovulation. We found that repeated superovulation markedly reduced meiotic maturation and disrupted spindle organization and chromosome alignment. Furthermore, we observed reduced mitochondrial content and enhanced early apoptosis in oocytes from mice subjected to repeated superovulation. In addition, 5-methylcytosine (5mc) fluorescence intensity was lower in oocytes from experimental mice than in those from control mice, indicating that repeated superovulation disrupts genomic DNA methylation, and elevations in reactive oxygen species levels indicated that repeated superovulation also induces oxidative stress. Conversely, melatonin administration improved oocyte maturation and attenuated the observed defects. Interestingly, supplementation with melatonin during in vitro maturation had the same protective effects on oocytes as in vivo melatonin administration. In summary, our results show that melatonin can improve oocyte quality after repeated superovulation and thus provide a potential strategy to improve ART efficiency.

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development.

NEK5, a member of never in mitosis-gene A-related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA-mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin-dependent kinase 1 in oocytes, resulting in a decrease of maturation-promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1-cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.

Maturation of buffalo oocytes in vitro with acetyl-L-carnitine improves cryotolerance due to changes in mitochondrial function and the membrane lipid profile.

The effects of acetyl-l-carnitine (ALC) supplementation during IVM on subsequently vitrified buffalo oocytes were evaluated, followed by determination of the mitochondrial DNA copy number, measurement of mitochondrial membrane potential (MMP) and identification of the lipid profile of oocyte membranes as markers of oocyte quality after vitrification. Supplementation with ALC during IVM significantly improved the rates of oocyte cleavage and morula and blastocyst formation, and increased MMP after vitrification compared with unsupplemented vitrified oocytes (P<0.05). Using a bidirectional orthogonal projection to latent structures discriminant analysis based on positive ion matrix-assisted laser desorption ionisation time-of-flight mass spectrometry data, five phospholipid ions (m/z 728.7 (phosphatidylcholine (PC) 32:3), 746.9 (PC 32:5), 760.6 (PC 34:1), 768.8 (PC P-36:3) and 782.6 (PC 36:4); P<0.05) were identified as significantly more abundant in fresh oocytes than in unsupplemented vitrified oocytes. Meanwhile, three phospholipid ions (m/z 734.6 (PC 32:0), 760.6 (PC 34:1), and 782.6 (PC 36:4); P<0.05) were more abundant in ALC-supplemented vitrified oocytes than in unsupplemented vitrified oocytes. Therefore, supplementation with ALC during IVM may improve buffalo oocyte quality after vitrification by enhancing mitochondrial function and altering the phospholipid composition of vitrified oocyte membranes.

Effects of resveratrol on in vitro maturation of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer.

As a natural plant-derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 µM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti-apoptotic B-cell lymphoma 2 (BCL-2) gene and significant downregulation of the pro-apoptotic BCL2-associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 µM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 µM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT.

The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini-pig.

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.

Generation of transgenic-cloned Huanjiang Xiang pigs systemically expressing enhanced green fluorescent protein.

Huanjiang Xiang pig is a unique native minipig breed originating in Guangxi, China, and has great utility value in agriculture and biomedicine. Reproductive biotechnologies such as somatic cell nuclear transfer (SCNT) and SCNT-mediated genetic modification show great potential value in genetic preservation and utilization of Huanjiang Xiang pigs. Our previous work has successfully produced cloned and transgenic-cloned embryos using somatic cells from a Huanjiang Xiang pig. In this study, we firstly report the generation of transgenic-cloned Huanjiang Xiang pigs carrying an enhanced green fluorescent protein (eGFP) gene. A total of 504 SCNT-derived embryos were transferred to two surrogate recipients, one of which became pregnant and gave birth to three live piglets. Exogenous eGFP transgene had integrated in all of the three Huanjiang Xiang piglets identified by genotyping. Furthermore, expression of eGFP was also detected from in vitro cultured skin fibroblast cells and various organs or tissues from positive transgenic-cloned Huanjiang Xiang pigs. The present work provides a practical method to preserve this unique genetic resource and also lays a foundation for genetic modification of Huanjiang Xiang pigs with improved values in agriculture and biomedicine.

Generation of chimeric minipigs by aggregating 4- to 8-cell-stage blastomeres from somatic cell nuclear transfer with the tracing of enhanced green fluorescent protein.

BACKGROUND: Blastocyst complementation is an important technique for generating chimeric organs in organ-deficient pigs, which holds great promise for solving the problem of a shortage of organs for human transplantation procedures. Porcine chimeras have been generated using embryonic germ cells, embryonic stem cells, and induced pluripotent stem cells; however, there are no authentic pluripotent stem cells for pigs. In previous studies, blastomeres from 4- to 8-cell-stage parthenogenetic embryos were able to generate chimeric fetuses efficiently, but the resulting fetuses did not produce live-born young. Here, we used early-stage embryos from somatic cell nuclear transfer (SCNT) to generate chimeric piglets by the aggregation method. Then, the distribution of chimerism in various tissues and organs was observed through the expression of enhanced green fluorescent protein (EGFP). METHODS: Initially, we determined whether 4- to 8- or 8- to 16-cell-stage embryos were more suitable to generate chimeric piglets. Chimeras were produced by aggregating two EGFP-tagged Wuzhishan minipig (WZSP) SCNT embryos and two Bama minipig (BMP) SCNT embryos. The chimeric piglets were identified by coat color and microsatellite and swine leukocyte antigen analyses. Moreover, the distribution of chimerism in various tissues and organs of the piglets was evaluated by EGFP expression. RESULTS: We found that more aggregated embryos were produced using 4- to 8-cell-stage embryos (157/657, 23.9%) than 8- to 16-cell-stage embryos (100/499, 20.0%). Thus, 4- to 8-cell-stage embryos were used for the generation of chimeras. The rate of blastocysts development after aggregating WZSP with BMP embryos was 50.6%. Transfer of 391 blastocysts developed from 4- to 8-cell-stage embryos to five recipients gave rise to 18 piglets, of which two (11.1%) were confirmed to be chimeric by their coat color and microsatellite examination of the skin. One of the chimeric piglets died at 35 days and was subsequently autopsied, whereas the other piglet was maintained for the following observations. The heart and kidneys of the dead piglet showed chimerism, whereas the spinal cord, stomach, pancreas, intestines, muscle, ovary, and brain had no chimerism. CONCLUSIONS: To our knowledge, this is the first report of porcine chimeras generated by aggregating 4- to 8-cell-stage blastomeres from SCNT. We detected chimerism only in the skin, heart, and kidneys. Collectively, these results indicate that aggregation using 4- to 8-cell-stage SCNT embryos offers a practical approach for producing chimeric minipigs. Furthermore, it also provides a potential platform for generating interspecific chimeras between pigs and non-human primates for xenotransplantation.

Andrographolide disrupts meiotic maturation by blocking cytoskeletal reorganisation and decreases the fertilisation potential of mouse oocytes.

Andrographolide (AG) is a diterpenoid lactone isolated from the stem and leaves of Andrographis paniculata Nees that is used for the effective treatment of infectious diseases in Asian countries. Previous studies have reported adverse effects of AG on female fertility in rodents; however, the underlying mechanisms are unknown. The aim of the present study was to investigate the effects of AG on the IVM of mouse oocytes and their fertilisation potential. Immature oocytes incubated for 6, 14 or 24h in medium containing 5, 10 or 20µM AG showed time- and dose-dependent decreases in maturation rates compared with the control group. Immunostaining revealed that AG exposure disrupted spindle organisation and migration, as well as actin cap formation and cytokinesis. Furthermore, most oocytes exposed to 20µM AG underwent apoptosis, and the few oocytes exposed to 5 or 10µM AG that reached MII exhibited lower fertilisation rates after intracytoplasmic sperm injection. The findings of the present study suggest that AG may disrupt mouse oocyte meiotic maturation by blocking cytoskeletal reorganisation, and may thus have an adverse effect on female fertility.

Role of linker histone H1c during the reprogramming of Chinese swamp buffalo (Bubalus Bubalis) embryos produced by somatic cell nuclear transfer.

During reprogramming, there is exchange of histone H1c and the oocyte-specific linker histone, and H1c may play a critically important role in the reprogramming process of somatic cell nuclear transfer (SCNT). The aim of the present study was to investigate the role of the H1c gene in SCNT reprogramming in Chinese swamp buffalo (Bubalus bubalis) using RNA interference (RNAi). Chinese swamp buffalo H1c gene sequences were obtained and H1c-RNAi vectors were designed, synthesised and then transfected into a buffalo fetal skin fibroblast cell line. Expression of H1c was determined by real-time polymerase chain reaction to examine the efficiency of vector interference. These cells were then used as a nuclear donor for SCNT so as to observe the further development of SCNT embryos. Inhibition of H1c gene expression in donor cells significantly improved the developmental speed of embryos from the 1-cell to 8-cell stage. Furthermore, compared with the control group, inhibition of H1c gene expression significantly reduced the blastocyst formation rate. It is concluded that linker histone H1c is very important in SCNT reprogramming in Chinese swamp buffalo. Correct expression of the H1c gene plays a significant role in preimplantation embryonic development in B. bubalis.

ZPAC is required for normal spermatogenesis in mice.

The ubiquitin-proteasome pathway, involved in genetic recombination and sex-chromosome silencing during meiosis, plays critical roles in the specification of germ-line stem cells and the differentiation of gametes from gonocytes. Zygote-specific proteasome assembly chaperone (ZPAC) is expressed in the early mouse embryo, where it is important for progression of the mouse maternal-to-zygotic transition. The role of ZPAC during spermatogenesis in the adult gonads, however, remains unknown. In this study, rapid amplification of cDNA ends was used to determine the Zpac cDNA sequence, a 1584-bp transcript that includes a putative 1122-bp open reading frame coding for a 373 amino acid protein. Western blot and immunohistochemistry revealed that ZPAC was specifically expressed in gonads. To further dissect the function of ZPAC during spermatogenesis, we employed PiggyBac-based RNA interference vectors for transgenesis combined with cell transplantation to deplete Zpac during spermatogenesis. This RNAi-mediate depletion in Zpac expression disrupted normal spermatogenesis from spermatogonial stem cells. Two independent yeast two-hybrid screens further revealed an interaction between ZPAC and SYCE1. Together, these data suggest that ZPAC is required for normal spermatogenesis in mice.

[Role of parathyroid hormone-like hormone during embryonic development].

Parathyroid hormone-like hormone (PTHLH), also known as parathyroid hormone-related protein (PTHrP), was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcaemia in malignancies. Subsequent studies demonstrated that PTHLH has multiple physiological functions in many fetal and adult tissues, including the regulation of morphogenesis, cell proliferation and differentiation, and transplacental calcium transport. This review focuses on the biological characteristics of PTHLH and its function during embryonic development. The signaling pathway and potential mechanism involved are further discussed.

Mogroside V alleviates the heat stress-induced disruption of the porcine oocyte in vitro maturation.

Heat stress (HS) is a stressor that negatively affect female reproduction. Specially, oocytes are very sensitive to HS. It has been demonstrated that some active compounds can protect oocyte from HS. We previously found that Mogroside V (MV), extracted from Siraitia grosvenorii (Luo Han Guo), can protect oocyte from many kinds of stresses. However, how MV alleviates HS-induced disruption of oocyte maturation remains unknown. In this study, we treated the HS-induced porcine oocytes with MV to examine their maturation and quality. Our findings demonstrate that MV can effectively alleviate HS-induced porcine oocyte abnormal cumulus cell expansion, decrease of first polar body extrusion rate, spindle assembly and chromosome separation abnormalities, indicating MV attenuates oocyte mature defects. We further observed that MV can effectively alleviate HS-induced cortical granule distribution abnormality and decrease of blastocyst formation rate after parthenogenesis activation. In addition, MV treatment reversed mitochondrial dysfunction and lipid droplet content decrease, reduced reactive oxygen species levels, early apoptosis and DNA damage in porcine oocytes after HS. Collectively, this study suggests that MV can effectively protect porcine oocytes from HS.

Incubation of sperm heads impairs fertilization and early embryo development following intracytoplasmic sperm injection (ICSI) by decreasing oocyte activation in mice.

When intracytoplasmic sperm injection (ICSI) is performed in mice, isolation of sperm heads is usually performed prior to injections in order to increase the efficiency of the procedure. Consequently, the isolated sperm heads undergo an inevitable incubation in vitro. However, little is known about the effects of this incubation step on fertilization and embryo development following ICSI. When we incubated sperm heads at 37 °C, there was a significant time-dependent decrease in fertilization and blastocyst formation. Moreover, the DNA integrity of the sperm heads was maintained over 12 h incubation. Using assisted oocyte activation, these defects in fertilization and embryo development were rescued. Taken together, incubation of sperm heads following isolation can affect the oocyte-activating capacity of sperm thereby compromising fertilization and embryo development associated with ICSI.

Methylation characteristics and developmental potential of Guangxi Bama minipig (Sus scrofa domestica) cloned embryos from donor cells treated with trichostatin A and 5-aza-2'-deoxycytidine.

Summary Reprogramming of DNA methylation in somatic cell nuclear transfer (SCNT) embryos is incomplete, and aberrant DNA methylation patterns are related to the inefficiency of SCNT. To facilitate nuclear reprogramming, this study investigated the effect of treating Guangxi Bama minipig donor cells with trichostatin A (TSA), 5-aza-2'-deoxycytine (5-aza-dC), or combination of TSA and 5-aza-dC prior to nuclear transfer. Analyses showed that there were no major changes in cell-cycle status among all groups. We monitored the transcription of DNMT1, DNMT3a, HDAC1 and IGF2 genes in donor cells. Transcription levels of HDAC1 were decreased significantly after treatment with a combination of TSA and 5-aza-dC, along with a significantly increased level of IGF2 (P < 0.05). Although treatment of donor cells with either TSA or 5-aza-dC alone resulted in non-significant effects in blastocyst formation rate and DNA methylation levels, a combination of TSA and 5-aza-dC significantly improved the development rates of minipig SCNT embryos to blastocyst (25.6% vs. 16.0%, P < 0.05). This change was accompanied by decreased levels of DNA methylation in somatic cells and blastocyst (P < 0.05). Thus in combination with TSA, lower concentrations of 5-aza-dC may produce a potent demethylating activity, and lead to the significantly enhanced blastocyst development percentage of Bama minipig SCNT embryos.