Cyclophosphamide reduces gene transcriptional activity and embryo in vitro development by inhibiting NF-κB expression through decreasing AcH4K12.

Cyclophosphamide (CTX), a widely used chemotherapeutic agent for cancer treatment, has been associated with long-term toxicity and detrimental effects on oocytes and ovaries, resulting in female reproductive dysfunction. This study aimed to investigate the potential impact of CTX on in vitro maturation (IVM) injury of porcine oocytes and subsequent embryonic development, as well as its effects on epigenetic modification and gene activation during early embryonic development. The results demonstrated that CTX treatment caused aberrant spindle structure and mitochondrial dysfunction during oocyte maturation, inducing DNA damage and early apoptosis, which consequently disrupted meiotic maturation. Indeed, CTX significantly reduced the in vitro developmental capacity of porcine embryos, and induced DNA damage and apoptosis in in vitro fertilization (IVF) blastocysts. Importantly, CTX induced abnormal histone modification of AcH4K12 in early porcine embryos. Moreover, addition of LBH589 before zygotic genome activation (ZGA) effectively increased AcH4K12 levels and restored the protein expression of NF-κB, which can effectively enhance the in vitro developmental potential of IVF embryos. The DNA damage and apoptosis induced by CTX compromised the quality of the blastocysts, which were recovered by supplementation with LBH589. This restoration was accompanied by down-regulation of BAX mRNA expression and up-regulation of BCL2, POU5F1, SOX2 and SOD1 mRNA expression. These findings indicated that CTX caused abnormal histone modification of AcH4K12 in early porcine embryos and reduced the protein expression of NF-κB, a key regulator of early embryo development, which may block subsequent ZGA processes.

Ginsenoside F2-Mediated Intestinal Microbiota and Its Metabolite Propionic Acid Positively Impact the Gut-Skin Axis in Atopic Dermatitis Mice.

Atopic dermatitis (AD) is a complex inflammatory skin disease induced by multiple factors. AD can also cause intestinal inflammation and disorders of the gut microbiota. Ginseng is a kind of edible and medicinal plant; its main active components are ginsenosides. Ginsenosides have a variety of anti-inflammatory effects and regulate the gut microbiota; however, their role in AD and the underlying mechanisms are unclear. In this study, we found that intragastric administration of ginsenoside F2 improved AD-like skin symptoms and reduced inflammatory cell infiltration, serum immunoglobulin E levels, and mRNA expression of inflammatory cytokines in AD mice. 16s rRNA sequencing analysis showed that ginsenoside F2 altered the intestinal microbiota structure and enriched the short-chain fatty acid-producing microbiota in AD mice. Metabolomic analysis revealed that ginsenoside F2 significantly increased the propionic acid (Pa) content of feces and serum in AD mice, which was positively correlated with significant enrichment of Parabacteroides goldsteinii and Lactobacillus plantarum in the intestines. Pa inhibits inflammatory responses in the gut and skin of AD mice through the G-protein-coupled receptor43/NF-κB pathway, thereby improving skin AD symptoms. These results revealed, for the first time, the mechanism by which ginsenoside F2 improves AD through the Pa (a metabolite of intestinal microbiota)-gut-skin axis.

Ginsenoside Rg1 activates brown adipose tissue to counteract obesity in high-fat diet-fed mice by regulating gut microbes and bile acid composition.

Obesity is a global health problem strongly linked to gut microbes and their metabolites. In this study, ginsenoside Rg1 (Rg1) reduced lipid droplet size and hepatic lipid accumulation by activating uncoupling protein 1 expression in brown adipose tissue (BAT), which in turn inhibited high-fat diet (HFD)-induced weight gain in mice. Furthermore, the intestinal flora of mice was altered, the abundance of Lachnoclostridium, Streptococcus, Lactococcus, Enterococcus and Erysipelatoclostridium was upregulated, and the concentrations of fecal bile acids were altered, with cholic acid and taurocholic acid concentrations being significantly increased. In addition, the beneficial effects of Rg1 were eliminated in mice treated with a combination of antibiotics. In conclusion, these results suggest that Rg1 activates BAT to counteract obesity by regulating gut microbes and bile acid composition in HFD-fed mice.

Fecal transplant from myostatin deletion pigs positively impacts the gut-muscle axis.

The host genome may influence the composition of the intestinal microbiota, and the intestinal microbiota has a significant effect on muscle growth and development. In this study, we found that the deletion of the myostatin (MSTN) gene positively regulates the expression of the intestinal tight junction-related genes TJP1 and OCLN through the myosin light-chain kinase/myosin light chain pathway. The intestinal structure of MSTN-/- pigs differed from wild-type, including by the presence of a thicker muscularis and longer plicae. Together, these changes affect the structure of intestinal microbiota. Mice transplanted with the intestinal microbiota of MSTN-/- pigs had myofibers with larger cross-sectional areas and higher fast-twitch glycolytic muscle mass. Microbes responsible for the production of short-chain fatty acids (SCFAs) were enriched in both the MSTN-/- pigs and recipient mice, and SCFAs levels were elevated in the colon contents. We also demonstrated that valeric acid stimulates type IIb myofiber growth by activating the Akt/mTOR pathway via G protein-coupled receptor 43 and ameliorates dexamethasone-induced muscle atrophy. This is the first study to identify the MSTN gene-gut microbiota-SCFA axis and its regulatory role in fast-twitch glycolytic muscle growth.

Dynamic changes in intestinal microbiota and metabolite composition of pre-weaned beef calves.

Gut microbes and their metabolites are essential for maintaining host health and production. The intestinal microflora of pre-weaned calves gradually tends to mature with growth and development and has high plasticity, but few studies have explored the dynamic changes of intestinal microbiota and metabolites in pre-weaned beef calves. In this study, we tracked the dynamics of faecal microbiota in 13 new-born calves by 16S rRNA gene sequencing and analysed changes in faecal amino acid levels using metabolomics. Calves were divided into the relatively high average daily gain group (HA) and the relatively low average daily gain group (LA) for comparison. The results demonstrated that the alpha diversity of the faecal microbiota increased with calf growth and development. The abundance of Porphyromonadaceae bacterium DJF B175 increased in the HA group, while that of Lactobacillus reuteri decreased. The results of the LEfSe analysis showed that the microbiota of faeces of HA calves at eight weeks of age was enriched with P. bacterium DJF B175, while Escherichia coli and L. reuteri were enriched in the microbiota of faeces of LA calves. Besides, the total amino acid concentration decreased significantly in the eighth week compared with that in the first week (P < 0.05). Overall, even under the same management conditions, microorganisms and their metabolites interact to play different dynamic regulatory roles. Our results provide new insights into changes in the gut microbiota and metabolites of pre-weaned calves.

Pathological features in 'humanized' neonatal pig.

Cytidine monophosphate-Nacetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) and glycoprotein, alpha1, 3-galactosyltransferase (GGTA1) double knockout (DKO) pig models were produced to reduce immune reaction for xenotransplantation. However, the role of Neu5Gc and α-Gal in pigs has not been fully elucidated and it is necessary to consider the after-effect of inactivation of GGTA1 and CMAH in pigs. Hematological profiles of DKO pigs were analyzed through complete blood count (CBC). Histology of liver and spleen of DKO were investigated, and lectin blotting and mass spectrometry (MS) were performed to explore glycosylation changes in red blood cell (RBC) membranes of DKO pigs. DKO pigs showed common clinical signs such as weakness (100%), dyspnea (90%) and constipation (65%). DKO pigs revealed a significant decrease in RBC, hemoglobin (HGB) and hematocrit (HGB), and an increase in white blood cell (WBC), lymphocyte (LYM), monocyte (MON), and erythrocyte mean corpuscular volume (MCV). DKO piglets showed swollen liver and spleen, and exhibited raised deposition of hemosiderin and severe bleeding. Lectin assay and MS proved variations in glycosylation on RBC membranes. GGTA1/CMAH DKO pigs developed pathological features which are similar to anemic symptoms, and the variations in glycosylation on RBC membranes of DKO pigs may be attributed to the pathologies observed.

Gastrodia elata Blume extract improves high-fat diet-induced type 2 diabetes by regulating gut microbiota and bile acid profile.

In this study, we aimed to characterize the anti-type 2 diabetes (T2D) effects of Gastrodia elata Blume extract (GEBE) and determine whether these are mediated through modification of the gut microbiota and bile acids. Mice were fed a high-fat diet (HFD), with or without GEBE, and we found that GEBE significantly ameliorated the HFD-induced hyperglycemia, insulin resistance, and inflammation by upregulating glucose transporter 4 (GLUT4) and inhibiting the toll-like receptor 4-nuclear factor kappa-B signaling pathway in white adipose tissue (WAT). In addition, we found that GEBE increased the abundance of Faecalibaculum and Lactobacillus, and altered the serum bile acid concentrations, with a significant increase in deoxycholic acid. The administration of combined antibiotics to mice to eliminate their intestinal microbiota caused a loss of the protective effects of GEBE. Taken together, these findings suggest that GEBE ameliorates T2D by increasing GLUT4 expression in WAT, remodeling the gut microbiota, and modifying serum bile acid concentrations.

Effect of myostatin gene mutation on erythrocyte osmotic fragility, hematological parameters and fatty acid composition of serum and erythrocyte membranes in piglets.

Fatty acid composition of serum and erythrocyte membrane, erythrocyte osmotic fragility and hematological parameters were estimated with the objective of determining effects of the gene mutation in one-week-old MSTN homozygous mutant (KO, MSTN-/-), heterozygous mutant (MSTN-/+) and wild type (WT, MSTN+/+) piglets (n = 4 each). Erythrocyte osmotic fragility, complete blood count (CBC), and fatty acid composition of serum and erythrocyte membrane were determined by flow cytometric analysis, automated hematology analyzer system, and liquid chromatography, respectively. Mean of median corpuscular fragility (MCF) was lower (P < 0.05, 0.001) in KO than MSTN-/+ and WT piglets. KO piglets had decreased (P < 0.05) white blood cell (WBC) count, lymphocyte (LYM) count, platelet (PLT) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width-standard deviation (RDW-SD), red cell distribution width-coefficient volume (RDW-CV), plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR), and an increased red blood cell (RBC) count when compared with MSTN-/+ and WT piglets. The ratios of unsaturated fatty acid (UFA) to saturated fatty acid (SFA) concentrations in serum and erythrocyte membranes of MSTN KO piglets were 2-fold and 4-fold higher compared to WT piglets (P < 0.001), respectively. In conclusion, MSTN KO piglets had a decreased erythrocyte osmotic fragility, and altered hematological profile and fatty acid composition of serum and erythrocyte membranes, as characteristic phenotype.

Altered fibrinogen level and fibrin clot structure in myostatin homozygous mutant pig.

Obesity is associated with increased serum fibrinogen level. Myostatin (MSTN), a strong inhibitor of skeletal muscle growth, is recognized as a potential target for obesity. However, the effect of MSTN inhibition on fibrinogen is not largely known. The objective of the present study was to explore fibrinogen levels after MSTN inhibition. Fibrinogen levels and the fibrin clot structure of MSTN homozygous knockout (KO) and wild-type (WT) pigs (n = 4 in each group) were investigated. The protein expression of fibrinogen in the serum and liver of KO pigs decreased greatly (1.6-fold loss for serum and 2.5-fold loss for liver). KO pigs showed significantly decreased gene expression of fibrinogen chains: FGA (fibrinogen-α; 11-fold), FGB (fibrinogen-ß; 8-fold) and FGG (fibrinogen-γ; 7.4-fold). The basal transcriptional regulators of fibrinogen, HNF1 (hepatocyte nuclear factor 1) and CEBP-α (CCAAT/Enhancing-binding protein-alpha) were remarkably down-regulated after interruption of MSTN expression by siRNA (small interfering RNA) in cultured hepatocytes (about 2- and 4-fold, respectively). Compared with WT pigs, KO pigs displayed altered fibrin clot structure with thinner fibers, decreased turbidity and increased permeability. The findings indicate that the inhibition of MSTN could affect fibrinogen levels and the fibrin clot structure.

Association of myostatin deficiency with collagen related disease-umbilical hernia and tippy toe standing in pigs.

Herein, we investigate the high incidence of umbilical hernia and tippy-toe standing and their underlying changes in gene expression and proliferation in myostatin knockout (MSTN-/-) pigs. Thirty-six male MSTN-/- pigs were generated by somatic cell nuclear transfer (SCNT). These pigs presented a considerably high incidence of tippy-toe standing and umbilical hernia (69.4% and 61.1%, respectively). The tendon to body weight ratio was significantly lower than wild-type pigs (0.202 ± 0.017 vs 0.250 ± 0.004, respectively). The crimp length of the MSTN-/- tendon was significantly longer than that of wild-type pigs. The expression of MSTN and the activin type IIB (ACVR2B) was detected in the tendon and linea alba of MSTN-/- pigs. MSTN treatment significantly increased the phosphorylation of Smad2/3 in both tendon and linea alba fibroblasts. Type I collagen (Col1A) and Scleraxis (Scx) expression levels in the tendon and linea alba of MSTN-/- pigs were significantly lower than those in wild-type in vivo, whereas and cyclin-dependent kinase inhibitor 1 (p21) expression levels were higher. Treatment of tendon and linea alba fibroblasts with recombinant MSTN increased Col1A and Scx and decreased p21 expression in vivo. Moreover, there was a significant increase in fibroblast proliferation after treatment. The results indicated that MSTN regulates collagen expression and proliferation in tendon and linea alba fibroblasts; thus, MSTN deficiency causes collagen-related pathological features in MSTN-/- pigs. Hence, MSTN could be used as a therapeutic target for treating UH and tendon abnormalities.

Ginsenoside Rb1 protects porcine oocytes against methylglyoxal damage thus it improves the quality of parthenogenetic activation and in vitro fertilization embryos.

Panax ginseng, a functional food, has been widely used as an edible nourishment and medicinal supplement. Ginsenoside Rb1 is a major bioactive ingredient of ginseng, which shows very specific anti-apoptosis and anti-oxidant activities. Methylglyoxal (MGO) is one of intermediate products of glucose metabolism, which is absorbed easily from high sugar foods or carbonated beverages. It may involve in a variety of detrimental processes in vivo. However, it has not been fully explored the effects of ginsenoside Rb1 on MGO-induced oocytes damage. This study found that MGO-induced DNA damage and mitochondrial dysfunction result in the failure of porcine oocytes maturation and low in vitro development capacity of parthenogenetic activation (PA) and in vitro fertilization (IVF) embryos. Conversely, Rb1 supplementation recovered the rate of maturation, and improved in vitro development capacity of PA and IVF embryos. Rb1 also provided porcine oocytes a lower level of reactive oxygen species production, higher level of ATP content and mitochondrial membrane potential, and stimulated pluripotency gene expression in blastocysts. The findings of this study reveal ginsenoside Rb1 protects porcine oocyte from the cytotoxicity effects of methylglyoxal and provides novel perspectives for the protection of reproduction system by functional food of ginseng.

The protective role of ginsenoside compound K in porcine oocyte meiotic maturation failed caused by benzo(a)pyrene during in vitro maturation.

Benzo(a)pyrene (BaP) is a pollutant and carcinogen derived from air pollution. It causes serious damage to reproductive system, especially ovary. Ginseng is always used in food and traditional medicine as a nutraceuticals or herbal medicine. Ginsenoside compound K (CK) is a major bioactive ingredient of ginseng, that shows very specific anti-apoptosis, anti-oxidant, and anti-inflammatory activities and thus, it protects cells from damage. The aim of this study was to investigate the effects of CK on the BaP-induced inhibition of the in vitro maturation of porcine oocytes and their subsequent embryonic development capacity. We found that supplementation with 10 µg mL-1 CK during in vitro maturation significantly increased maturation rate (P < 0.05) and the expression level of related genes after damage induced by 40 µM BaP treatment. In addition, reactive oxygen species (ROS) levels significantly decreased and ATP content and mitochondrial membrane potential (MMP) increased after CK supplementation (P < 0.05). The competence for embryonic development was improved by the induction of pluripotency gene expression and the inhibition of apoptosis after CK supplementation of BaP-treated oocytes. Supplementation with 10 µg mL-1 CK improved porcine oocyte maturation and subsequent embryonic development of parthenogenetic activation (33.01 vs. 20.92, P < 0.05) and in vitro fertilization (24.01 vs. 16.52, P < 0.05) by increasing antioxidant activity and improving mitochondrial function after BaP-induced damage.

Myristoleic acid produced by enterococci reduces obesity through brown adipose tissue activation.

OBJECTIVE: Dietary fibre has beneficial effects on energy metabolism, and the majority of studies have focused on short-chain fatty acids produced by gut microbiota. Ginseng has been reported to aid in body weight management, however, its mechanism of action is not yet clear. In this study, we focused on the potential modulating effect of ginseng on gut microbiota, aiming to identify specific strains and their metabolites, especially long-chain fatty acids (LCFA), which mediate the anti-obesity effects of ginseng. DESIGN: Db/db mice were gavaged with ginseng extract (GE) and the effects of GE on gut microbiota were evaluated using 16S rDNA-based high throughput sequencing. To confirm the candidate fatty acids, untargeted metabolomics analyses of the serum and medium samples were performed. RESULTS: We demonstrated that GE can induce Enterococcus faecalis, which can produce an unsaturated LCFA, myristoleic acid (MA). Our results indicate that E. faecalis and its metabolite MA can reduce adiposity by brown adipose tissue (BAT) activation and beige fat formation. In addition, the gene of E. faecalis encoding Acyl-CoA thioesterases (ACOTs) exhibited the biosynthetic potential to synthesise MA, as knockdown (KD) of the ACOT gene by CRISPR-dCas9 significantly reduced MA production. Furthermore, exogenous treatment with KD E. faecalis could not reproduce the beneficial effects of wild type E. faecalis, which work by augmenting the circulating MA levels. CONCLUSIONS: Our results demonstrated that the gut microbiota-LCFA-BAT axis plays an important role in host metabolism, which may provide a strategic advantage for the next generation of anti-obesity drug development.

Baicalin improves the in vitro developmental capacity of pig embryos by inhibiting apoptosis, regulating mitochondrial activity and activating sonic hedgehog signaling.

Baicalin, a traditional Chinese medicinal monomer whose chemical structure is known, can be used to treat female infertility. However, the effect of baicalin on embryonic development is unknown. This study investigated the effects of baicalin on in vitro development of parthenogenetically activated (PA) and in vitro fertilized (IVF) pig embryos and the underlying mechanisms involved. Treatment with 0.1 µg/ml baicalin significantly improved (P < 0.05) the in vitro developmental capacity of PA pig embryos by reducing the reactive oxygen species (ROS) levels and apoptosis and increasing the mitochondrial membrane potential (ΔΨm) and ATP level. mRNA and protein expression of sonic hedgehog (SHH) and GLI1, which are related to the SHH signaling pathway, in PA pig embryos at the 2-cell stage, were significantly higher in the baicalin-treated group than in the control group. To confirm that the SHH signaling pathway is involved in the mechanism by which baicalin improves embryonic development, we treated embryos with baicalin in the absence or presence of cyclopamine (Cy), an inhibitor of this pathway. Cy abolished the effects of baicalin on in vitro embryonic development. In conclusion, baicalin improves the in vitro developmental capacity of PA and IVF pig embryos by inhibiting ROS production and apoptosis, regulating mitochondrial activity and activating SHH signaling.

Comparison of internal organs between myostatin mutant and wild-type piglets.

BACKGROUND: Myostatin (MSTN) negatively regulates skeletal muscle development; however, its functions in internal organs have not been thoroughly investigated. Here, we compared the morphological, molecular, and biological characteristics of the heart, liver, spleen, lungs, kidneys, and tongue of homozygous MSTN mutant (MSTN-/- ), heterozygous MSTN mutant (MSTN+/- ), and wild-type (WT) piglets. RESULTS: The heart and liver were lighter in MSTN-/- piglets than in MSTN+/- piglets, while the tongue was heavier in MSTN-/- piglets than in WT piglets (P < 0.05). Furthermore, the tongue was longer in MSTN-/- piglets than in WT piglets, and myofibers of the tongue were significantly larger in the former piglets than in the latter ones (P < 0.01). mRNA expression of MSTN in all organs was significantly lower in MSTN-/- and MSTN+/- piglets than in WT piglets (P < 0.05). Meanwhile, mRNA expression of follistatin, which is closely related to MSTN, in the heart and liver was significantly higher in MSTN-/- piglets than in MSTN+/- and WT piglets (P < 0.05). In addition, protein expression of MSTN in the heart, kidneys, and tongue was significantly lower in MSTN-/- piglets than in WT piglets (P < 0.01). CONCLUSION: These results suggest that MSTN is widely expressed and has marked effects in multiple internal organs. Myostatin has crucial functions in regulating internal organ size, especially the tongue. © 2019 Society of Chemical Industry.

Hairless-knockout piglets generated using the clustered regularly interspaced short palindromic repeat/CRISPR-associated-9 exhibit abnormalities in the skin and thymus.

The nuclear receptor corepressor Hairless (HR) interacts with nuclear receptors and controls expression of specific target genes involved in hair morphogenesis and hair follicle cycling. Patients with HR gene mutations exhibit atrichia, and in rare cases, immunodeficiency. Pigs with HR gene mutations may provide a useful model for developing therapeutic strategies because pigs are highly similar to humans in terms of anatomy, genetics, and physiology. The present study aimed to knockout the HR gene in pigs using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated-9 (Cas9) system and to investigate the molecular and structural alterations in the skin and thymus. We introduced a biallelic mutation into the HR gene in porcine fetal fibroblasts and generated nine piglets via somatic cell nuclear transfer. These piglets exhibited a lack of hair on the eyelids, abnormalities in the thymus and peripheral blood, and altered expression of several signaling factors regulated by HR. Our results indicate that introduction of the biallelic mutation successfully knocked out the HR gene, resulting in several molecular and structural changes in the skin and thymus. These pigs will provide a useful model for studying human hair disorders associated with HR gene mutations and the underlying molecular mechanisms.

Semen quality and fertilization ability of myostatin-knockout boars.

There is no data currently available on the semen quality and fertility of myostatin-knockout (MSTN-/-) boars. We showed that sexually mature adult homozygous MSTN mutant boars have an obvious "double muscling" phenotype, along with a MSTN-/- boar head, back, abdomen, eyes, and oral cavity. Additionally, no abnormalities were found in the reproductive organs. The semen color, odor, and pH also had no abnormalities. The MSTN-/- boars showed good sexual desire. The concentration, motility, plasma membrane integrity, deformity, acrosome integrity, and mitochondrial activity of the semen presented no significant differences from those of the control semen (Duroc). The ejaculation volume of the MSTN-/- boars was significantly lower than that of the control (168.78 ±â€¯6.70 and 223.11 ±â€¯21.21 mL, respectively). The rate of cleavage and blastocyst between the MSTN-/- and control boar semen were compared by in vitro fertilization. The results showed that in the eggs fertilized by the MSTN-/- boar semen, the two-cell and blastocyst rates were similar to those of the control semen (69.1 ±â€¯0.7% vs 65.2 ±â€¯1.6% and 20.2 ±â€¯1.2% vs 22.8 ±â€¯1.4% for the two-cell and blastocyst rates, respectively). In this study, nine healthy offspring were successfully produced through artificial insemination using the MSTN-/- boar semen. Thus, an MSTN-/- boar can be used as the terminal male parent and is expected to be developed into new super lean meat varieties in the future.

Shift from slow- to fast-twitch muscle fibres in skeletal muscle of newborn heterozygous and homozygous myostatin-knockout piglets.

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily that negatively regulates skeletal muscle development. A lack of MSTN induces muscle hypertrophy and increases formation of fast-twitch (Type II) muscle fibres. This study investigated muscle development in newborn heterozygous (MSTN+/-) and homozygous (MSTN-/-) MSTN-knockout piglets. Detailed morphological and gene and protein expression analyses were performed of the biceps femoris, semitendinosus and diaphragm of MSTN+/-, MSTN-/- and wild-type (WT) piglets. Haematoxylin-eosin staining revealed that the cross-sectional area of muscle fibres was significantly larger in MSTN-knockout than WT piglets. ATPase staining demonstrated that the percentage of Type IIb and IIa muscle fibres was significantly higher in MSTN-/- and MSTN+/- piglets respectively than in WT piglets. Western blotting showed that protein expression of myosin heavy chain-I was reduced in muscles of MSTN-knockout piglets. Quantitative reverse transcription-polymerase chain reaction revealed that, compared with WT piglets, myogenic differentiation factor (MyoD) mRNA expression in muscles was 1.3- to 2-fold higher in MSTN+/- piglets and 1.8- to 3.5-fold higher MSTN-/- piglets (P<0.05 and P<0.01 respectively). However, expression of myocyte enhancer factor 2C (MEF2C) mRNA in muscles was significantly lower in MSTN+/- than WT piglets (P<0.05). MSTN plays an important role in skeletal muscle development and regulates muscle fibre type by modulating the gene expression of MyoD and MEF2C in newborn piglets.

Baicalin improves IVM of pig oocytes and subsequent preimplantation embryo development by inhibiting apoptosis.

Baicalin, a monomer of flavonoids extracted from dried roots of Scutellaria baicalensis, is used to treat female infertility. However, the effect of baicalin on oocyte maturation is unknown. In this study we investigated the effects of baicalin on the IVM of pig oocytes and subsequent embryo development following parthenogenetic activation (PA). We found that 0.1µgmL-1 baicalin significantly (P<0.05) increased the IVM rate of oocytes compared with the non-treatment (control) group by reducing levels of reactive oxygen species (ROS). In addition, the mRNA expression of genes related to nuclear maturation and cumulus cell expansion, mitochondrial membrane potential and ATP content was significantly (P<0.05) higher in baicalin-treated than control oocytes. To determine whether baicalin treatment during IVM of pig oocytes improves subsequent development of PA embryos, we measured the cleavage and blastocyst formation rates, as well as the number of cells per blastocyst. All these parameters were significantly (P<0.05) higher in the baicalin-treated than control group. In conclusion, this study demonstrates that baicalin improves pig oocyte maturation and subsequent embryo development invitro by inhibiting production of ROS and reducing apoptosis in oocytes.

The Histone Deacetylase Inhibitor, CI994, Improves Nuclear Reprogramming and In Vitro Developmental Potential of Cloned Pig Embryos.

Epigenetic reprogramming and somatic cell nuclear transfer (SCNT) cloning efficiency were recently enhanced using histone deacetylase inhibitors (HDACis). In this study, we investigated the time effect of CI994, an HDACi, on the blastocyst formation rate, acetylation levels of H3K9 and H4K12, DNA methylation levels of anti-5-methylcytosine (5mC), and some mRNA expression of pluripotency-related genes in pig SCNT embryos. Treatment with 10 µM CI994 for 24 hours significantly improved the blastocyst formation rate of SCNT embryos in comparison with the untreated group (p < 0.05). Moreover, average fluorescence intensities of H3K9 and H4K12 in CI994-treated embryos were remarkably increased at the pseudo-pronuclear stage, but not at the blastocyst stage. The intensity of POU5F1 was higher in CI994-treated blastocysts than in control blastocysts, whereas that of 5mC did not differ between the two groups. The percentage of apoptotic cells in blastocysts was significantly higher in the untreated group than in the CI994-treated group. mRNA levels of POU5F1 and SOX2 were significantly increased in the CI994-treated group. These observations suggest that optimum exposure (10 µM for 24 hours) to CI994 after activation elevates the level of histone acetylation and subsequently improves the in vitro development of pig SCNT embryos.