Interference with DGAT Gene Inhibited TAG Accumulation and Lipid Droplet Synthesis in Bovine Preadipocytes.

Triacylglycerol (TGA) is the primary component of intramuscular fat. Expression of diacylglyceryl transferase (DGAT) determines the polyester differentiation ability of precursor adipocytes. The two DGAT isoforms (DGAT1 and DGAT2) play different roles in TAG metabolism. This study investigates the roles of DGAT1 and DGAT2 in signaling pathways related to differentiation and lipid metabolism in Yanbian bovine preadipocytes. sh-DGAT1 (sh-1), sh-DGAT2 (sh-2), and sh-DGAT1 + sh-DGAT2 (sh-1 + 2) were prepared using short interfering RNA (siRNA) interference technique targeting DGAT1 and DGAT2 genes and infected bovine preadipocytes. Molecular and transcriptomic techniques, including differentially expressed genes (DEGs) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis, were used to investigate the effects on the differentiation of Yanbian bovine preadipocytes. After interference with DGAT1 and DGAT2 genes, the contents of TAG and adiponectin were decreased. The TAG content in the sh-2 and sh-1 + 2 groups was significantly lower than that in the sh-NC group. RNA sequencing (RNA-seq) results showed 2070, 2242, and 2446 DEGs in the sh-1, sh-2, and sh-1 + 2 groups, respectively. The DEGs of the sh-2 group were mainly concentrated in the PPAR, AMPK, and Wnt signaling pathways associated with adipocyte proliferation and differentiation. These results demonstrated that at the mRNA level, DGAT2 plays a more important role in lipid metabolism than DGAT1.

Overexpression of DGAT2 Regulates the Differentiation of Bovine Preadipocytes.

Triacylglycerols (TAGs) are a major component of intramuscular fat. Diacylglycerol O-acyltransferase 2(DGAT2) expression determines the rate of TAG synthesis. The purpose of this study was to investigate the role of DGAT2 in the differentiation of Yanbian cattle preadipocytes and lipid metabolism-related signalling pathways. Bovine preadipocytes were infected with overexpression and interfering adenovirus vectors of DGAT2. The effects on the differentiation of Yanbian cattle preadipocytes were examined using molecular and transcriptomic techniques, including differentially expressed genes (DEGs) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. DGAT2 overexpression significantly increased (p < 0.05) intracellular TAG, adiponectin, and lipid droplet (LD) contents. Moreover, it upregulated (p < 0.05) peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α, and fatty acid binding protein 4 mRNA expression. In contrast, DGAT2 knockdown reduced intracellular TAG and LD content and downregulated (p < 0.05) C/EBPß, mannosyl (alpha-1,3-)-glycoproteinbeta-1,2-N-acetylglucosaminyltransferase, lipin 1,1-acylglycerol-3-phosphate O-acyltransferase 4, and acetyl-CoA carboxylase alpha mRNA expression. Between DGAT2-overexpressing preadipocytes and normal cells, 208 DEGs were identified, including 106 upregulated and 102 downregulated genes. KEGG pathway analysis revealed DEGs mainly enriched in PPAR signalling and AMP-activated protein kinase pathways, cholesterol metabolism, and fatty acid biosynthesis. These results demonstrated that DGAT2 regulated preadipocyte differentiation and LD and TAG accumulation by mediating the expression of adipose differentiation-, lipid metabolism-, and fatty acid synthesis-related genes.

Effect of microencapsulation of egg yolk immunoglobulin Y by sodium alginate/chitosan/sodium alginate on the growth performance, serum parameters, and intestinal health of broiler chickens.

OBJECTIVE: Egg yolk immunoglobulin (IgY) is an antibiotic alternative to prevent and fight intestinal pathogenic infections. This study aimed to investigate the effects of sodium alginate/chitosan/sodium alginate IgY microcapsules on the growth performance, serum parameters, and intestinal health of broiler chickens. METHODS: One-day-old broilers (Ross 308) were divided into five treatments, each with 10 replicates of five chickens. The dietary treatments were maintained for 28 days and consisted of a basal diet (NC), basal diet + 500 mg chlortetracycline/kg diet (CH), basal diet + 50 mg non-microencapsulated IgY/kg diet (NM), basal diet + 600 mg low levels microencapsulated IgY/kg diet (LM), and basal diet + 700 mg high levels microencapsulated IgY/kg diet (HM). RESULTS: Throughout the 28-day trial period, the NM, LM, HM, and CH groups increased average daily gain compared with the NC group (p<0.05), and the HM group reduced feed conversion ratio compared with the CH group (p<0.05). The LM and HM groups increased relative organ weights of thymus and spleen compared with the CH and NM groups (p<0.05). The HM group improved the duodenal, jejunal and ileum villi height (VH) and villus height to crypt depth ratio (VH:CD) compared with the CH and NM groups (p<0.05). Compared with the CH group, the HM group increased serum immunoglobulin (IgA), immunoglobulin G (IgG), superoxide dismutase, total antioxidant capacity, and glutathione peroxidase levels (p<0.05), and decreased serum malondialdehyde levels (p<0.05). Compared with the NC group, the NM, LM, HM, and CH groups reduced colonic Escherichia coli and Salmonella levels (p<0.05). and the HM group promoted the levels of lactic acid bacteria and bifidobacteria compared with the CH group (p<0.05). CONCLUSION: Microencapsulation could be considered as a way to improve the efficiency of IgY. The 700 mg high levels microencapsulated IgY/kg diet could potentially be used as an alternative to antibiotics to improve the immune performance and intestinal health, leading to better performance of broiler chickens.

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.

Population genomics reveals that natural variation in PRDM16 contributes to cold tolerance in domestic cattle.

Environmental temperature serves as a major driver of adaptive changes in wild organisms. To discover the mechanisms underpinning cold tolerance in domestic animals, we sequenced the genomes of 28 cattle from warm and cold areas across China. By characterizing the population structure and demographic history, we identified two genetic clusters, i.e., northern and southern groups, as well as a common historic population peak at 30 kilo years ago. Genomic scan of cold-tolerant breeds determined potential candidate genes in the thermogenesis-related pathways that were under selection. Specifically, functional analysis identified a substitution of PRDM16 (p.P779L) in northern cattle, which maintains brown adipocyte formation by boosting thermogenesis-related gene expression, indicating a vital role of this gene in cold tolerance. These findings provide a basis for genetic variation in domestic cattle shaped by environmental temperature and highlight the role of reverse mutation in livestock species.

Ubiquinol-10 delays postovulatory oocyte aging by improving mitochondrial renewal in pigs.

Ubiquinol-10, the reduced form of coenzyme Q10, protects mammalian cells from oxidative damage and enhances mitochondrial activity. However, the protective effect of ubiquinol-10 on mammalian oocytes is not well understood. In this study, we investigated the effect of ubiquinol-10 on porcine oocytes during postovulatory aging. Metaphase II oocytes were selected as fresh oocytes and further cultured for 48 h with different concentrations of ubiquinol-10 (0-400 µM) in vitro as a postovulatory aging model. After choosing the optimal concentration of ubiquinol-10 (100 µM) that maintained oocyte morphology and developmental competence during the progression of aging, the oocytes were randomly divided into five groups: fresh, control-24 h, ubiquinol-24 h, control-48 h, and ubiquinol-48 h. The results revealed that ubiquinol-10 significantly prevented aging-induced oxidative stress, GSH reduction, cytoskeleton impairment, apoptosis, and autophagy. Mitochondrial biogenesis (SIRT1 and PGC-1α) and mitophagy (PINK1 and PARKIN)-related proteins were decreased during aging. Addition of ubiquinol-10 prevented the aging-induced reduction of these proteins. Consequently, although mitochondrial content was decreased, the number of active mitochondria and ATP level were significantly increased upon treatment with ubiquinol-10. Thus, ubiquinol-10 has beneficial effects on porcine postovulatory aging oocytes owing to its antioxidant properties and ability to promote mitochondrial renewal.

Nuclear accumulation of pyruvate dehydrogenase alpha 1 promotes histone acetylation and is essential for zygotic genome activation in porcine embryos.

Porcine zygotic genome activation (ZGA) occurs along with global epigenetic remodeling at the 4-cell stage. These processes are regulated by histone acetylation, which requires acetyl-coenzyme A (CoA). Pyruvate dehydrogenase complex (PDC) is a crucial enzyme in glucose metabolism that converts pyruvate into acetyl-CoA. In mammalian cells, acetyl-CoA is produced by pyruvate dehydrogenase alpha 1 (PDHA1) translocated into the nucleus in special conditions. To determine whether zygotic PDHA1 plays a critical role in promoting histone acetylation during ZGA, a CRISPR/Cas9 genome editing system using multiple guide RNAs was employed to generate a PDHA1-targeted parthenogenetic embryo model. Results of immunofluorescent staining showed that the nuclear accumulation of PDHA1 during ZGA was significantly inhibited by PDHA1 targeting. Meanwhile, the 4-cell arrest rate significantly increased at 72 h after activation, indicating impeded embryonic development. In addition, nuclear histone acetylation significantly decreased when PDHA1 was targeted, and quantitative PCR showed that expression of several zygotic genes was significantly decreased in the PDHA1-targeting group compared to the control group. Overexpression of PDHA1 recovered the nuclear PDHA1, H3K9Ac and H3K27Ac and EIF1A expression levels. Moreover, the 5-to-8-cell-stage embryo development rate was only partially rescued. In conclusion, expression of zygotic origin PDHA1 contributes to porcine ZGA by maintaining histone acetylation in porcine embryos.

l-carnitine supplementation during in vitro culture regulates oxidative stress in embryos from bovine aged oocytes.

Aging oocytes undergo various molecular, cellular, and biochemical changes. Aging of oocytes results in reduced embryo developmental capacity and blastocyst quality, which is thought to be caused partly by the accumulation of reactive oxygen species (ROS). This study aimed to determine the effect of l-carnitine (LC) on the development of embryos formed from aged oocytes in vitro. The development and quality of the blastocysts in the LC-treated group were significantly higher than those in the untreated aged group after in vitro fertilization (IVF). In addition, after LC treatment, the level of intracellular ROS in aged group significantly decreased, and glutathione (GSH) levels significantly increased compared with those in the untreated aged group. There was no significant difference in the mitochondrial membrane potential among the three groups. Moreover, ROS could induce autophagy and LC3 antibody was widely used as a marker for detecting autophagy. The fluorescence intensity of LC3 in the aged group was significantly higher than that of LC3 in the LC-treated group. Furthermore, Real-time reverse transcriptase-polymerase chain reaction showed that the mRNA levels of antioxidation genes GPX4 and SOD1 were significantly higher in embryos from LC-treated group than in those from the untreated aged group. In summary, our results indicated that LC can improve the developmental capacity of embryos from aging oocytes in vitro by reducing oxidative stress.

Reduced adiposity by compensatory WAT browning upon iBAT removal in mice.

The strong effects of classic brown adipose tissue (BAT) and recruited beige adipocytes in treatment of obesity and metabolic syndrome have been attracting increasing research interest. Cold treatment is an effective, convenient approach to stimulate BAT activity and induce white adipose tissue (WAT) browning. Here, we utilized prolonged cold exposure (from 2 h to 2 weeks in a 4° cold chamber) to elucidate dynamic changes in BAT and in WAT browning during acute and chronic cold exposure in mice. BAT mass decreased quickly, with reduced lipid droplet sizes within 8 h of cold exposure owing to the utilization of BAT pre-storage triglycerides, and subsequently increased during prolonged cold exposure. These dynamic morphological changes in BAT were confirmed by gene expression changes in ADRB3 and PGC1α, while UCP1 and ELOVL3 expression was continuously up-regulated throughout the entire cold exposure period. Additionally, cold treatment increased BAT secretion of FGF21, which has been reported to activate beige adipocyte formation. Thus, to illustrate potential crosstalk between secreted BAT proteins (so-called BATokines) and beige adipogenesis during cold stress, we performed an interscapular BAT (iBAT) removal experiment in mice. Surprisingly, loss of classic iBAT enhanced WAT browning due to compensatorily increased sympathetic WAT input. Unexpectedly, we observed significantly reduced adiposity in the iBAT removal group compared with the control group. These results further suggest that WAT browning plays an important role in whole-body energy metabolism during cold acclimation, even without iBAT. Furthermore, our data imply that enhanced WAT browning may be an efficient therapeutic tool to combat obesity and related syndromes.

A New Method of Producing a Natural Antibacterial Peptide by Encapsulated Probiotics Internalized with Inulin Nanoparticles as Prebiotics.

Synbiotics are a combination of probiotics and prebiotics, which lead to synergistic benefits in host welfare. Probiotics have been used as an alternative to antibiotics. Among the probiotics, Pediococcus acidilactici (PA) has shown excellent antimicrobial activity against Salmonella Gallinarum (SG) as a major poultry pathogen and has improved the production performances of animals. Inulin is widely used as a prebiotic for the improvement of animal health and growth. The main aim of this study is to investigate the effect of the antimicrobial activity of inulin nanoparticles (INs)-internalized PA encapsulated into alginate/chitosan/alginate (ACA) microcapsules (MCs) in future in vivo application. The prepared phthalyl INs (PINs) were characterized by DLS and FE-SEM. The contents of phthal groups in phthalyl inulin were estimated by ¹H-NMR measurement as 25.1 mol.-%. The sizes of the PINs measured by DLS were approximately 203 nm. Internalization into PA was confirmed by confocal microscopy and flow cytometry. Antimicrobial activity of PIN-internalized probiotics encapsulated into ACA MCs was measured by co-culture antimicrobial assays on SG. PIN-internalized probiotics had a higher antimicrobial ability than that of ACA MCs loaded with PA/inulin or PA. Interestingly, when PINs were treated with PA and encapsulated into ACA MCs, as a natural antimicrobial peptide, pediocin was produced much more in the culture medium compared with other groups inulin-loaded ACA MCs and PA-encapsulated into ACA MCs.

Oral Immunization of FMDV Vaccine Using pH-Sensitive and Mucoadhesive Thiolated Cellulose Acetate Phthalate Microparticles.

Several barriers such as gastric pH, enzymatic degradation and rapid transit should be overcome to orally deliver antigens for taking up by epithelial microfold cells in Peyer's patches of small intestine. To solve the above mentioned problems, we designed pH-sensitive and mucoadhesive polymeric microparticles (MPs) prepared by double emulsion technique using cellulose acetate phthalate (CAP) to enhance immune response of foot-and-mouth disease (FMD) virus (FMDV) subunit vaccine. Thiolation of CAP improved mucoadhesive property of CAP to prolong the MPs transit time through the gastrointestinal tract. Thiolated CAP (T-CAP) also slowed down antigen release in acidic pH of stomach but released more antigens in neutral pH of small intestine due to the pH-sensitivity of the T-CAP. Oral immunization of a chimerical multi-epitope recombinant protein as the FMD subunit vaccine via T-CAP MPs effectively delivered the vaccine to Peyer's patches eliciting mucosal IgA response. It will make a step forward into a promising oral subunit vaccine development in livestock industry.

Cold adaptation in pigs depends on UCP3 in beige adipocytes.

Pigs lack functional uncoupling protein 1 (UCP1) making them susceptible to cold. Nevertheless, several pig breeds are known to be cold resistant. The molecular mechanism(s) enabling such adaptation are currently unknown. Here, we show that this resistance is not dependent on shivering, but rather depends on UCP3 and white adipose tissue (WAT) browning. In two cold-resistant breeds (Tibetan and Min), but not a cold-sensitive breed (Bama), WAT browning was induced after cold exposure. Beige adipocytes from Tibetan pigs exhibited greater oxidative capacity than those from Bama pigs. Notably, UCP3 expression was significantly increased only in cold-resistant breeds, and knockdown of UCP3 expression in Tibetan adipocytes phenocopied Bama adipocytes in culture. Moreover, the eight dominant pig breeds found across China can be classified into cold-sensitive and cold-resistant breeds based on the UCP3 cDNA sequence. This study indicates that UCP3 has contributed to the evolution of cold resistance in the pig and overturns the orthodoxy that UCP1 is the only thermogenic uncoupling protein.

Generation of large pig and bovine blastocysts by culturing in human induced pluripotent stem cell medium.

We investigated the effect of human induced pluripotent stem cell (hiPS) medium on porcine somatic cell nuclear transfer and bovine in vitro fertilized early blastocysts, in comparison with North Carolina State University (NCSU)-37 medium and in vitro culture (IVC)-II medium. After 2 days of culture, the diameter of the portion of the blastocyst that was extruded from the zona pellucid dramatically differed between porcine blastocysts cultured in hiPS medium and those cultured in NCSU-37 medium (221.47 ± 38.94 µm versus 481.87 ± 40.61 µm, P < 0.01). Moreover, the diameter of the portion of the blastocyst significantly differed between bovine blastocysts cultured in hiPS medium and those cultured in IVC-II medium (150.30 ± 29.49 µm versus 195.58 ± 41.59 µm, P < 0.01). Furthermore, the total number of cells per porcine and bovine blastocyst was more than two-fold higher in blastocysts cultured in hiPS medium than in those cultured in NCSU-37 medium (44.33 ± 5.28 and 143.33 ± 16.05, P < 0.01) or IVC-II medium (172.12 ± 45.08 and 604.83 ± 242.64, P < 0.01), respectively. These results indicate that hiPS medium markedly improves the quality of porcine and bovine blastocysts.

PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos.

In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101 for 24h. Treatment with 0.5 µM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P<0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 µM PXD101 for various amounts of times following activation. Treatment for 24h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P<0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.

Differential Expression of PPARγ, FASN, and ACADM Genes in Various Adipose Tissues and Longissimus dorsi Muscle from Yanbian Yellow Cattle and Yan Yellow Cattle.

The objective of this study was to investigate the correlation between cattle breeds and deposit of adipose tissues in different positions and the gene expressions of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), and Acyl-CoA dehydrogenase (ACADM), which are associated with lipid metabolism and are valuable for understanding the physiology in fat depot and meat quality. Yanbian yellow cattle and Yan yellow cattle reared under the same conditions display different fat proportions in the carcass. To understand this difference, the expression of PPARγ, FASN, and ACADM in different adipose tissues and longissimus dorsi muscle (LD) in these two breeds were analyzed using the Real-time quantitative polymerase chain reaction method (qRT-PCR). The result showed that PPARγ gene expression was significantly higher in adipose tissue than in LD in both breeds. PPARγ expression was also higher in abdominal fat, in perirenal fat than in the subcutaneous fat (p<0.05) in Yanbian yellow cattle, and was significantly higher in subcutaneous fat in Yan yellow cattle than that in Yanbian yellow cattle. On the other hand, FASN mRNA expression levels in subcutaneous fat and abdominal fat in Yan yellow cattle were significantly higher than that in Yanbian yellow cattle. Interestingly, ACADM gene shows greater fold changes in LD than in adipose tissues in Yan yellow cattle. Furthermore, the expressions of these three genes in lung, colon, kidney, liver and heart of Yanbian yellow cattle and Yan yellow cattle were also investigated. The results showed that the highest expression levels of PPARγ and FASN genes were detected in the lung in both breeds. The expression of ACADM gene in kidney and liver were higher than that in other organs in Yanbian yellow cattle, the comparison was not statistically significant in Yan yellow cattle.

Effect of demecolcine-assisted enucleation on the MPF level and cyclin B1 distribution in porcine oocytes.

Demecolcine (DEM) treatment of oocytes induces formation of a membrane protrusion containing a mass of condensed maternal chromosomes, which can be removed with minimal damage prior to somatic cell nuclear transfer (SCNT). However, the effect of this method on the distribution of maturation-promoting factor (MPF) in porcine oocytes has not been reported. Here, the level of MPF and the distribution of cyclin B1 were assessed in porcine oocytes following DEM treatment. In addition, the efficiencies of DEM-assisted and mechanical enucleation were compared, as were the development (in vitro and in vivo) of these oocytes following SCNT. MPF was uniformly distributed in oocytes that had been treated with 0.4 µg/ml DEM for 1 h. Immunofluorescence microscopy showed that in untreated oocytes, cyclin B1, the regulatory subunit of MPF, accumulated around the spindle, and was lowly detected in the cytoplasm. DEM treatment disrupted spindle microtubules, induced chromosome condensation, and reduced the level of cyclin B1 in the nuclear region. Cyclin B1 was uniformly distributed in DEM-treated oocytes and the level of MPF was increased. The potential of embryos generated from DEM-treated oocytes to develop in vivo was significantly greater than that of embryos generated from mechanically enucleated oocytes. This is the first study to report the effects of DEM-assisted enucleation of porcine oocytes on the distribution of cyclin B1. MPF in mature oocytes is important for the development of reconstructed embryos and for efficient SCNT.

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer.

Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P>0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P<0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2mM valproic acid for 24h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

CUDC-101, a histone deacetylase inhibitor, improves the in vitro and in vivo developmental competence of somatic cell nuclear transfer pig embryos.

The aim of the present study was to examine the effects of CUDC-101, a novel histone deacetylase inhibitor, on the in vitro development and expression of the epigenetic marker histone H3 at lysine 9 (AcH3K9) in pig SCNT embryos. We found that treatment with 1 µmol/L CUDC-101 for 24 hours significantly improved the development of pig SCNT embryos. Compared with the control group, the blastocyst rate was higher (18.5% vs. 10.3%; P < 0.05). To assess in vivo developmental potency, CUDC-101-treated SCNT embryos were transferred into two surrogate mothers, resulting in one pregnancy with six fetuses. We then investigated the acetylation level of histone H3K9 in SCNT embryos treated with CUDC-101 and compared them only against untreated embryos. The acetylation level of control SCNT embryos was lower than that of CUDC-101-treated embryos at pseudo-pronuclear stages, and immunofluorescent signal for H3K9ac in CUDC-101-treated embryos in a pattern similar to that of control group. In conclusion, we demonstrated that CUDC-101 can significantly improve in vitro and in vivo developmental competence and enhance the nuclear reprogramming of pig SCNT embryos.

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer.

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality.

Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.