Construction of a co-expression network affecting intramuscular fat content and meat color redness based on transcriptome analysis.

Introduction: Intramuscular fat content (IFC) and meat color are vital indicators of pork quality. Methods: A significant positive correlation between IFC and redness of meat color (CIE a* value) indicates that these two traits are likely to be regulated by shared molecular pathways.To identify candidate genes, hub genes, and signaling pathways that regulate these two traits, we measured the IFC and CIE a* value in 147 hybrid pigs, and selected individuls with extreme phenotypes for transcriptome analysis. Results: The results revealed 485 and 394 overlapping differentially expressed genes (DEGs), using the DESeq2, limma, and edgeR packages, affecting the IFC and CIE a* value, respectively. Weighted gene co-expression network analysis (WGCNA) identified four modules significantly correlated with the IFC and CIE a* value. Moreover, we integrated functional enrichment analysis results based on DEGs, GSEA, and WGCNA conditions to identify candidate genes, and identified 47 and 53 candidate genes affecting the IFC and CIE a* value, respectively. The protein protein interaction (PPI) network analysis of candidate genes showed that 5 and 13 hub genes affect the IFC and CIE a* value, respectively. These genes mainly participate in various pathways related to lipid metabolism and redox reactions. Notably, four crucial hub genes (MYC, SOX9, CEBPB, and PPAGRC1A) were shared for these two traits. Discussion and conclusion: After functional annotation of these four hub genes, we hypothesized that the SOX9/CEBPB/PPARGC1A axis could co-regulate lipid metabolism and the myoglobin redox response. Further research on these hub genes, especially the SOX9/CEBPB/PPARGC1A axis, will help to understand the molecular mechanism of the co-regulation of the IFC and CIE a* value, which will provide a theoretical basis for improving pork quality.

Mulberry leaf supplementation inhibits skatole deposition by regulating gut microbiota and upregulating liver cytochrome P450 1A1 expression in finishing pigs.

Skatole, a strong fecal odor substance, is generated through microbial degradation of tryptophan in the animal hindgut. It easily accumulates in adipose tissue and affects meat quality. In this study, the effect of mulberry leaf supplementation on skatole in finishing pigs was studied. In a 35-day trial, 20 finishing pigs (barrows and gilts) were fed with a basal diet or basal diet with 6% mulberry leaves. Growth performance of the pigs (n = 10) was automatically recorded by a performance-testing feeder system and 8 pigs in each treatment were slaughtered and sampled for the remaining tests. Skatole and short-chain fatty acids were detected using HPLC and gas chromatography, respectively. Fecal microbiota were analyzed using 16S rRNA gene sequencing. The metabolomics analysis of feces and serum was performed with UHPLC-MS/MS. The major cytochrome P450 (CYP) enzymes that catalyze skatole degradation in the liver were tested by using RT-PCR and Western blot. Effects of major bioactive compounds in mulberry leaves on the CYP genes were verified in the hepatic cell line HepG2 in an in vitro test (n = 3). In finishing pigs, mulberry leaf supplementation had no significant effect on the average daily gain, average daily feed intake, and feed conversion ratio (P > 0.05), but reduced skatole levels in feces, serum, and backfat (P < 0.05), and increased acetic acid levels in feces (P = 0.027). Mulberry leaf supplementation decreased the relative abundance of the skatole-producing bacteria Megasphaera and Olsenella (P < 0.05). Indole-3-acetic acid, the intermediate that is essential for skatole production, was significantly reduced in feces by mulberry leaf supplementation (P < 0.05) and was positively correlated with skatole content in feces (P = 0.004). In pigs treated with mulberry leaves, liver CYP1A1 expression was increased (P < 0.05) and was negatively correlated with skatole content in backfat (P = 0.045). The in vitro test demonstrated that mulberry leaf polyphenols and polysaccharides could directly stimulate CYP1A1 expression in hepatic cells. These findings suggest that mulberry leaf supplementation reduces skatole production and deposition in finishing pigs by regulating the gut microbiota and promoting skatole degradation in liver.

Effects of Growth Stage and Rearing Pattern on Pig Gut Microbiota.

Understanding the developmental period or the patterns of gut microbiota is important for nutritionists when designing a feed formula or adjusting a feeding strategy. The effects of growth stage or rearing pattern on pig gut microbiota have not been fully investigated. Herein, 39 fecal samples from pigs aged 3-9 months under two rearing patterns were collected to analyze the gut microbiome. Samples were clustered into three distinct groups, namely, early (3 months), middle (5 months), and late (7 and 9 months) stages, using principal coordinate analysis and analysis of similarities test. The rearing-pattern effects were very minimal, and no differences were observed in the alpha diversity [observed operational taxonomic units (OTUs) and Shannon index] of gut microbiota. From early and middle to late stage, Shannon index gradually decreased and OTUs gradually increased. Pigs at early stage were enriched with bacteria from family Prevotellaceae, including the genera Prevotella_9 and Prevotellaceae_NK3B31, whereas pigs at late stage were enriched with family Ruminococcaceae, including genera Ruminococcaceae_UCG-005 and Oscillospira. Pigs in the semi-free-grazing farm group were significantly enriched with bacteria from order Clostridiales. Growth stage better explained the changes in porcine gut microbiota than rearing patterns.

Low Crude Protein Diet Affects the Intestinal Microbiome and Metabolome Differently in Barrows and Gilts.

Low protein diets are commonly used in the growing-finishing pig stage of swine production; however, the effects of low dietary protein on the intestinal microbiota and their metabolites, and their association with pig sex, remain unclear. The present study aimed to assess the impact of a low crude protein (CP) diet on the gut microbiome and metabolome, and to reveal any relationship with sex. Barrows and gilts (both n = 24; initial body = 68.33 ± 0.881 kg) were allocated into two treatments according to sex. The four groups comprised two pairs of gilts and barrows fed with a high protein diet (CP 17% at stage I; CP 13% at stage II) and a low protein diet (CP 15% at stage I; CP 11% at stage II), respectively, for 51 d. Eight pigs in each group were slaughtered and their colon contents were collected. Intestinal microbiota and their metabolites were assessed using 16S rRNA sequencing and tandem mass spectrometry, respectively. The low protein diet increased intestinal microbiota species and richness indices (P < 0.05) in both sexes compared with the high protein diet. The sample Shannon index was different (P < 0.01) between barrows and gilts. At the genus level, unidentified Clostridiales (P < 0.05), Neisseria (P < 0.05), unidentified Prevotellaceae (P < 0.01) and Gracilibacteria (P < 0.05) were affected by dietary protein levels. The relative abundance of unidentified Prevotellaceae was different (P < 0.01) between barrows and gilts. The influence of dietary protein levels on Neisseria (P < 0.05), unidentified Prevotellaceae (P < 0.01) and Gracilibacteria (P < 0.05) were associated with sex. Metabolomic profiling indicated that dietary protein levels mainly affected intestinal metabolites in gilts rather than barrows. A total of 434 differentially abundant metabolites were identified in gilts fed the two protein diets. Correlation analysis identified that six differentially abundant microbiota communities were closely associated with twelve metabolites that were enriched for amino acids, inflammation, immune, and disease-related metabolic pathways. These results suggested that decreasing dietary protein contents changed the intestinal microbiota in growing-finishing pigs, which selectively affected the intestinal metabolite profiles in gilts.

FNDC5 expression closely correlates with muscle fiber types in porcine longissimus dorsi muscle and regulates myosin heavy chains (MyHCs) mRNA expression in C2C12 cells.

BACKGROUND: Irisin (a glycosylated protein) is cleaved from fibronectin type III domain-containing protein 5 (FNDC5), which is expressed mainly in animal muscle tissues and has multiple metabolic regulatory activities. However, their roles in controlling myofiber types in skeletal muscle remain unclear. METHODOLOGY: Two different commercial hybridized pigs, LJH (a crossed pig containing Chinese native pig genotypes) and DLY (Duroc × Landrace × Yorkshire) were selected to analyze FNDC5 mRNA expression and the mRNA composition of four adult myosin heavy chain (MyHC) isoforms (IIIaIIxIIb) in the longissimus dorsi (LD) muscle. C2C12 myoblasts were cultured to investigate the effects of FNDC5 on the four MyHCs mRNA expressive levels, using small interfering RNA for depletion and a eukaryotic expression vector carrying FNDC5 for overexpression. ZLN005 (a small molecule activator of FNDC5's upstream control gene PGC1α) or recombinant human irisin protein were also used. RESULTS: In LD muscle, LJH pigs had the higher FNDC5 mRNA level, and MyHC I or IIa proportion than DLY pigs (P <  0.05). For C2C12 cells in vitro, small interfering RNA (si-592) silencing of FNDC5 expression markedly reduced MyHC IIa mRNA levels (P <  0.05), while FNDC5 overexpression significantly increased MyHC IIa mRNA levels (P <  0.05). Exogenous irisin increased the mRNA levels of PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), FNDC5, MyHCI, MyHCIIa, NRF1 (nuclear respiratory factor 1), VEGF (vascular endothelial growth factor), and TFAM (mitochondrial transcription factor A,) (P <  0.05), and the enzyme activities of SDH (succinate dehydrogenase), CK (creatine kinase), and MDH (malate dehydrogenase) in C2C12 myotubes (P <  0.05). These results showed that FNDC5 mRNA expression had a significant association with the characteristics of myofiber types in porcine muscle, and participated in regulating MyHCs mRNA expression of C2C12 myogenic differentiation cells in vitro. FNDC5 could be an important factor to control muscle fiber types, which provides a new direction to investigate pork quality via muscle fiber characteristics.

Effects of Bacillus subtilis on growth performance, serum parameters, digestive enzyme, intestinal morphology, and colonic microbiota in piglets.

The present study was conducted to investigate effects of Bacillus subtilis on growth performance, serum parameters, digestive enzymes, intestinal morphology, and colonic microbiota in piglets. A total of 72 piglets were weighed and randomly allotted into three treatments (four replication pens per treatment with six piglets/pen) for a 28-day experiment. The dietary treatments were as follows: basal diet (control group, CTR), basal diet supplementation with antibiotic (antibiotic group, ABT), and basal diet supplementation with 0.1% Bacillus subtilis (probiotic group, PBT). The average daily gain of body weight increased in both the ABT and PBT groups, and dietary antibiotics decreased the feed:gain ratio (F:G), as compared to the CTR group (P < 0.05). Both ABT and PBT piglets had increased serum triglycerides and lipase, amylase, maltase activities and villus height:crypt depth ratio (V/C) in ileum (P < 0.05). The PBT group also showed an increase in serum glucose and villus height in the ileum (P < 0.05). Dietary antibiotics increased Lactobacillus johnsonii, as compared to the CTR group, but decreased bacterial diversity and increased Escherichia coli, as compared to the PBT group (P < 0.05). Piglets dietary with B. subtilis modulated the microbiota by increasing the abundance of Firmicutes (L. johnsonii, L. reuteri) and decreasing the abundance of E. coli, as compared to the control group (P < 0.05). These results indicate that dietary of B. subtilis improves growth performance and intestinal health and can be a promising alternative to antibiotics in piglets diet.

A compound plant extract and its antibacterial and antioxidant properties in vitro and in vivo.

The present study was conducted to prepare a compound plant extract as a candidate animal feed additive. Firstly, Evodia rutaecarpa (ER), Schisandra sphenanthera (SS), Punica granatum (PG) and Artemisia argyi (AA) were screened out from 17 plants as materials of candidate compound plant extracts by measuring the antibacterial rate on Escherichia coli and Salmonella paratyphoid, and the scavenging capability on 2,2 diphenyl-1-picrylhydrazine radical in vitro. Secondly, proportions of the four materials were optimized with an L9 (43) orthogonal experiment. By range analysis of experimental results, two compound extracts (named as F1 and F2) with the strongest antibacterial and antioxidant functions were obtained. The ratio of ER: SS: PG: AA is 9:9:1:3 in F1 and 9:9:9:3 in F2, respectively. Finally, the effects of F1 and F2 on security and efficacy in vivo were evaluated. In healthy mice, F1 had no significant effects (p > 0.05) on all blood parameters and viscera indices, and at 1000 mg/kg bw dose significantly increased (p < 0.05) the average daily gain (ADG). F2 decreased (p < 0.05) white blood cell count at 3000 mg/kg bw and increased (p < 0.05) red blood cell count at 333 mg/kg bw. In immunosuppressed mice, both F1 and F2 improved ADG (p < 0.05) and the feed intake to gain ratio (p < 0.01), and increased the activities of hepatic superoxide dismutase (p < 0.05), catalase (p < 0.05) and total antioxygen capacity (p < 0.05), and the content of malonaldehyde (p < 0.01). In mice challenged with Escherichia coli, the antidiarrhea and reducing mortality effects of F1 were equivalent to the antibiotic. F2 failed to protect the experimental mice. These results suggested F1, a compound plant extract, show a great potential as a substitute for antibiotics in animal feed.

Rearing pattern alters porcine myofiber type, fat deposition, associated microbial communities and functional capacity.

BACKGROUND: The Chinese believe that the meat of pigs reared in the past with free range tastes better than that of the pigs reared indoor on a large scale today. Gastrointestinal microflora is closely associated with the main factor of meat flavour, including fibre characteristics and lipid metabolism. Our method in this study involved different raising patterns within the semi free-grazing farm (FF) or indoor feeding farm (DF), the measurement of fat deposition and myofiber type by paraffin section and reverse transcription polymerase chain reaction and the identification of microbiome and functional capacities associated with meat quality through metagenomic sequencing. RESULTS: Results showed that the fat area in muscle and adipose tissue and the myofiber density significantly increased in the pigs of the FF group. The relative abundance of bacteria associated with lipid metabolism, such as g_Oscillibacter, in the feces of the FF group was higher than that in DF group, and the relative abundance of some bacteria with probiotic function, including g_Lactobacillus and g_Clostridium, was lower than that in DF group. The abundance of g_Clostridium was significantly positively correlated with the intramuscular fat area, whereas health-related bacteria, such as g_Butyricicoccus, g_Eubacterium, g_Phascolarctobacterium and g_Oribacterium, was significantly negatively correlated with abdominal fat area, myofiber density and adipose triglyceride lipase (ATGL) mRNA expression. KEGG analysis showed that pigs raised in semi free-grazing farm can activate the pathway of inosine monophosphate (IMP) biosynthesis, glycolysis/gluconeogenesis and alanine, aspartate and glutamate metabolism. CONCLUSIONS: Free range feeding improves meat quality by changing the fibre type, myofiber density and metabolic pathways related to flavour amino acids, IMP or glycolysis/gluconeogenesis in muscle. However, prolonged feeding cycle increases fat deposition and associated microbial communities.

Over-expression of miR-146b and its regulatory role in intestinal epithelial cell viability, proliferation, and apoptosis in piglets.

BACKGROUND: Weaning stress affects the small intestine of piglets. MiR-146b is differentially expressed in suckling and weaned piglets. In this study, we evaluated the effects of miR-146b on cell viability, proliferation, and apoptosis in IPEC-J2 cells. RESULTS: Transfection with miR-146b mimics successfully increased miR-146b levels by 1000× (P < 0.001). The over-expression of miR-146b significantly promoted the apoptosis (P < 0.01) of IPEC-J2 cells, with no significant effects on cell viability or proliferation. MiR-146b suppressed the luciferase activity of the miR-TLR4-wt by 57% compared with the negative control, while mutation of the miR-146b binding site significantly blocked the suppressive effect (P < 0.05). Western blot results showed that TLR4 levels decreased in IPEC-J2 cells transfected with miR-146b mimics (P < 0.05). CONCLUSIONS: The over-expression of miR-146b promotes IPEC-J2 cell apoptosis. TLR4 is a direct target of miR-146b in IPEC-J2 cells. REVIEWERS: This article was reviewed by Eugene Berezikov and Jan B Hoek.

Probiotics and Probiotic Metabolic Product Improved Intestinal Function and Ameliorated LPS-Induced Injury in Rats.

In the present study, we sought to determine the effects of Bacillus subtilis (BAS) and Bacillus licheniformis (BAL) in rats after lipopolysaccharide (LPS)-induced acute intestinal inflammation. We also determined whether the B. subtilis metabolic product (BASM) is as effective as the live-cell probiotic. 60 male SD rats were randomly assigned to five groups and administered a diet containing 0.05% B. licheniformis (BAL group), 0.05% B. subtilis (BAS group), 0.5% B. subtilis metabolic product (BASM group), or a basic diet (PC group and NC group) for 40 days. On day 40, BAL, BAS, BASM, and NC groups were injected with 4 mg/kg body weight LPS. 4 h later, all rats were anesthetized and sacrificed. The results showed that the administration of B. licheniformis and B. subtilis improved intestinal function as evidenced by histology, increased enzyme activity, and mucosal thickness. They also increased the number of intraepithelial lymphocytes and decreased mucosal myeloperoxidase activity and plasma TNF-α. In addition, the cecal content of B. subtilis-treated rats had significantly increased microbial diversity, decreased numbers of Firmicutes, and increased numbers of Bacteroidetes as compared to rats fed basic diets. Similar to BAS group, the cecal content of B. licheniformis-treated rats decreased the number of Firmicutes. Administration of B. subtilis metabolic product had similar effects on intestinal function, inflammation response, and microbial diversity as B. subtilis but these effects were attenuated. In conclusion, administration of probiotic strains B. licheniformis or B. subtilis improved intestinal function, ameliorated the inflammation response, and modulated microflora after LPS-induced acute inflammation in rats. Non-living cells also exerted probiotic properties but live cells tended to function better.

Analysis of Serum microRNA Expression Profiles and Comparison with Small Intestinal microRNA Expression Profiles in Weaned Piglets.

Weaning stress induces tissue injuries and impairs health and growth in piglets, especially during the first week post-weaning. MicroRNAs (miRNAs) play vital roles in regulating stresses and diseases. Our previous study found multiple differentially expressed miRNAs in small intestine of piglets at four days post-weaning. To better understand the roles of miRNAs during weaning stress, we analyzed the serum miRNA expressional profile in weaned piglets (at four days post-weaning) and in suckling piglets (control) of the same age using miRNA microarray technology. We detected a total of 300 expressed miRNAs, 179 miRNAs of which were differentially expressed between the two groups. The miRNA microarray results were validated by RT-qPCR. The biological functions of these differentially expressed miRNAs were predicted by GO terms and KEGG pathway annotations. We identified 10 highly expressed miRNAs in weaned piglets including miR-31, miR-205, and miR-21 (upregulated) and miR-144, miR-30c-5p, miR-363, miR-194a, miR-186, miR-150, and miR-194b-5p (downregulated). Additionally, miR-194b-5p expression was significantly downregulated in serum and small intestine of weaned piglets. Our results suggest that weaning stress affects serum miRNA profiles in piglets. And serum miR-194b-5p levels can reflect its expressional changes in small intestine of piglets by weaning stress.

Transient changes of enzyme activities and expression of stress proteins in the small intestine of piglets after weaning.

To determine the transient effects of weaning on the small intestine, 16 piglets were slaughtered at days 0, 1, 4 and 7 after weaning. Jejunal samples were collected to examine different enzyme activities and mRNA expressions of two stress protein families, namely, heat-shock proteins (HSP) and trefoil factors (TFF). Results showed that the activities of ceruloplasmin, alkaline phosphatase and lactate dehydrogenase, were significantly changed at Day 1 and/or Day 4. The mRNA expressions of HSP10, HSP60 and HSP90 showed a pattern of increased expression with time after weaning. Expression significantly differed between Day 0 and Day 7 after weaning. The mRNA expression of HSP70 was significantly increased on Day 1 only. Similarly, the mRNA expressions of TFF1 and TFF2 were significantly increased on Day 7 compared with those on Day 0. Expression of TFF3 was not affected by time after weaning. In conclusion, the present study indicated that weaning induced transient injury to small intestinal morphology and function. Particularly it changed enzyme activities and gene expression of stress proteins in the small intestine of piglets. At first time, a change in the gene expression of HSP10 and a gene overexpression of TFF1 in the small intestine of piglets after weaning was found.

Bioinformatic analysis of the ssc-miR-146b upstream promoter region.

Sus Scrofa microRNA-146b-5p (ssc-miR-146b) was found to be one of differentially expressional microRNAs (miRNA) in our previous study. Not only it is highly expressed but also it maintains the largest up-regulated differences on the expressional level at different time points in the small intestinal mucosa of weaned piglets. To further explore the regulation mechanism of microRNA-146b-5p (miR-146b) during the stressful progress in weaned piglets, the present study predicted the functions of the ssc-miR-146b upstream promoter region using biological analysis. The analytical results showed that ssc-miR-146b is an intergenic miRNA. The length of the promoter region of ssc-miR-146b was predicted to be 2,249 bp using the Ensemble database. The length of the CpG island in the ssc-miR-146b promoter region was found to be 167 bp and it was located from 464 to 630 bp. Twenty six binding sites of 9 transcription factors in the upstream promoter region, including the sites of genes such as Sp1, AP-1, MyoD, GATA etc, were discovered using different kinds of analytical software. The predictions of the CpG island and transcription factor binding sites provided significant information for further studying the transcriptional regulation mechanism of ssc-miR-146b on the small intestinal injury due to weaning stress.

Purified PEGylated porcine glucagon-like peptide-2 reduces the severity of colonic injury in a murine model of experimental colitis.

The rapid degradation of porcine glucagon-like peptide-2 (pGLP-2) by the enzyme dipeptidyl peptidase-IV (DPP-IV) is the main impediment in the development of pGLP-2 as a potential therapeutic agent for intestinal dysfunction and damage. In this study, one mono-modified Lys(30)-polyethylene glycol (PEG)-pGLP-2 was prepared using mPEG-succinimidyl propionate. To determine the optimized condition for PEGylation, the reactions were monitored by RP-HPLC and MALDI-TOF-MS. Stability was tested in purified DPP-IV in vitro. In vivo, the protective effects for colonic injury were measured in dextran sulfate sodium (DSS)-induced colitis in mice. The monoPEGylated products reached the maximum yield at 4:1 ratio of mPEG5k-SPA to pGLP-2. An effective method of successfully separating PEGylated pGLP-2 from mPEG-SPA5kD using CM Sepharose Fast Flow resin was established. The half-life of Lys(30)-PEG-pGLP-2 was 16-fold longer than that of pGLP-2 in DPP-IV. The DSS mice exhibited marked weight loss), which was significantly reduced by Lys(30)-PEG-pGLP-2 therapy. DSS treatment significantly increased colonic damage score, which was significantly reduced by administration of Lys(30)-PEG-pGLP-2 in DSS-mice. DSS-induced colitis clearly induced Myeloperoxidase activity in the colon, which was significantly reduced by treatments with 3% DSS-pGLP-2 or 3% DSS-PEG-pGLP-2. These results showed that site-specific Lys(30)-PEG-GLP-2 was resistant to degradation and reduced the severity of colonic injury in murine colitis. The enhanced biological potency of this product highlighted its potential as a therapeutic agent for intestinal diseases.