Deoxynivalenol induced intestinal barrier injury, mitochondrial dysfunction and calcium overload by inositol 1,4,5-triphosphate receptors (IP3Rs)-mitochondrial calcium uniporter (MCU) calcium axis.

Deoxynivalenol (DON) contamination is widespread in crops and could easily cause intestinal injury, which brings hazards to animals. Mitochondria are considered as an important target of DON, nevertheless, the mechanism is still unclear. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) have gained arousing interest and are recognized as critical signaling hubs that control calcium signaling transduction between ER and mitochondria. This study aims to investigate the effects of DON on intestinal barrier, mitochondria, MAMs and inositol 1,4,5-triphosphate receptors (IP3Rs)-mitochondrial calcium uniporter (MCU) calcium axis in piglets and porcine intestinal epithelial cells (IPEC-J2). Furthermore, inhibition of IP3Rs or MCU was used to explore whether IP3Rs-MCU axis of MAMs was involved in the mitochondria dysfunction and intestinal epithelium barrier injury induced by DON in IPEC-J2. The data showed that DON induced intestinal barrier injury, mitochondrial dysfunction and ERS in piglets' jejunum and IPEC-J2. Moreover, DON increased MAMs by upregulating the protein level of Mitofusin 2 (Mfn2), increasing the percentage of mitochondria with MAMs/total mitochondria and the ratio of MAMs length/mitochondrial perimeter and shortening the distance between mitochondria and ER of MAMs. Importantly, DON influenced IP3Rs-glucose-regulated protein 75 (GRP75)-voltage-dependent anion channel 1 (VDAC1)-MCU calcium axis by increasing the protein levels of GRP75 and MCU and the interaction of VDAC1-GRP75-IP3Rs complex, which in turn induced mitochondrial calcium overload. Furthermore, inhibition of IP3Rs or MCU alleviated DON-induced intestinal epithelium barrier injury, mitochondrial dysfunction and mitochondrial calcium overload of IPEC-J2. The current investigation proposed that DON induced intestinal injury, mitochondrial dysfunction and calcium overload via IP3Rs-GRP75-VDAC1-MCU calcium axis.

Effects of DON on Mitochondrial Function, Endoplasmic Reticulum Stress, and Endoplasmic Reticulum Mitochondria Contact Sites in the Jejunum of Piglets.

Recent research has emphasized the significance of investigating the interplay between organelles, with endoplasmic reticulum mitochondria contact sites (ERMCSs) being recognized as critical signaling hubs between organelles. The objective of the current study was to assess the impact of deoxynivalenol (DON) on jejunal mitochondria, ER, and ERMCSs. Twelve piglets (35 d, 10.22 ± 0.35 kg) were randomized into two groups: control group, basal diet; the DON group, basal diet + 1.5 mg/kg DON. The findings revealed that DON decreased growth performance, induced jejunal oxidative stress, and impaired jejunal barrier function. DON was also found to induce mitochondrial dysfunction, trigger endoplasmic reticulum stress (ERS) in the piglets' jejunum, and activate mitochondrial and ER apoptosis pathways by upregulating apoptosis-related proteins (Caspase-8, Caspase-12, Bax, and CHOP). To investigate the involvement of ERMCSs in DON-induced intestinal injury, we measured the protein levels of ERMCS proteins, such as mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and glucose-regulated protein 75 (GRP75) and Pearson's correlation coefficient of ERMCS proteins and ERMCS ultrastructure. Our finding showed that DON upregulated the protein level of Mfn2 and GRP75 and increased the percentage of mitochondria with ERMCSs/total mitochondria, the length of ERMCSs compared to the perimeter of mitochondria, and the Pearson's correlation coefficient of voltage-dependent anion-selective channel protein 1 (VDAC1) and inositol 1,4,5-triphosphate receptors (IP3Rs) in piglets' jejunum. Furthermore, DON shortened the distance between mitochondria and ER at ERMCSs. These findings suggested that DON impaired mitochondrial function, triggered ERS, and increased ERMCSs, indicating that the increased ERMCSs could be related to mitochondrial dysfunction and ERS involved in the intestinal injury of piglets induced by DON.

Phylogenetic and expression analysis of the angiopoietin-like gene family and their role in lipid metabolism in pigs.

OBJECTIVE: The objective of this study was to investigate the phylogenetic and expression analysis of the angiopoietin-like (ANGPTL) gene family and their role in lipid metabolism in pigs. METHODS: In this study, the amino acid sequence analysis, phylogenetic analysis, and chromosome adjacent gene analysis were performed to identify the ANGPTL gene family in pigs. According to the body weight data from 60 Jinhua pigs, different tissues of 6 pigs with average body weight were used to determine the expression profile of ANGPTL1-8. The ileum, subcutaneous fat, and liver of 8 pigs with distinct fatness were selected to analyze the gene expression of ANGPTL3, ANGPTL4, and ANGPTL8. RESULTS: The sequence length of ANGPTLs in pigs was between 1,186 and 1,991 bp, and the pig ANGPTL family members shared common features with human homologous genes, including the high similarity of the amino acid sequence and chromosome flanking genes. Amino acid sequence analysis showed that ANGPTL1-7 had a highly conserved domain except for ANGPTL8. Phylogenetic analysis showed that each ANGPTL homologous gene shared a common origin. Quantitative reverse-transcription polymerase chain reaction analysis showed that ANGPTL family members had different expression patterns in different tissues. ANGPTL3 and ANGPTL8 were mainly expressed in the liver, while ANGPTL4 was expressed in many other tissues, such as the intestine and subcutaneous fat. The expression levels of ANGPTL3 in the liver and ANGPTL4 in the liver, intestine and subcutaneous fat of Jinhua pigs with low propensity for adipogenesis were significantly higher than those of high propensity for adipogenesis. CONCLUSION: These results increase our knowledge about the biological role of the ANGPTL family in this important economic species, it will also help to better understand the role of ANGPTL3, ANGPTL4, and ANGPTL8 in lipid metabolism of pigs, and provide innovative ideas for developing strategies to improve meat quality of pigs.

Resveratrol Improves Intestinal Morphology and Anti-Oxidation Ability in Deoxynivalenol-Challenged Piglets.

This study aimed to investigate the potential effects of resveratrol (RES) on intestinal function and oxidative stress in deoxynivalenol (DON)-challenged piglets. Twenty-four healthy Duroc × Yorkshire × Landrace weaned piglets at the age of 28 ± 1 days were randomly divided into four groups with six repetitions per group. The four groups were as follows: the control group (CON), fed with a basic diet; the RES group, fed with a basal diet + 300 mg/kg RES; the DON group, fed with a basal diet containing 2.65 mg/kg DON; and the DON + RES group, fed with a basal diet containing 2.65 mg/kg DON + 300 mg/kg RES. The results showed that the growth performance and intestinal function of DON-challenged piglets were significantly decreased (p < 0.05). Compared with the DON group, the average daily feed intake of piglets in the DON + RES group was significantly increased (p < 0.05). Additionally, dietary RES ameliorated DON-induced intestinal morphology impairment, as indicated by the increased (p < 0.05) jejunal villi height and the ratio of the jejunal villi height/crypt depth. Furthermore, after the addition of RES, the activities of superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in the jejunum mucosa were significantly increased, and the content of malondialdehyde (MDA) was significantly declined (p < 0.05). In addition, the level of reactive oxygen species (ROS) in the mitochondria was significantly reduced by RES, while the mitochondrial membrane potential in jejunum was significantly increased by RES (p < 0.05). However, there was no obvious difference between DON + RES and DON groups on average daily gain and the ratio of feed togain, except for the significant inhibition of average daily feed intake (p < 0.05). In conclusion, RES could effectively alleviate the DON-induced oxidative stress on weaned piglets, and reduce the damage to mitochondria and intestinal morphology, so as to improve the growth performance of piglets.

Lactic acid bacteria strains selected from fermented total mixed rations improve ensiling and in vitro rumen fermentation characteristics of corn stover silage.

OBJECTIVE: This study identified the major lactic acid bacteria (LAB) strains from different fermented total mixed rations (FTMRs) via metataxonomic analysis and evaluated the ability of their standard strain as ensiling inoculants for corn stover silage. METHODS: The bacterial composition of eight FTMRs were analyzed by 16S rDNA sequencing. Corn stover was ensiled without LAB inoculation (control) or with 1×106 cfu/g LAB standard strain (Lactobacillus vaginalis, Lactobacillus reuteri, Lactobacillus helveticus, or Lactobacillus paralimentarius) selected from the FTMRs or 10 g/t commercial silage inoculant (CSI) around 25°C for 56 days. For each inoculation, a portion of the silage was sampled to analyze ensiling characteristics at time intervals of 0, 1, 3, 7, 14, 28, and 56 days, gas production (GP), microbial crude protein and volatile fatty acids as the measurements of rumen fermentation characteristics were evaluated in vitro with the silages of 56 days after 72 h incubation. RESULTS: Lactobacillus covered >85% relative abundance of all FTMRs, in which L. pontis, L. vaginalis, L. reuteri, L. helveticus, and L. paralimentarius showed >4% in specific FTMRs. CSI, L. helveticus, and L. paralimentarius accelerated the decline of silage pH. Silage inoculated with L. paralimentarius and CSI produced more lactic acid the early 14 days. Silage inoculated with L. paralimentarius produced less acetic acid and butyric acid. For the in vitro rumen fermentation, silage inoculated with CSI produced more potential GP, isobutyric acid, and isovaleric acid; silage inoculated with L. helveticus produced more potential GP and isovaleric acid, silage inoculated with L. paralimentarius or L. reuteri produced more potential GP only. CONCLUSION: The standard strain L. paralimentarius (DSM 13238) is a promising ensiling inoculant for corn stover silage. The findings provide clues on strategies to select LAB to improve the quality of silage.

Gut Microbiota Influence Lipid Metabolism of Skeletal Muscle in Pigs.

Gut microbiota is recognized as a strong determinant of host physiology including fat metabolism and can transfer obesity-associated phenotypes from donors to recipients. However, the relationship between gut microbiota and intramuscular fat (IMF) is still largely unknown. Obese Jinhua pigs (JP) have better meat quality that is associated with higher IMF content than lean Landrace pigs (LP). The present study was conducted to test the contribution of gut microbiota to IMF properties by transplanting fecal microbiota of adult JP and LP to antibiotics-treated mice. Similar to JP donors, the mice receiving JP's microbiota (JM) had elevated lipid and triglyceride levels and the lipoprotein lipase activity, as well as reduced mRNA level of angiopoietin-like 4 (ANGPTL4) in the gastrocnemius muscles, compared to those in mice receiving LP's microbiota (LM). High-throughput 16S rRNA sequencing confirmed that transplantation of JP and LP feces differently reconstructed the gut microbiota in both jejunum and colon of mouse recipients. In colonic samples, we observed an elevated ratio of Firmicutes to Bacteroidetes and increased abundance of genus Romboutsia in JM, which were positively correlated with obesity. Furthermore, the abundance of Akkermansia decreased in JM, which is positively correlated with lean. Colonic concentrations of acetate (P = 0.047) and butyrate (P = 0.014) were significantly lower in JM than in LM, and consistently, the terminal genes for butyrate synthesis, butyryl CoA: acetate CoA transferase were less abundant in colonic microbiota of JM. Taken together, these gut microbiota of obese JP intrinsically promotes IMF accumulation and can transfer the properties to mouse recipients. Manipulation of intestinal microbiota will, therefore, have the potential to improve the meat quality and flavor of pigs and even to ameliorate the metabolic syndrome in human.

Dietary copper/zinc-loaded montmorillonite improved growth performance and intestinal barrier and changed gut microbiota in weaned piglets.

The effects of copper/zinc-loaded montmorillonite (Cu/Zn-Mt) on growth performance, intestinal barrier and gut microbiota of weaned pigs were investigated in the present study. A total of 108 piglets (Duroc × Landrace × Yorkshire; 6.36 kg; weaned at 21 ± 1 d age) were used in this experiment. The pigs were randomly assigned to three treatments with six replicates, six pigs in each replicate. The three treatments were as follows: (1) control group: basal diet; (2) Cu/Zn-Mt group: basal diet supplemented with 39 mg/kg Cu and 75 mg/kg Zn as Cu/Zn-Mt; and (3) Cu +Zn +Mt group: basal diet supplemented with the mixture of copper sulphate, zinc sulphate and montmorillonite (equivalent to the copper and zinc in the Cu/Zn-Mt treatment). The results indicated that, compared with the pigs from control group, average daily gain and gain: feed ratio were increased and the faecal score on days 7 and 14 after weaning was decreased by supplementation of Cu/Zn-Mt; intestinal transepithelial electrical resistance (TER) and expressions of tight junction protein claudin-1 and zonula occludens-1 were increased, and intestinal permeability of fluorescein isothiocyanate-dextran 4 kDa was decreased by supplementation with Cu/Zn-Mt. According to the Illumina-based sequencing results, Cu/Zn-Mt supplementation increased the relative abundance of core bacteria (Lactococcus, Bacillus) at genus level and decreased the potentially pathogenic bacteria (Streptococcus and Pseudomonas) in colon of weaned piglets. However, the piglets fed with the mixture of copper sulphate, zinc sulphate and montmorillonite showed no effects in above parameters in comparison with the pigs from control group. In conclusion, dietary Cu/Zn-Mt could improve growth performance, decrease the diarrhoea and improve intestinal barrier and bacterial communities of weaned pigs. The results indicated that 'loading' of montmorillonite with Zn and Cu changed not only its chemical but also its nutritional properties.

Effects of dietary tributyrin on intestinal mucosa development, mitochondrial function and AMPK-mTOR pathway in weaned pigs.

BACKGROUND: The objective of this experiment was to investigate the influence of dietary tributyrin on intestinal mucosa development, oxidative stress, mitochondrial function and AMPK-mTOR signaling pathway. METHODS: Seventy-two pigs were divided into two treatments and received either a basal diet or the same diet supplemented with 750 mg/kg tributyrin. Each treatment has six replicates of six pigs. After 14 days, 6 pigs from each treatment were selected and the jejunal samples were collected. RESULTS: Results showed that supplemental tributyrin increased (P < 0.05) villus height and villus height: crypt depth of weaned pigs. Pigs fed tributyrin had greater (P < 0.05) RNA/DNA and protein/DNA ratios than pigs on the control group. The mRNA levels of sodium glucose transport protein-1 and glucose transporter-2 in the jejunum were upregulated (P < 0.05) in pigs fed the tributyrin diet. Dietary tributyrin supplementation lowered (P < 0.05) the malondialdehyde and hydrogen peroxide (H2O2) content in jejunum, enhanced (P < 0.05) the mitochondrial function, as demonstrated by decreased (P < 0.05) reactive oxygen species level and increased (P < 0.05) mitochondrial membrane potential. Furthermore, tributyrin increased (P < 0.05) mitochondrial DNA content and the mRNA abundance of genes related to mitochondrial functions, including peroxisomal proliferator-activated receptor-γ coactivator-1α, mitochondrial transcription factor A, nuclear respiratory factor-1 in the jejunum. Supplementation with tributyrin elevated (P < 0.05) the phosphorylation level of AMPK and inhibited (P < 0.05) the phosphorylation level of mTOR in jejunum compared with the control group. CONCLUSIONS: These findings suggest that dietary supplementation with tributyrin promotes intestinal mucosa growth, extenuates oxidative stress, improves mitochondrial function and modulates the AMPK-mTOR signal pathway of weaned pigs.

Dietary Tributyrin Attenuates Intestinal Inflammation, Enhances Mitochondrial Function, and Induces Mitophagy in Piglets Challenged with Diquat.

The study evaluated the effects of butyric acid, in the form of tributyrin on the oxidative stress, inflammation, and mitochondrial function in diquat-challenged pigs. Twenty-four weaned pigs were allocated to four treatments in a 2 × 2 factorial arrangement with the main effects of tributyrin supplementation and diquat challenge. The results showed that supplemental tributyrin increased ( P < 0.05) average daily gain and average daily feed intake of diquat-challenged pigs. Tributyrin elevated ( P < 0.05) the activities of total antioxidant capacity and superoxide dismutase, reduced ( P < 0.05) malondialdehyde content, and increased ( P < 0.05) mRNA levels of copper and zinc superoxide dismutase and manganese-containing superoxide dismutase of diquat-challenged pigs. Tributyrin relieved ( P < 0.05) intestinal inflammation reflected by decreased mRNA abundances of tumor necrosis factor-α, interferon-γ, and interleukin-6 in the intestine. Tributyrin reduced ( P < 0.05) serum diamine oxidase activity and d-lactate content, increased ( P < 0.05) transepithelial electrical resistance, decreased paracellular flux of dextran (4 kDa), and prevented the diquat-induced decrease ( P < 0.05) in the expressions of claudin-1, occludin, and zonula occludens-1. Tributyrin alleviated ( P < 0.05) diquat-induced mitochondrial dysfunction shown by lowered reactive oxygen species, increased mitochondrial membrane potential, and increased adenosine triphosphate content. Furthermore, tributyrin increased ( P < 0.05) expressions of mitophagy proteins (PTEN-induced putative kinase 1 and Parkin), and ratio of light chain 3-II to light chain 3-I in intestine. Collectively, tributyrin attenuated oxidative stress and intestinal inflammation, improved mitochondrial function, and induced mitophagy in diquat-challenged pigs.

Resveratrol improves intestinal barrier function, alleviates mitochondrial dysfunction and induces mitophagy in diquat challenged piglets1.

This study evaluated whether resveratrol can alleviate intestinal injury and enhance the mitochondrial function and the mitophagy level in diquat induced oxidative stress of piglets. Twenty-four 35 day old piglets were randomly allocated to four groups: (1) nonchallenged control; (2) control + 100 mg kg-1 resveratrol diet; (3) diquat-treated control and (4) diquat + 100 mg kg-1 resveratrol diet. At the beginning of the experiment, the piglets were injected with diquat or saline (10 mg per kg bodyweight). On day 14, the piglets were killed to obtain the jejuna segments. The results showed that resveratrol increased (P < 0.05) the total antioxidant capacity (T-AOC) and decreased (P < 0.05) the hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels in the jejunal mucosa, in comparison with the diquat group. Resveratrol improved (P < 0.05) the intestinal barrier function, as indicated by the increased transepithelial electrical resistance and the decreased paracellular permeability of fluorescein isothiocyanate dextran 4 kDa in the jejunum. Moreover, resveratrol prevented (P < 0.05) the diquat induced decline of occludin, claudin-1 and ZO-1 levels in the jejunal mucosa. Resveratrol ameliorated mitochondria swelling, vacuolation and cracked cristae induced by diquat. Resveratrol decreased (P < 0.05) the ROS generation and increased (P < 0.05) the membrane potential of intestinal mitochondria, the content of mitochondrial DNA and the activity of mitochondrial complexes I-IV in the jejunum. Finally, resveratrol enhanced (P < 0.05) the level of PTEN induced putative kinase 1 (PINK1) and Parkin in intestinal mitochondria; meanwhile it increased (P < 0.05) the LC3-II/LC3-I ratio in the jejunum. These data indicate that resveratrol is effective in protecting the intestinal barrier, improving the redox status, alleviating mitochondrial damage and inducing mitophagy in piglets challenged with diquat.

Association of MyoD1 Gene Polymorphisms with Meat Quality Traits in Domestic Pigeons (Columba livia).

Myogenic differentiation 1 (MyoD1) belongs to the MyoD family and plays a key role in myogenesis and consequently, in determining muscle fiber characteristics. In this study, single nucleotide polymorphisms (SNPs) in the exons of MyoD1 were identified in 200 domestic pigeons (Columba livia) by direct DNA sequencing, and the association between MyoD1 polymorphisms and meat quality traits was analyzed. We found four novel variations (A2967G, G3044A, A3164C, and C3311G) in exon 3. The SNP A2967G is a synonymous mutation, while the other 3 SNPs are located in the 3' untranslated region. The analysis revealed 3 genotypes, in which allele A was the predominant allele in the SNP A2967G, while allele B was the predominant allele in the SNPs G3044A and A3164C. The mutations A2967G and G3044A were significantly associated with meat quality traits in pigeon. Pigeons with AA or AB genotypes had higher breast muscle concentrations of inosinic acid and intramuscular fat than those of BB genotype. Moreover, these 2 SNPs had significant effects on MyoD1 mRNA expression. The SNPs A2967G and G3044A were organized into 4 haplotypes, which formed 7 diplotypes. Association analysis showed that the diplotypes were not significantly associated with meat quality traits. Our results implied that associations do exist between MyoD1 gene polymorphisms and meat quality traits in domestic pigeons, and the AA and AB genotypes could be applied as genetic markers in marker-aid pigeon breeding.

LPS challenge increased intestinal permeability, disrupted mitochondrial function and triggered mitophagy of piglets.

Here we investigated the influence of LPS-induced gut injury on antioxidant homeostasis, mitochondrial (mt) function and the level of mitophagy in piglets. The results showed that LPS-induced intestinal injury decreased the transepithelial electrical resistance, increased the paracellular permeability of F1TC dextran 4 kDa, and decreased the expression of claudin-1, occludin and zonula occludens-1 in the jejunum compared with the control group. LPS decreased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and increased the content of malondialdehyde in the jejunum. Meanwhile, the expression of SOD-related genes ( Cu/Zn-SOD, Mn-SOD) and GSH-Px-related genes ( GPX-1, GPX-4) declined in LPS-challenged pigs compared with the control. LPS also increased TNF-α, IL-6, IL-8 and IL-1ß mRNA expression. LPS induced mt dysfunction, as demonstrated by increased reactive oxygen species production and decreased membrane potential of intestinal mitochondria, intestinal content of mt DNA and activities of the intestinal mt respiratory chain. Furthermore, LPS induced an increase in expression of mitophagy related proteins, PTEN-induced putative kinase (PINK1) and Parkin in the intestinal mitochondria, as well as an enhancement of the ratio of light chain 3-II (LC3-II) to LC3-I content in the jejunal mucosa. These results suggested that LPS-induced intestinal injury accompanied by disrupted antioxidant homeostasis, caused mt dysfunction and triggered mitophagy.

Response to weaning and dietary L-glutamine supplementation: metabolomic analysis in piglets by gas chromatography/mass spectrometry.

A novel metabolomic method based on gas chromatography/mass spectrometry (GC-MS) was applied to determine the metabolites in the serum of piglets in response to weaning and dietary L-glutamine (Gln) supplementation. Thirty-six 21-d-old piglets were randomly assigned into three groups. One group continued to suckle from the sows (suckling group), whereas the other two groups were weaned and their diets were supplemented with 1% (w/w) Gln or isonitrogenous L-alanine, respectively, representing Gln group or control group. Serum samples were collected to characterize metabolites after a 7-d treatment. Results showed that twenty metabolites were down-regulated significantly (P<0.05) in control piglets compared with suckling ones. These data demonstrated that early weaning causes a wide range of metabolic changes across arginine and proline metabolism, aminosugar and nucleotide metabolism, galactose metabolism, glycerophospholipid metabolism, biosynthesis of unsaturated fatty acid, and fatty acid metabolism. Dietary Gln supplementation increased the levels of creatinine, D-xylose, 2-hydroxybutyric acid, palmitelaidic acid, and α-L-galactofuranose (P<0.05) in early weaned piglets, and were involved in the arginine and proline metabolism, carbohydrate metabolism, and fatty acid metabolism. A leave-one-out cross-validation of random forest analysis indicated that creatinine was the most important metabolite among the three groups. Notably, the concentration of creatinine in control piglets was decreased (P=0.00001) compared to the suckling piglets, and increased (P=0.0003) in Gln-supplemented piglets. A correlation network for weaned and suckling piglets revealed that early weaning changed the metabolic pathways, leading to the abnormality of carbohydrate metabolism, amino acid metabolism, and lipid metabolism, which could be partially improved by dietary Gln supplementation. These findings provide fresh insight into the complex metabolic changes in response to early weaning and dietary Gln supplementation in piglets.