| The piglets are commonly weaned early to enhance the productive efficiency in modern pig farms. However, early weaning often causes stress in piglets, which exerts serious influence on the growth and metabolism. In the present study, time-related changes of amino acid (AA) profiles in weanling piglets were assessed based on metabonomic method, the effects of dietary L-glutamine (Gln) supplementation on nutrient digestibility and metabolism were estimated, and the mechanism regarding Gln inhibiting oxidant-induced apoptosis in enterocyte was also discussed. The main contents and results in the current study are as follows:Trial1. The time-course of serum AA following response to early weaning was investigated over a30d period in weanling piglets using AA analyzer. Eight21-d old piglets were selected and their blood samples were collected on d0,1,3,5,7,10,15, and30post-weaning. Serum concentrations of arginine (Arg) and its immediate precursors (citrulline and ornithine) decreased (P<0.05) from d0to5post-weaning, and increased from d7to30post-weaning. Serum concentrations of leucine, proline (Pro), tyrosine (Tyr), and taurine decreased (P<0.05) on d1and3post-weaning, and increased to the level of d0post-weaning on d30. Serum concentrations of isoleucine, lysine (Lys), glycine, and serine were lowest on d5post-weaning; histidine, phenyalaine, valine, aspartate, and serine were lowest on d10post-weaning, which then increased thereafter. Methionine concentration changed slightly (P>0.05) from d0to3post-weaning, while it increased by43.51%(P<0.05) and41.11%(P<0.05) on d5compared with that on d1and3post-weaning, and kept constant thereafter. Serum concentration of threonine increased by31.67%(P<0.05) and79.06%(P<0.05) on d3post-weaning compared with that on d0and1, and then decreased on d15and30post-weaning. Serum concentration of Gln and dutamatc (Glu) were relatively higher (P<0.05) from d3to5, and d15to30than that on d0and1post-weaning. Total AA was relatively higher (P<0.05) on d0than those from d1to30post-weaning, and it decreased from d1to5, then increased on d7and keep constant thereafter. In score plot of principal component analysis, the profiles of AA were gradually away from the profile of AA on d0post-weaning, and then were closed to that, and tended to be stable. On d3and5postweaning, the profiles of AA were robustly altered. The pattern of partial least-square discriminant analysis demonstrated that Gln and Glu played the most important role in differentiating the profiles of AA at different time point post-weaning.Trial2. 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 Gln supplementation. Thirty-six21-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 with1%Gln (wt:wt) or isonitrogenous L-alanino (Ala), respectively, representing Gln group or control group. Serum samples were collected to characterize metabolites after a7-d treatment. Results showed twenty metabolites were down-regulated significantly (P<0.05) in control piglets compared with suckling ones. These data demonstrate that early weaning causes a wide range of metabolic changes across Arg and Pro 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 a-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 (LOOCV) 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 decreased754.58%(P=0.00001) compared to the suckling piglets, and increased447.39%(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, AA 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.Trial3. Gln has an essential role with a beneficial function in improving the nutrition status of young mammals. The influence of Gln on the coefficient of total tract apparent digestibility (CTTAD) and apparent ileal digestibility (AID), the jejunal enzyme activity associated with nutrient absorption, and the energy production in weaned piglets has not been sufficiently studied. The aim of the present paper is to provide a profile of the effects of Gln on CTTAD, AID, the activities of jejunal enzymes in connection with nutrient digestion and absorption and energy production. The piglets were weaned at21d of age. There were two groups in Exp.1and Exp.2, representing supplementation with0or1%Gln to the basal diet. In Exp.1, the CTTAD of the dietary components and energy was assessed at3,5,10,15, and30d after weaning. In Exp.2, productive performance, AID, jejunal enzyme activities, and expression of peroxisome proliferator-activated receptor gamma (PPARy), mammalian target of rapamycin (mTOR), and pyruvate kinase (PK) were measured at10and30d post-weaning. Results showed that dietary Gln supplementation significantly improved (P<0.05) the CTTAD of DM, OM, GE, and AA, and the CTTAD increased significantly (P<0.05) with the increasing days after weaning. For the entire experiment, the average daily gain increased by12.40%(P=0.049) in the Gln group. Dietary Gln supplementation increased the AID of GE, Leu, Lys, Cys, and Pro by12.50%(P=0.047),7.03%(P=0.041),5.95%(P=0.036),9.30%(P=0.025),11.17%(P=0.009), respectively, at10d post-weaning; Pro by6.11%(P=0.044) at30d post-weaning. Jejunal brush border membrane-bound alkaline phosphatase activity increased in the Gln-supplemented pigs by30.36%(P=0.048) and6.21%(P=0.30) at10and30d post-weaning, respectively. Compared with the control pigs, the mRNA level of PPARy decreased by10.85%(P=0.14) and41.88%(P=0.023) after the administration of1%Gln for10and30d, respectively. The mRNA level of mTOR increased by22.10%(P=0.061) and22.28%(P=0.042), respectively. The activity of Gln synthetase decreased by48.89%(P=0.044) at10d post-weaning, and pyruvate kinase (PK) by13.13%(P=0.036) at30d post-weaning in the Gln-supplemented pigs. The Ala aminotransferase activity was numerically elevated (P=0.076) in the Gln group. The mRNA level of PK decreased by29.75%(P0.062) and44.40%(P=0.039) for the Gln supplementation for10and30d, respectively. In conclusion,1%Gln supplementation to the post-weaned piglet diet enhanced the CTTAD and AID of diet, improved intestinal absorption, and modified jejunal enzyme activities related to Gln metabolism and energy production.Trial4. A novel metabolomic method based on GC-MS was applied to investigate serum metabolites in response to dietary Gln supplementation in piglets. Sixteen21-d-old pigs were weaned and randomly assigned into treatments, which were1) Gkn supplementation and2) control, representing1%Gln (wt:wt) and isonitrogenous Ala, respectively. Serum samples were collected to characterize metabolites after a30-d treatment. Additionally,4liver samples per treatment were collected to examine enzyme activity and gene expression involved in metabolic regulation. Results indicated that12metabolites were altered (P<0.05) by Gln treatment, including carbohydrates, AA, and fatty acids. A LOOCV of random forest analysis indicated that Pro was the most important metabolites among the12different metabolites. These data demonstrate that the control and Gln-supplemented pigs differed (P<0.05) in terms of metabolism of carbohydrates, Pro.Tvr. and glycerophospholipids. Principal component analysis yielded separate clustering of profiles between the Gin and control groups. Metabolic enzyme activities of Ala aminotransferase and hexokinase increased by26.77%(P=0.026) and26.18%(P=0.004) in liver of Gln-supplemented pigs over the control, respectively, whereas PK activity decreased by29.08%(P=0.001). The gene expression of PK in liver deereased by66.10%(P=0.034) by Gln treatment for30d. No differences were observed for the mRNA levels of mTOR and PPARy. Based on the data, correlation network for the Gln-supplemented pigs and the control pigs indicated that Gin treatment affected carbohydrate, lipid and△A metabolism in the whole body of the early weaned piglets. These findings provide fresh insight into specific metabolic pathways and lay the groundwork for the complex metabolic alteration in response to dietary Gln supplementation in pigs.Trial5. This experiment tested the hypothesis that Gln prevents oxidant-induced death of enterocytes. HT-29cells were cultured for12h,24h, and32h, respectively, in Gln-free Dulbecco’s modified Eagle’s-F12Ham medium containing0,0.5,2.0or10.0mM Gln, and0.35mM H2O2. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) formation were slightly changed (P>0.05) after administration of Gln for12h, while SOD activity increased and MDA formation decreased (P<0.05) for24h and32h. Administration of0.5and2.0mM Gln decreased mRNA level of FAS, Caspase-3, NF-kB, and Bax (P<0.05), and increased Bcl-2mRNA level, whereas administration of10.0mM Gln increased mRNA level of FAS, Caspase-3, NF-kB, and Bax (P<0.05) at32h after incubation. The live cells increased by5.32%～11.97%at24h after administration of Gln, and by1.39%～7.63%at32h. Therefore, these results suggest that Gln can promote antioxidant capacity and anti-apoptosis gene expression in enterocytes, and reduce pro-apoptosis gene expression to inhibit oxidant-induced apoptosis.In conclusion, early weaning influences AA, protein, carbohydrate, and lipid metabolism in piglets. Dietary1%Gln supplementation inhibits oxidant-induced apoptosis of enterocytes, increases nutrient digestibility, regulate the metabolic activities of intestine and liver, which results in the improvement of body metabolism in young pigs. |
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