Effect Of Fermented Cottonseed Meal With Yeast On Lipid Metabolism Of Broiler Chickens And Its Metabolomic Research

Object:This study was conducted to investigate the effect of fermented cottonseed meal(FCSM) with yeast treatment on lipid metabolism of broiler chickens, and to research the metabolic mechanism of regulation from levels of small molecular metabolism.Methods:The present paper consists of effect of fermented feed on lipid metabolism of broilers and metabolomics studies. Part one. Based on growth performance, apparent digestibility and carcass traits, the effect of fermented feed on lipid metabolism of broiler chickens was study from the levels of organization, cellular and molecule. A total of 300 1-d-old Chinese yellow-feathered chickens were raised in a brooder house for 14 d and fed a commercial diet. Then, the brooders were fed the control diet for seven days to acclimatize powder experimental feeds. Then, 180 birds were selected according to body weight and gender, and randomly divided into three groups consisting of six replicates(cages) each with 10 individuals. The three groups were fed three different diets: either 6% unfermented CSM(groupⅠ); 6% FCSM by C. tropicalis treatment(group Ⅱ); or 6% FCSM by C. tropicalis and S. cerevisae treatment(group Ⅲ). This experiment consisted of a starter phase(21-42 days) and a finisher phase(43-64 days). At the raising end of each stage, serum, liver tissue, and abdominal fat tissue were sampled for related research. Part two. Six birds were randomly selected according to body weight used to obtain the serum, liver, and cecum contents for metabolomic analysis based on gas chromatography time of flight mass spectrometry(GC-TOF/MS). The dates obtained from GC-TOF/MS were used for the principal component analysis(PCA), partial least squares-discriminant analysis(PLS-DA), and orthogonal partial least squares-discriminant analysis(OPLS-DA). Both the variable importance in the projection(VIP) values(VIP>1) obtained from the OPLS-DA and the Student’s T test(P<0.05) were employed to find deferentially expressed metabolites between the two comparison groups. The difference metabolites were identified and analysis of metabolic process by LECO/Fiehn database, NIST, Wiley library and KEGG database.Results:Part one.(1) FCSM supplementation improved the average daily gain(ADG) during the 43 rd to 64th(P<0.05), and decreased feed-to-gain ratio during the whole stage(P<0.05). The dietary nutrient digestibility of dry matter(DM), crude protein(CP), and crude ash(Ash) significantly increased(P<0.05) by FCSM supplementation, and significant difference was observed between treated groups(P<0.05).(2) The contents of abdominal fat(d 42) were lower in treatment group than in control group(P<0.05). Compared with control group, the contents breast muscle(d 42) and linoleic acid in thigh muscle(d 64) increased in treatment group(P<0.05). FCSM supplementation decreased the CP content, and increased EE in thigh muscle(d 42 and d 64), respectively(P<0.05). In addition, composition of fatty acid was no significant difference after adding FCSM(d 42)(P>0.05). The contents of oleic acid in the breast muscle(d 42) and thigh muscle(d 64) were lower in group than in groupⅢ Ⅰ(P<0.05), and EE of breast muscle(d 42 and d 64) in group Ⅲ increased compared to control(P<0.05). The contents stearic acid and linoleicacid of breast muscle and oleic acid of thigh muscle in group Ⅱ were lower than in control group at 64 days of age(P<0.05). The content of oleic acid, ratio of unsaturated fatty acid to saturated fatty acid, and CP of breast muscle were higher in group Ⅱ than in groupⅠduring the whole stage(P<0.05).(3) FCSM supplementation decreased significantly triglyceride(TG) level in serum(P<0.05). Compared with control group, the concentration of total cholesterol(TC) and low density lipoprotein cholesterol(LDL-C) in serum decreased and increase at the 42 rd and 64 th days of age in treatment groups, respectively(P<0.05). At 42 days of age, enzyme activity of acetyl-coa carboxylase(ACC), lipoprotein lipase(LPL), and hormone sensitive esterase(HSL) increased significantly in experimental group than in control group(P<0.05).(4) Dietary FCSM supplementation significantly down-regulated the m RNA levels of ACC and fatty acid synthase(FAS) in liver and LPL in abdominal fat tissue(d 42)(P<0.05). The gene expression of peroxisome proliferator-activated receptor alpha(PPAR-α) and LPL in liver tissue were significantly up-regulated at 64 days of age(P<0.05). Part two.(1) Dietary FCSM supplementation caused significant changes of small molecule metabolites in the serum of broiler chickens. Total of 21 metabolites(d 42) and 25 metabolites(d 64) were altered by adding FCSM by C. tropicalis treatment compared with control. In addition, total of 22 metabolites(d 42) and 20 metabolites(d 64) were altered in FCSM by C. tropicalis and S. cerevisae treatment group than in control group. These metabolites involved multiple biochemical metabolism pathways such as metabolism of carbohydrates, lipids, amino acids, vitamins, nucleotide, and microbe. Alteration of multiple metabolic pathways in carbohydrates metabolism was mainly involved in fructose and mannose metabolism, galactose metabolism, glycolysis and tricarboxylic acid cycle. Amino acid metabolism was altered including glycine, tyrosine, ornithine, and phenylalanine. In terms of lipid metabolism, contents of palmitoleic acid(d 42) increased, and levels of D-erythro-sphingosine(d 64) and stearic acid during whole period(d 42 and d 64) decreased. In addition, the levels of dihydroxyacetone, glyceric acid, glycerol-1-phosphate, myo-inositol, ethanolamine, and O-phosphorylethanolamine were significant difference between two groups of adding FCSM(P<0.05).(2) FCSM caused the significant alteration of small molecule metabolites in the liver tissue of broiler chickens. Total of 9 metabolites(d 42) and 11 metabolites(d 64) were altered by adding FCSM by C. tropicalis treatment compared with control. In addition, total of 9 metabolites(d 42) and 12 metabolites(d 64) were altered in FCSM by C. tropicalis and S. cerevisae treatment group than in control group. These metabolites involved different metabolic pathway including the carbohydrates metabolism(e.g. fructose and mannose metabolism, galactose metabolism, Kreb’s cycle, starch and sucrose metabolism), amino acid(serinem, tryptophan, ornithine, and creatine) metabolism, and lipid metabolism. In terms of lipid metabolism, the contents of 2-monoolein and myo-inositol decreased, and 1-monopalmitin and ethanolamine increased at 42 days of age. The levels of arachidonic acid, prostaglandin E2, and β-glycerophosphoric acid were significant difference between two groups of adding FCSM(P<0.05).(3) Metabolomics analysis of the cecum contents showed that FCSM supplementation mainly caused metabolites alteration of amino acids and 2-oxocarboxylic acid metabolism(e.g. 2-ketobutyric acid, 2-oxoadipate, isoleucine, and citraconic acid). The difference metabolites(citraconic acid, isoleucine, lipoic acid, and N-methyl-L-glutamic) between two treatment groups were also presented because of different strains used for fermentation.Conclusion:FCSM with yeast treatment has a potential regulation effect on lipid metabolism for broiler chickens, especially can reduce the contents of abdominal fat and TG in stater growth stage(d 42), and enhance the EE content in whole period. Dietary FCSM supplementation decrease the fat synthesis by down-regulating m RNA expression of hepatic ACC and FAS during the starter period. Fatty acid oxidation and TG catabolism are regulated by increasing PPAR-α and LPL expression throughout the finisher stage.Small molecule metabolic analysis of serum, liver, and cecum content showed that FCSM alter the metabolic process including carbohydrates metabolic pathway(fructose and mannose metabolism, galactose metabolism, glycolysis and tricarboxylic acid cycle), lipids(fatty acid, glycerolipids and glyceryl phosphatide) metabolism, and amino acids(serine and tyrosine, ornithine and phenylalanine) metabolism accompanied with strengthening gut microbial metabolism, which mainly reflects in the increase of energy and amino acid,and lipid utilization. Therefore, the regulation of fermented feed on lipid metabolism for poultry is a complicated process involving multiple biochemical pathways, and the control effect is difference for different strains used for fermentation.