Tissue lipid metabolism and hepatic metabolomic profiling in response to supplementation of fermented cottonseedmeal in the diets of broiler chickens

Tissue lipid metabolism and hepatic metabolomic profiling in response to supplementation of fermented cottonseedmeal in the diets of broiler chickens 肉鸡日粮中添加发酵棉粕对其组织脂类代谢及肝脏代谢谱的影响 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhangwj1022@sina.com †E-mail:zhangwj1022@sina.com zhangwj1022@sina.com College of Animal Science and Technology, Shihezi University, Shihezi 832003, China College of Animal Science and Technology, Shihezi University, Shihezi 832003, China This study investigated the effects of fermented cottonseed meal (FCSM) on lipid metabolites, lipid metabolism-related gene expression in liver tissues and abdominal adipose tissues, and hepatic metabolomic profiling in broiler chickens. One hundred and eighty 21-d-old broiler chickens were randomly divided into three diet groups with six replicates of 10 birds in each group. The three diets consisted of a control diet supplemented with unfermented cottonseed meal, an experimental diet of cottonseed meal fermented by Candida tropicalis , and a second experimental diet of cottonseed meal fermented by C. tropicalis plus Saccharomyces cerevisae . The results showed that FCSM intake significantly decreased the levels of abdominal fat and hepatic triglycerides ( P < 0.05 for both). Dietary FCSM supplementation down-regulated the mRNA expression of fatty acid synthase and acetyl CoA carboxylase in liver tissues and the lipoprotein lipase expression in abdominal fat tissues ( P < 0.05 for both). FCSM intake resulted in significant metabolic changes of multiple pathways in the liver involving the tricarboxylic acid cycle, synthesis of fatty acids, and the metabolism of glycerolipid and amino acids. These findings indicated that FCSM regulated lipid metabolism by increasing or decreasing the expression of the lipid-related gene and by altering multiple endogenous metabolites. Lipid metabolism regulation is a complex process, this discovery provided new essential information about the effects of FCSM diets in broiler chickens and demonstrated the great potential of nutrimetabolomics in researching complex nutrients added to animal diets. 目的 2 旨在研究发酵棉粕对肉鸡脂类代谢的影响。 创新点 2 将荧光定量聚合酶链反应PCR和代谢组学方法相结合，就采食发酵饲料（发酵棉粕）肉鸡的组织脂类代谢相关基因及肝脏代谢谱进行分析，发现发酵棉粕具有调控肉鸡脂类代谢的作用。该研究为研究日粮中复杂营养素对动物机体代谢的调控提供了新思路。 方法 2 将180只21日龄肉鸡随机分为三组，每组6个重复，每个重复10只鸡。对照组饲喂未发酵棉粕底物，试验组分别饲喂热带假丝酵母发酵棉粕和热带假丝酵母与酿酒酵母复合发酵棉粕。饲喂21天后，采集肉鸡腹部脂肪和肝脏进行组织基因表达和肝脏代谢组学分析。 结论 2 发酵棉粕可通过下调肝脏FAS、ACC和腹脂LPL等基因的表达，改变肝脏多种内源性代谢的代谢途径，进而降低肉鸡腹部脂肪沉积和肝脏甘油三酯含量。 Fermented cottonseed meal Lipid metabolism Broiler Gene expression Metabolomics 发酵棉粕 脂类代谢 肉鸡 基因表达 代谢组学 Introduction 1 Cottonseed meal (CSM), a major protein source used in animal feed for many years in China, has good nutritive value (Yin et al., 1993 1993 ; 1994 1994 ). The utilization of CSM is limited in poultry diets due to the presence of gossypol, a toxic polyphenol. However, to solve this problem, solid-state fermentation is an effective option (Zhang et al., 2006 2006 ; 2007 2007 ). Additionally, CSM by solid-state fermentation improves nutrition characteristics (Zhang et al., 2006 2006 ; Sun et al., 2012 2012 ; Yang et al., 2012 2012 ) and produces many essential nutrients such as amino acids, small-size peptides, vitamins, and oligosaccharides (Tang et al., 2012 2012 ). Yeast, a unicellular fungus, can be used as a microbial feed additive for preventing disease and encouraging growth (Koh et al., 2002 2002 ). Candida tropicalis and Saccharomyces cerevisae were used in CSM fermentation by Zhang et al. ( 2007 2007 ), who found that the two yeast strains were very effective in detoxifying free gossypol in CSM. In addition, CSM fermented by C. tropicalis increased the levels of total amino acids, essential amino acids, and in vitro digestible crude protein and amino acids (Zhang et al., 2007 2007 ). Koh et al. ( 2002 2002 ) reported that S. cerevisae and fermented rice bran resulted in various biological activities in feed additives such as strengthening the immune system and reinforcing stress-fighting capabilities. Some studies have reported that fermented CSM (FCSM) improves growth performance (Tang et al., 2012 2012 ) and gut microbes (Sun et al., 2013 2013 ) in broiler chickens. However, little information is known about the influence of fermented CSM on lipid metabolism in broilers. Lipid metabolism is closely associated with the nutritional value of meat, the yield of lean meat, or efficient feed utilization (de Genova Gaya et al., 2005 2005 ; Zhao et al., 2007 2007 ). Furthermore, excess fat deposition results in waste for consumers or economic loss for producers. Previous studies have indicated that fermented products can decrease fat deposition and improve lipid profiles (Park et al., 2012 2012 ; Cha et al., 2013 2013 ). In broiler chickens, the liver is the most important organ for lipid metabolism, and lipids are primarily stored in adipose tissue (Huang et al., 2008 2008 ). Fat deposition results from a complex interactive process that involves genetic and environmental factors (Hausman et al., 2009 2009 ; An et al., 2013 2013 ). Metabolomics can comprehensively reveal the change rule and related mechanism of the biosystem affected by exogenous substances (Patti et al., 2012 2012 ); thus, it is useful to understand the lipid metabolism of broilers when fermented feed is added to their diets. Metabolomics has been successfully applied to better understand the effects of organic feed on chickens (Huber et al., 2010 2010 ), and has also been used to research the metabolism of adipose tissue of chickens (Ji et al., 2012 2012 ). This study investigated the effects of fermented CSM on lipid-related gene expression in tissue and liver metabolomics changes based on gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) in yellow-feathered broiler chickens. This valuable commercial broiler species has good meat quality and its production approaches three billion animals every year in China (Tang et al., 2012 2012 ). The aim of this study was to understand the effects of fermented CSM intake on the lipid metabolism of broiler chicken tissue and its mechanisms. Materials and methods 1 Substrate treatment and strains 2 The CSM used in this study was obtained from the Shihezi District (Xinjiang Uygur Autonomous Region of China). The substrate treatment material was performed according to the methods of Zhang et al. ( 2007 2007 ). The strains C. tropicalis and S. cerevisae were bred and collected by the Feed Science Institute of Zhejiang University (Hangzhou, China). These two strains were maintained in yeast extract peptone dextrose medium. Preparation of fermented CSMs 2 The treated substrate (1000 g) was inoculated with 80 ml of liquid yeast inocula (10 8 cells/ml) of C. tropicalis or both strains (the ratio of C. tropicalis to S. cerevisae was 3:7). Every inoculated substrate was blended evenly, encased in a plastic container (50 cm×30 cm×12 cm) and incubated at 30 °C for 48 h in an incubator. After fermentation, the substrate was dried at 40 °C for 48 h in a draught drying cabinet. The same treatment process was performed for the unfermented CSM except that the same volume of sterile culture medium was inoculated. The chemical compositions of the unfermented CSM, the CSM fermented by C. tropicalis (Ct CSM), and the CSM fermented by C. tropicalis plus S. cerevisae (Ct-Sc CSM) were analyzed (AOAC, 1999 1999 ) on a dry matter basis as follows: dry matter, 953, 946, and 947 g/kg; crude protein, 342, 376, and 375 g/kg; ether extract, 7.1, 7.2, and 7.4 g/kg; crude ash, 53.2, 57.6, and 58.5 g/kg in unfermented CSM, Ct CSM, and Ct-Sc CSM, respectively. The free gossypol contents, determined by a standard method (AOCS, 2009 2009 ), were 126.7, 44.9, and 33.0 mg/kg, respectively. The concentration of free gossypol was less than 30 mg/kg in the diet and had no effect on the broilers’ health (Özdoğan et al., 2012 2012 ; Tang et al., 2012 2012 ). Broiler breeding 2 The animal care and use protocol was approved by the Animal Welfare Committee of Shihezi University (Xinjiang Uygur Autonomous Region of China). A total of 300 1-d-old Chinese yellow-feathered chickens were raised in a brooder house for 14 d fed a commercial diet. Then, the birds were fed the control diet for 7 d to acclimatize the feeds because the trial diets were afforded in mash form. Next, the birds underwent fasting for 12 h and were then weighed. After that, 180 birds were chosen from the 300 total birds according to their similar weight and randomly divided into three groups, each with 60 chickens. The chickens were raised in wire-floored cages (120 cm×80 cm×50 cm) located in the same room. Ten chickens were kept in each cage of six cages from each group, and each cage was equipped with two nipple water bottles and one feeder. The diets of the three groups included the unfermented CSM (control group, Con), the Ct CSM (treatment group 1, Ct), and the Ct-Sc CSM (treatment group 2, Ct-Sc). The yeast levels were 2×10 6 colony-forming units (CFU) per g in the FCSM diet. Composition of the diets and nutrient levels are presented in Table 1 1 . The birds were housed in conditions with continuous light and ad libitum access to feed and water throughout the rearing period, and the temperature was maintained at 23 to 25 °C during the ages of 21 to 42 d. CSM: cottonseed meal; ME: metabolizable energy; AP: available phosphorus; Met: methionine; Cys: cysteine; Lys: lysine; Con: control group with unfermented cottonseed meal; Ct: treatment group with cottonseed meal fermented by C. tropicalis ; Ct-Sc: treatment group with cottonseed meal fermented by C. tropicalis plus S. cerevisiae . Premix (per kg of diet): L -lysine·HCl 2 g, D L -methionine 1.1 g, NaCl 3 g, choline chloride (50%) 1 g, Cu 6 mg, Fe 100 mg, Mn 150 mg, Zn 100 mg, Se 0.3 mg, I 0.4 mg, vitamin A 12 500 IU, vitamin D 3 4000 IU, vitamin E 18 IU, menadione 3.0 mg, thiamin 2.0 mg, riboflavin 7.5 mg, pyridoxine 4.5 mg, vitamin B 12 0.02 mg, pantothenic acid 12 mg, niacin 50 mg, folic acid 1.2 mg, biotin 0.15 mg. Nutrient contents are calculated values except for crude protein and calcium Data for ME are in MJ/kg 2 Ingredient 3 Percentage (%) 2 Nutrient content 3 Percentage (%) Con Ct Ct-Sc Con Ct Ct-Sc  Yellow corn 64.35 64.70 64.65  ME (MJ/kg) 11.94 * * 11.96 * * 11.95 * *  Soybean meal 17.15 16.80 16.85  Crude protein 18.70 18.73 18.75  Rapeseed meal 2.00 2.00 2.00  Calcium 0.87 0.85 0.84  Ordinary CSM 6.00 6.00 6.00  AP 0.36 0.34 0.35  Unfermented CSM 6.00  Met+Cys 0.68 0.68 0.68  Fermented CSM 6.00 6.00  Lys 1.04 1.03 1.03  Cottonseed oil 1.00 1.00 1.00  Dicalcium phosphate 1.20 1.20 1.20  Limestone 1.30 1.30 1.30  Premix 1.00 1.00 1.00 Sample collection 2 Six birds near the average weight (1276.81 g) were randomly selected from each group, and the 18 birds were killed by cervical dislocation to sample the abdominal fat (from the gizzard to the cloaca) and livers. Abdominal fat was calculated as the percentage of abdominal fat weight relative to the total weight of the carcass. About 5 g abdominal fat pared from the left sides and liver tissues sampled from left lobes were collected from birds, immediately frozen in liquid nitrogen, and then stored at −80 °C for further analysis. Lipid metabolite assays of the livers 2 The concentrations of triglycerides (TG), total cholesterol (TC), and total protein in the livers were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions with a microplate reader (Thermo Labsystems MK3, Helsinki, Finland). Total protein content was used to calculate the concentrations of TG and TC. RNA isolation and real-time quantitative polymerase chain reaction (qPCR) analysis 2 Total RNAs of the livers and abdominal fat were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and electrophoresed on a 1% (0.01 g/ml) denaturing agarose gel to test the integrity. All RNA samples were adjusted to the same concentration. The RNA was reverse transcribed to complementary DNA (cDNA) by PrimeScript™ RT reagent kits (TaKaRa, Shiga, Japan) according to the instructions. A real-time quantitative PCR was carried out in a 7500 Real Time PCR systems (Applied Biosystems, Foster City, CA, USA) using a SYBR-Green PCR kit (Roche Diagnostics, Laval, Quebec, Canada) according to optimized PCR protocols. The PCR programme was as follows: 95 °C for 2 min, followed by 32 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and 72 °C for 10 min. The gene primers of fatty acid synthase (FAS), lipoprotein lipase (LPL), acetyl CoA carboxylase(ACC), peroxisome proliferator-activated receptor α (PPAR-α), peroxisome proliferator-activated receptor γ (PPAR-γ), and β-actin were designed as described by Wu ( 2012 2012 ), and liver fatty acid-binding protein (L-FABP) was designed as the chicken sequences from GenBank. The sequences of the primers were follows: FAS forward, 5'-TCAGGGTGTTCTGGAATGCAA-3', reverse, 5'-AATCCTGGTGGGCAATCGTAG-3'; LPL forward, 5'-AGTCAGAGTGAAGTCAGGCGAAAC-3', reverse, 5'-CTGCTCCAGGCACTTCACAAATA-3'; ACC forward, 5'-CTGATGGTCTTTGCCAACTGGA-3', reverse, 5'-CACGATGTAGGCACCAAACTTGA-3'; PPAR-α forward, 5'-TGCACTGGAACTGGATGATAGTGA-3', reverse, 5'-TCCTACATTTACAAGACCAGGACGA-3'; PPAR-γ forward, 5'-TGTGAAGTTCAACGCACTGGAATTA-3', reverse, 5'-GGAGCTCCAAAGCTTGCAACA-3'; L-FABP forward, 5'-ATGAGCTTCACTGGAAAGTACGAG-3', reverse, 5'-TCTTGATGTCCTTACCCTTCTGG-3'; β-actin forward, 5'-ATTGTCCACCGCAAATGCTTC-3', reverse, 5'-AAATAAAGCCATGCCAATCTCGTC-3'. All samples were analyzed in triplicate and results were expressed as 2 −∆∆ C T according to a previous method (Livak and Schmittgen, 2001 2001 ). Liver metabolomics profiling analysis 2 The samples of liver tissues (100 mg) were weighed in 2-ml tubes. Chilled extraction liquid (400 μl, the volume ratio of methanol to chloroform was 3:1) at −80 °C and internal standard L -2-chlorophenylalanine (50 μl, 0.1 mg/ml) were added to each tube and homogenized in a ball mill for 5 min at 70 Hz. Then, the mixture was centrifuged at 12 000 g for 15 min at 4 °C and 0.35 ml of supernatant was transferred into a fresh 2-ml GC vial, followed by evaporation to dryness under vacuum in a speedvac concentrator (Thermo Fisher Scientific, Asheville, USA). Methoxyamine (80 μl) in pyridine (20 mg/ml) was added to the dried residue and vigorously vortex-mixed for 2 min. Methoximation reaction was performed at 37 °C for 2 h, followed by trimethylsilylation for 1 h by adding 100 μl of bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane as catalyst. At last, the solution was shaken for 1 h at 70 °C and cooled to room temperature. Each sample (1 μl) was injected into an Agilent 7890 GC with an Agilent 7683 Series autosampler (Agilent, Atlanta, GA, USA). Mass spectra were detected and obtained by using Pegasus HT TOF/MS (LECO Corporation, St. Joseph, MI, USA). Helium served as the carrier gas through the column. The optimized GC/MS operating conditions were as follows: the front inlet purge flow was 3 ml/min, and the gas flow rate through the column was 1 ml/min. The initial temperature was kept at 80 °C for 0.2 min, increased to 180 °C at a rate of 10 °C/min, then increased to 240 °C at a rate of 5 °C/min and finally increased to 280 °C at a rate of 20 °C/min for 18.8 min. The injection, transfer line, and ion source temperatures were 280, 270, and 220 °C, respectively. The MS data were acquired in full scan mode over the range between 35 and 600 m/z at a rate of 100 spectra/s. Data collection and processing 2 ChromaTOF 4.3X software (LECO, St. Joseph, MI, USA) and Leco-Fiehn Rtx5 database were used for raw peaks extracting, the data baselines filtering and calibration of the baseline, peak alignment, deconvolution analysis, peak identification, and integration of the peak area (Kind et al., 2009 2009 ). In addition, an internal standard normalization method was employed in the data analysis. The normalized peak area was fed to SIMCA-P 11.5 software (Umetrics, AB, Sweden) for the 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 differentially expressed metabolites between the two comparison groups. The LECO/Fiehn Metabolomics Library for GC/MS metabolome date (similarity value of 70%) was used for discriminating compound annotation. One-way analysis of variance was performed for the data of body weight, lipid content, and gene expression via statistical software SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Differences among means were tested using Duncan’s multiple range tests. A significance level of 0.05 was used. Results 1 Effects of FCSM on body weight and lipid metabolite contents 2 All the broiler chickens remained healthy in appearance throughout the experimental period of the FCSM-supplemented diet. The contents of abdominal fat and hepatic TG were significantly lower in the treatment groups than in the control group ( P < 0.05 for both; Table 2 2 ). However, the body weights and TC concentrations in the liver tissues did not change after adding FCSM to the diet ( P > 0.05 for both). FCSM: fermented cottonseed meal; Con: control group with unfermented cottonseed meal; Ct: treatment group with cottonseed meal fermented by C. tropicalis ; Ct-Sc: treatment group with cottonseed meal fermented by C. tropicalis plus S. cerevisiae . Values are expressed as mean±standard deviation (SD) ( n =6) Means with different letters are significantly different ( P < 0.05) Means with different letters are significantly different ( P < 0.05) Group Initial body weight (g) Final body weight (g) Abdominal fat (%) Triglycerides (mmol/g protein) Total cholesterol (mmol/g protein) Con 540.23±16.26 1 280.00±26.71 2.48±0.11 a a 0.85±0.09 a a 0.57±0.13 Ct 532.03±14.29 1 273.98±22.51 2.01±0.26 b b 0.56±0.09 b b 0.50±0.08 Ct-Sc 535.94±18.66 1 276.45±28.48 2.03±0.39 b b 0.69±0.14 b b 0.59±0.11 Effects of FCSM on gene expression related to lipid metabolism 2 Fig. 1 1 presents the results of gene expression. The gene expression of hepatic ACC and FAS in the treatment groups was significantly decreased compared with that in the controls ( P < 0.05). The FCSM-supplemented diet significantly decreased the LPL mRNA level in abdominal fat tissues ( P < 0.05). However, the expression levels of L-FABP, PPAR-α, and LPL in liver tissue, and PPAR-γ in abdominal fat tissues showed no significant changes among the three groups ( P > 0.05 for all). FCSM: fermented cottonseed meal; Con: control group with unfermented cottonseed meal; Ct: treatment group with cottonseed meal fermented by C. tropicalis ; Ct-Sc: treatment group with cottonseed meal fermented by C. tropicalis plus S. cerevisiae . a, b Means with different letters are significantly different ( P < 0.05). Values are expressed as mean±SD ( n =6) Effects of FCSM on metabolomics profiling in liver tissue 2 FCSM intake caused metabolic changes of endogenous metabolites in the liver tissues of broiler chickens. Results indicated that FCSM supplementation significantly increased the levels of 1-monopalmitin, ethanolamine, and O -phosphoserine but decreased the levels of 2-mono-olein and myo-inositol ( P < 0.05 for all; Tables 3 3 and 4 4 ). The levels of arachidonic acid and glutamine were higher in the Ct group than in the Con group ( P < 0.05 for both; Table 3 3 ), while the levels of citric acid and prostaglandin E2 in the Ct group increased compared with the Con group ( P < 0.05 for both; Table 3 3 ). The Ct-Sc CSM group showed significantly elevated levels of fructose-6-phosphate and glycine, while the levels of 3-hydroxypyruvate and mannose declined ( P < 0.05 for all; Table 4 4 ). Ct CSM: cottonseed meal fermented by C. tropicalis ; VIP: variable importance in the projection. Up arrow (↑) and down arrow (↓) indicate elevation and decrease of metabolite level after adding Ct CSM, respectively No. Metabolite Trend P -value VIP 1 1-Monopalmitin ↑ <0.001 3.56 2 2-Mono-olein ↓ <0.001 1.25 3 Arachidonic acid ↑ 0.039 2.35 4 Citric acid ↓ 0.032 1.29 5 Ethanolamine ↑ 0.007 3.18 6 Myo-inositol ↓ 0.029 1.15 7 Glutamine ↑ 0.028 1.33 8 O -phosphoserine ↑ 0.002 2.15 9 Prostaglandin E2 ↓ 0.028 1.57 Ct-Sc CSM: cottonseed meal fermented by C. tropicalis plus S. cerevisiae ; VIP: variable importance in the projection. Up arrow (↑) and down arrow (↓) indicate elevation and decrease of metabolite level after adding Ct-Sc CSM, respectively No. Metabolite Trend P -value VIP 1 1-Monopalmitin ↑ <0.001 2.08 2 2-Mono-olein ↓ <0.001 1.73 3 3-Hydroxypyruvate ↓ 0.016 3.26 4 Ethanolamine ↑ 0.018 1.72 5 Fructose-6-phosphate ↑ 0.006 2.73 6 Glycine ↑ 0.046 1.03 7 Mannose ↓ 0.020 2.26 8 Myo-inositol ↓ 0.050 1.01 9 O -phosphoserine ↑ 0.049 1.46 Discussion 1 In the present study, the CSM fermented with yeast ( C. tropicalis and S. cerevisiae ) decreased the abdominal fat levels of the broiler chickens, which may have been caused by the fermented products and yeast probiotics of the CSM. Similar results of decreased abdominal fat by adding fermented products and yeast in the broiler chicken diets have been reported by Yamamoto et al. ( 2007 2007 ) and Aluwong et al. ( 2013 2013 ), respectively. A diet containing 1.5%–2.0% yeast (Aluwong et al., 2013 2013 ) or 1% Koji-feed (fermented distillery by-product) (Yamamoto et al., 2007 2007 ) resulted in significantly lower amounts of abdominal fat in broiler chickens. The decreased content of abdominal fat resulted from a decrease of TG synthesis and/or enhancement of TG hydrolysis. The decreased concentrations of hepatic TG revealed that lipid synthesis was reduced when the broilers were fed the FCSM-supplemented diet, which can be associated with the reduction of abdominal fat. This notion is supported by a decrease of fatty acid synthesis because of the down-regulated mRNA expression of FAS and ACC. ACC, a rate-limiting enzyme of lipogenesis, plays a key role in the regulation of fatty acid synthesis (Numa et al., 1971 1971 ). All the steps of conversion from malonyl-CoA to palmitate were catalyzed by FAS, and the FAS gene was a critical target in the control of lipogenesis (Joseph et al., 2002 2002 ). The ACC and FAS have the highest correlation among lipogenic genes linked in lipogenesis (Huang et al., 2008 2008 ). In addition, FCSM intake was not found to alter the lipid transport and lipid oxidation in the present study because L-FABP is involved with lipid transport, and PPAR-α highly expressed in the liver plays a major role in the regulation of peroxisomal β-oxidation (Lee et al., 1995 1995 ). PPAR-γ is expressed at the highest level in adipose tissue and promotes adipocyte differentiation (Mandrup and Lane, 1997 1997 ), and its expression did not change after adding FCSM in the broiler chickens’ diet. LPL, a critical enzyme in the hydrolysis of TG, catalyzes TG from circulating chylomicrons and very-low-density lipoprotein in adipose tissue (Zhao et al., 2013 2013 ). In the present study, the down-regulated mRNA level of LPL in abdominal fat was associated with a decreased fatty acid synthesis, hepatic TG content, and fat deposition in abdominal fat in the chickens fed the diet containing FCSM. Voshol et al. ( 2001 2001 ) reported that overexpression of muscle LPL enhances the accumulation of intramuscular TG and fat in mammals. However, FCSM supplementation did not alter the hepatic LPL expression in the broilers, which may be the result of the decreased fatty acid synthesis. A more specific reason requires further research. Lipid metabolism is a complex process regulated by multiple metabolic pathways, such as the tricarboxylic acid (TCA) cycle, glycolysis, β-oxidation, lipogenesis, and metabolism of amino acids, which can be comprehensively revealed by metabolomics analysis (An et al., 2013 2013 ; Shi et al., 2013 2013 ). Metabolomics, as a systems approach, has been used to understand the lipid metabolism in the process of fat deposition in animal growth (He et al., 2012 2012 ). Comprehensive analysis of metabolite changes in liver metabolic profiling was performed to reveal alterations of the lipid metabolism when broilers were on the FCSM diet in this study. The metabolites involve a variety of metabolic pathways including the TCA cycle, fatty acid synthesis, and metabolism of glycerolipids and amino acids. The levels of citric acids were clearly lower in the Ct group, and the increased levels of fructose-6-phosphate with decreased levels of mannose were observed in the Ct-Sc group compared wtih those in the Con group. Citric acid and fructose-6-phosphate are important intermediates which entered into the TCA cycle and gluconeogenesis to produce energy (Yang et al., 2006 2006 ; Pilvi et al., 2008 2008 ). Citric acid also plays an important role in biosynthesis routes, such as fatty acid biosynthesis in the liver (Liu et al., 2011 2011 ). Mannose is converted into fructose-6-phosphate, which is an immediate precursor to glucose-6-phosphate in gluconeogenesis, and it also further metabolizes to enter the pentose phosphate pathway. This suggested that energy metabolism was altered by adding FCSM to the birds’ diets. However, differences of energy metabolism were also observed between the Ct and Ct-Sc groups associated with CSM fermented by different strains. FCSM was shown to improve the intestinal bacterial microbiota (Sun et al., 2013 2013 ) and further regulate energy metabolism by the gut microbiome (Donohoe et al., 2011 2011 ; Velagapudi et al., 2010 2010 ). The decreased levels of 2-mono-olein and myo-inositol were accompanied by increased levels of ethanolamine and 1-monopalmitin in the treatment groups compared with the Con group. The 2-mono-olein and 1-monopalmitin are monoacylglycerols formed by glycerol combined with a fatty acid molecule, and these changes are believed to be associated with fatty acid synthesis or absorption. Myo-inositol and ethanolamine are the key materials needed for the synthesis of glycerol phospholipid (phosphatidylinositol or phosphatidylethanolamine). The results indicated that the FCSM-supplemented diets caused the complex metabolic effects on the lipids due to the nutritional properties of fermented CSM (Sun et al., 2012 2012 ) or microbial metabolism within the host (Velagapudi et al., 2010 2010 ). A previous study showed that the addition of FCSM in broiler chickens’ diets increased the metabolites of glycerol phospholipids in their plasma, such as phosphatidylcholine, phophatidylethanolamine, cholesterol ester, and sphingomyelin, analyzed by metabolomics technology (Nie et al., 2013 2013 ). He et al. ( 2012 2012 ) reported that the serum of lean pigs had lower myo-inositol levels than that in obese pigs. Similarly, distinct variations of phospholipids and fatty acids were found in blood and liver tissues between obese and lean mice (Kim et al., 2011 2011 ). Moreover, the idea that Ct CSM caused the up-regulation of arachidonic acid is supported by the metabolic differences. Arachidonic acid is a fatty acid combined into phospholipids of the cell membrane (Khairallah et al., 2012 2012 ) or a precursor substance that forms prostaglandin E 2 (Lukivskaya et al., 2001 2001 ). Arachidonic acid suppresses liver fat by influencing the gene expression or activity of lipogenic enzymes (Oikawa et al., 2009 2009 ), and prostaglandin E 2 protects the liver against damage from alcohol use (Lukivskaya et al., 2001 2001 ). A novel finding from this work was that amino acids (glutamine and glycine) and amino acid products (3-hydroxypyruvate and O -phosphoserine) were altered in the broiler chickens’ livers while on the FCSM-supplemented diet. O -phosphoserine is a product of carbon metabolism, and it is a precursor substance that forms many amino acids, such as glycine, serine, threonine, cysteine, and methionine. The significant elevation of O -phosphoserine in the treatment groups showed that broilers fed the control diet needed to generate more amino acid by endogenous metabolism; that was different from the FCSM diet, which included lots of amino acid via microbial fermentation (Zhang et al., 2006 2006 ; Tang et al., 2012 2012 ). This was further supported by the decrease of 3-hydroxypyruvate and increase of glutamine or glycine. The 3-hydroxypyruvate can be converted to O -phosphoserine, which is catalyzed by phosphoserine aminotransferase. Comparison analysis of serum metabolomics indicated that obese pigs show signs of distinct metabolism of proteins and amino acids compared with lean pigs (He et al., 2012 2012 ). Furthermore, it has been found that amino acid supplementation in weaned piglets’ diets can alter multiple metabolite pathways associated with lipid metabolism based on serum metabolomic methods (Xiao et al., 2012 2012 ). Conclusions 1 This study has shown that dietary FCSM supplementation reduced the levels of abdominal fat and liver TG by down-regulating the expression of hepatic lipogenetic genes (ACC and FAS) and LPL in abdominal fat tissue. Moreover, liver metabolomics profiling analysis indicated that dietary FCSM supplementation resulted in the multiple pathway alterations in the chickens’ metabolism including the TCA cycle, fatty acid synthesis, and metabolism of glycerolipid and amino acids. Thus, the regulation of lipid metabolism was found to be a complex process involving multiple pathways. References T Aluwong , F Hassan , T Dzenda , 等 . Effect of different levels of supplemental yeast on body weight, thyroid hormone metabolism and lipid profile of broiler chickens . J Vet Med Sci , 2013 . 75 ( 3 ): 291 - 298 . 23100117 DOI: 10.1292/jvms.12-0368 http://doi.org/10.1292/jvms.12-0368 . Effect of different levels of supplemental yeast on body weight, thyroid hormone metabolism and lipid profile of broiler chickens Y An , W Xu , H Li , 等 . High-fat diet induces dynamic metabolic alterations in multiple biological matrices of rats . J Proteome Res , 2013 . 12 ( 8 ): 3755 - 3768 . 23746045 DOI: 10.1021/pr400398b http://doi.org/10.1021/pr400398b . High-fat diet induces dynamic metabolic alterations in multiple biological matrices of rats AOAC (Association of Official Analytical Chemists) Official Methods of Analysis , 1999 . 16th Ed : Washington, DC, USA AOAC , . Official Methods of Analysis AOCS (American Oil Chemists Society) Official Methods and Recommended Practices of the AOCS , 2009 . 6th Ed : Chicago, IL, USA AOCS , . Official Methods and Recommended Practices of the AOCS YS Cha , SR Kim , JA Yang , 等 . Kochujang , fermented soybean-based red pepper paste, decreases visceral fat and improves blood lipid profiles in overweight adults . Nutr Metab , 2013 . 10 ( 1 ): 24 DOI: 10.1186/1743-7075-10-24 http://doi.org/10.1186/1743-7075-10-24 . Kochujang, fermented soybean-based red pepper paste, decreases visceral fat and improves blood lipid profiles in overweight adults L de Genova Gaya , GB Mourão , FM de Rezende , 等 . Genetic trends of abdominal fat content in a male broiler chicken line . Genet Mol Res , 2005 . 4 ( 4 ): 760 - 764 . 16475123 . Genetic trends of abdominal fat content in a male broiler chicken line DR Donohoe , N Garge , X Zhang , 等 . The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon . Cell Metab , 2011 . 13 ( 5 ): 517 - 526 . 21531334 DOI: 10.1016/j.cmet.2011.02.018 http://doi.org/10.1016/j.cmet.2011.02.018 . The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon GJ Hausman , MV Dodson , K Ajuwon , 等 . Board-invited review: the biology and regulation of preadipocytes and adipocytes in meat animals . J Anim Sci , 2009 . 87 ( 4 ): 1218 - 1246 . 18849378 DOI: 10.2527/jas.2008-1427 http://doi.org/10.2527/jas.2008-1427 . Board-invited review: the biology and regulation of preadipocytes and adipocytes in meat animals Q He , P Ren , X Kong , 等 . Comparison of serum metabolite compositions between obese and lean growing pigs using an NMR-based metabolomic approach . J Nutr Biochem , 2012 . 23 ( 2 ): 133 - 139 . 21429726 DOI: 10.1016/j.jnutbio.2010.11.007 http://doi.org/10.1016/j.jnutbio.2010.11.007 . Comparison of serum metabolite compositions between obese and lean growing pigs using an NMR-based metabolomic approach J Huang , D Yang , S Gao , 等 . Effects of soy-lecithin on lipid metabolism and hepatic expression of lipogenic genes in broiler chickens . Livest Sci , 2008 . 118 ( 1-2 ): 53 - 60 . DOI: 10.1016/j.livsci.2008.01.014 http://doi.org/10.1016/j.livsci.2008.01.014 . Effects of soy-lecithin on lipid metabolism and hepatic expression of lipogenic genes in broiler chickens M Huber , LPL van de Vijver , H Parmentier , 等 . Effects of organically and conventionally produced feed on biomarkers of health in a chicken model . Br J Nutr , 2010 . 103 ( 5 ): 663 - 676 . 19860984 DOI: 10.1017/S0007114509992236 http://doi.org/10.1017/S0007114509992236 . Effects of organically and conventionally produced feed on biomarkers of health in a chicken model B Ji , B Ernest , JR Gooding , 等 . Transcriptomic and metabolomic profiling of chicken adipose tissue in response to insulin neutralization and fasting . BMC Genomics , 2012 . 13 ( 1 ): 441 22938590 DOI: 10.1186/1471-2164-13-441 http://doi.org/10.1186/1471-2164-13-441 . Transcriptomic and metabolomic profiling of chicken adipose tissue in response to insulin neutralization and fasting SB Joseph , BA Laffitte , PH Patel , 等 . Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors . J Biol Chem , 2002 . 277 ( 13 ): 11019 - 11025 . 11790787 DOI: 10.1074/jbc.M111041200 http://doi.org/10.1074/jbc.M111041200 . Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors RJ Khairallah , J Kim , KM O'Shea , 等 . Improved mitochondrial function with diet-induced increase in either docosahexaenoic acid or arachidonic acid in membrane phospholipids . PLoS ONE , 2012 . 7 ( 3 ): e34402 22479624 DOI: 10.1371/journal.pone.0034402 http://doi.org/10.1371/journal.pone.0034402 . Improved mitochondrial function with diet-induced increase in either docosahexaenoic acid or arachidonic acid in membrane phospholipids H Kim , JH Kim , S Noh , 等 . Metabolomic analysis of livers and serum from high-fat diet induced obese mice . J Proteome Res , 2011 . 10 ( 2 ): 722 - 731 . 21047143 DOI: 10.1021/pr100892r http://doi.org/10.1021/pr100892r . Metabolomic analysis of livers and serum from high-fat diet induced obese mice T Kind , G Wohlgemuth , DY Lee , 等 . FiehnLib-mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry . Anal Chem , 2009 . 81 ( 24 ): 10038 - 10048 . 19928838 DOI: 10.1021/ac9019522 http://doi.org/10.1021/ac9019522 . FiehnLib-mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry JH Koh , KW Yu , HJ Suh . Biological activities of Saccharomyces cerevisiae and fermented rice bran as feed additives . Lett Appl Microbiol , 2002 . 35 ( 1 ): 47 - 51 . 12081549 DOI: 10.1046/j.1472-765X.2002.01131.x http://doi.org/10.1046/j.1472-765X.2002.01131.x . Biological activities of Saccharomyces cerevisiae and fermented rice bran as feed additives SS Lee , T Pineau , J Drago , 等 . Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators . Mol Cell Biol , 1995 . 15 ( 6 ): 3012 - 3022 . 7539101 . Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators G Liu , Y Wang , Z Wang , 等 . Nuclear magnetic resonance (NMR)-based metabolomic studies on urine and serum biochemical profiles after chronic cysteamine supplementation in rats . J Agric Food Chem , 2011 . 59 ( 10 ): 5572 - 5578 . 21476594 DOI: 10.1021/jf104129k http://doi.org/10.1021/jf104129k . Nuclear magnetic resonance (NMR)-based metabolomic studies on urine and serum biochemical profiles after chronic cysteamine supplementation in rats KJ Livak , TD Schmittgen . Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔ C T method . Methods , 2001 . 25 ( 4 ): 402 - 408 . 11846609 DOI: 10.1006/meth.2001.1262 http://doi.org/10.1006/meth.2001.1262 . Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method OY Lukivskaya , AA Maskevich , VU Buko . Effect of ursodeoxycholic acid on prostaglandin metabolism and microsomal membranes in alcoholic fatty liver . Alcohol , 2001 . 25 ( 2 ): 99 - 105 . 11747979 DOI: 10.1016/S0741-8329(01)00171-9 http://doi.org/10.1016/S0741-8329(01)00171-9 . Effect of ursodeoxycholic acid on prostaglandin metabolism and microsomal membranes in alcoholic fatty liver S Mandrup , MD Lane . Regulating adipogenesis . J Biol Chem , 1997 . 272 ( 9 ): 5367 - 5370 . 9102400 DOI: 10.1074/jbc.272.9.5367 http://doi.org/10.1074/jbc.272.9.5367 . Regulating adipogenesis C Nie , W Zhang , L Yan , 等 . A metabolomics study on chicken plasma based on fermented feed from cottonseed meal mixed substrate . Acta Vet Zootech Sin , 2013 . 44 ( 5 ): 737 - 744 . (in Chinese) DOI: 10.11843/j.issn.0366-6964.2013.05.010 http://doi.org/10.11843/j.issn.0366-6964.2013.05.010 . A metabolomics study on chicken plasma based on fermented feed from cottonseed meal mixed substrate S Numa , S Nakanishi , T Hashimoto , 等 . Role of acetyl coenzyme A carboxylase in the control of fatty acid synthesis . Vitam Horm , 1971 . 28 213 - 243 . DOI: 10.1016/S0083-6729(08)60895-X http://doi.org/10.1016/S0083-6729(08)60895-X . Role of acetyl coenzyme A carboxylase in the control of fatty acid synthesis D Oikawa , S Tsuyama , Y Akimoto , 等 . Arachidonic acid prevents fatty liver induced by conjugated linoleic acid in mice . Br J Nutr , 2009 . 101 ( 10 ): 1558 - 1563 . 18947440 DOI: 10.1017/S0007114508091253 http://doi.org/10.1017/S0007114508091253 . Arachidonic acid prevents fatty liver induced by conjugated linoleic acid in mice M Özdoğan , K Wellmann , E Paksuz . Effect of gossypol on blood serum parameters and small intestinal morphology of male broilers . J Anim Physiol Anim Nutr , 2012 . 96 ( 1 ): 95 - 101 . DOI: 10.1111/j.1439-0396.2010.01126.x http://doi.org/10.1111/j.1439-0396.2010.01126.x . Effect of gossypol on blood serum parameters and small intestinal morphology of male broilers JA Park , PB Tirupathi Pichiah , JJ Yu , 等 . Anti-obesity effect of kimchi fermented with Weissella koreensis OK1-6 as starter in high-fat diet-induced obese C57BL/6J mice . J Appl Microbiol , 2012 . 113 ( 6 ): 1507 - 1516 . 22978326 DOI: 10.1111/jam.12017 http://doi.org/10.1111/jam.12017 . Anti-obesity effect of kimchi fermented with Weissella koreensis OK1-6 as starter in high-fat diet-induced obese C57BL/6J mice GJ Patti , O Yanes , G Siuzdak . Innovation: metabolomics: the apogee of the omics trilogy . Nat Rev Mol Cell Biol , 2012 . 13 ( 4 ): 263 - 269 . 22436749 DOI: 10.1038/nrm3314 http://doi.org/10.1038/nrm3314 . Innovation: metabolomics: the apogee of the omics trilogy TK Pilvi , T Seppänen-Laakso , H Simolin , 等 . Metabolomic changes in fatty liver can be modified by dietary protein and calcium during energy restriction . Word J Gastroenterol , 2008 . 14 ( 28 ): 4462 - 4472 . DOI: 10.3748/wjg.14.4462 http://doi.org/10.3748/wjg.14.4462 . Metabolomic changes in fatty liver can be modified by dietary protein and calcium during energy restriction X Shi , C Xiao , Y Wang , 等 . Gallic acid intake induces alterations to systems metabolism in rats . J Proteome Res , 2013 . 12 ( 2 ): 991 - 1006 . 23231653 DOI: 10.1021/pr301041k http://doi.org/10.1021/pr301041k . Gallic acid intake induces alterations to systems metabolism in rats H Sun , JW Tang , XH Yao , 等 . Improvement of the nutritional quality of cottonseed meal by Bacillus subtili s and the addition of papain . Int J Agric Biol , 2012 . 14 ( 4 ): 987 - 993 . . Improvement of the nutritional quality of cottonseed meal by Bacillus subtilis and the addition of papain H Sun , JW Tang , CL Fang , 等 . Molecular analysis of intestinal bacterial microbiota of broiler chickens fed diets containing fermented cottonseed meal . Poult Sci , 2013 . 92 ( 2 ): 392 - 401 . 23300306 DOI: 10.3382/ps.2012-02533 http://doi.org/10.3382/ps.2012-02533 . Molecular analysis of intestinal bacterial microbiota of broiler chickens fed diets containing fermented cottonseed meal JW Tang , H Sun , XH Yao , 等 . Effects of replacement of soybean meal by fermented cottonseed meal on growth performance, serum biochemical parameters and immune function of yellow-feathered broilers . Asian Australas J Anim Sci , 2012 . 25 ( 3 ): 393 - 400 . 25049578 DOI: 10.5713/ajas.2011.11381 http://doi.org/10.5713/ajas.2011.11381 . Effects of replacement of soybean meal by fermented cottonseed meal on growth performance, serum biochemical parameters and immune function of yellow-feathered broilers VR Velagapudi , R Hezaveh , CS Reigstad , 等 . The gut microbiota modulates host energy and lipid metabolism in mice . J Lipid Res , 2010 . 51 ( 5 ): 1101 - 1112 . 20040631 DOI: 10.1194/jlr.M002774 http://doi.org/10.1194/jlr.M002774 . The gut microbiota modulates host energy and lipid metabolism in mice PJ Voshol , MC Jong , VEH Dahlmans , 等 . In muscle-specific lipoprotein lipase-overexpressing mice, muscle triglyceride content is increased without inhibition of insulin-stimulated whole-body and muscle-specific glucose uptake . Diabetes , 2001 . 50 ( 11 ): 2585 - 2590 . 11679438 DOI: 10.2337/diabetes.50.11.2585 http://doi.org/10.2337/diabetes.50.11.2585 . In muscle-specific lipoprotein lipase-overexpressing mice, muscle triglyceride content is increased without inhibition of insulin-stimulated whole-body and muscle-specific glucose uptake XH Wu . Effect and Regulation of Linseed Oil on Meat Quality and Fat Metabolism of Broilers , 2012 . : Yangling, China Univ. Northwest A & F , PhD Thesis (in Chinese) . Effect and Regulation of Linseed Oil on Meat Quality and Fat Metabolism of Broilers YP Xiao , TX Wu , JM Sun , 等 . Response to dietary L -glutamine supplementation in weaned piglets: a serum metabolomic comparision and hepatic metabolic regulation analysis . J Anim Sci , 2012 . 90 ( 12 ): 4421 - 4430 . 23255817 DOI: 10.2527/jas.2012-5039 http://doi.org/10.2527/jas.2012-5039 . Response to dietary L-glutamine supplementation in weaned piglets: a serum metabolomic comparision and hepatic metabolic regulation analysis M Yamamoto , F Saleh , M Tahir , 等 . The effect of Koji-feed (fermented distillery by-product) on the growth performance and nutrient metabolizability in broiler . J Poul Sci , 2007 . 44 ( 3 ): 291 - 296 . DOI: 10.2141/jpsa.44.291 http://doi.org/10.2141/jpsa.44.291 . The effect of Koji-feed (fermented distillery by-product) on the growth performance and nutrient metabolizability in broiler L Yang , T Kasumov , L Yu , 等 . Metabolomic assays of the concentration and mass isotopomer distribution of gluconeogenic and citric acid cycle intermediates . Metabolomics , 2006 . 2 ( 2 ): 85 - 94 . DOI: 10.1007/s11306-006-0019-1 http://doi.org/10.1007/s11306-006-0019-1 . Metabolomic assays of the concentration and mass isotopomer distribution of gluconeogenic and citric acid cycle intermediates X Yang , JY Sun , JL Guo , 等 . Identification and proteomic analysis of a novel gossypol-degrading fungal strain . J Sci Food Agric , 2012 . 92 ( 4 ): 943 - 951 . 22002592 DOI: 10.1002/jsfa.4675 http://doi.org/10.1002/jsfa.4675 . Identification and proteomic analysis of a novel gossypol-degrading fungal strain YL Yin , HY Zhong , RL Huang , 等 . Nutritive value of feedstuffs and diets for pigs: I. chemical composition, apparent ileal and fecal digestibility . Anim Feed Sci Tech , 1993 . 44 ( 1-2 ): 1 - 27 . DOI: 10.1016/0377-8401(93)90034-H http://doi.org/10.1016/0377-8401(93)90034-H . Nutritive value of feedstuffs and diets for pigs: I. chemical composition, apparent ileal and fecal digestibility YL Yin , CM Chen , HY Zhong , 等 . Apparent digestibility of energy, cell wall constituents, crude protein and amino acids of the Chinese oil seed meals for growing pigs . Anim Feed Sci Tech , 1994 . 45 ( 3 ): 283 - 298 . DOI: 10.1016/0377-8401(94)90033-7 http://doi.org/10.1016/0377-8401(94)90033-7 . Apparent digestibility of energy, cell wall constituents, crude protein and amino acids of the Chinese oil seed meals for growing pigs WJ Zhang , ZR Xu , JY Sun , 等 . Effect of selected fungi on the reduction of gossypol levels and nutritional value during solid substrate fermentation of cottonseed meal . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 2006 . 7 ( 9 ): 690 - 695 . DOI: 10.1631/jzus.2006.B0690 http://doi.org/10.1631/jzus.2006.B0690 . Effect of selected fungi on the reduction of gossypol levels and nutritional value during solid substrate fermentation of cottonseed meal WJ Zhang , ZR Xu , SH Zhao , 等 . Development of a microbial fermentation process for detoxification of gossypol in cottonseed meal . Anim Feed Sci Tech , 2007 . 135 ( 1-2 ): 176 - 186 . DOI: 10.1016/j.anifeedsci.2006.06.003 http://doi.org/10.1016/j.anifeedsci.2006.06.003 . Development of a microbial fermentation process for detoxification of gossypol in cottonseed meal S Zhao , H Ma , S Zou , 等 . Effects of in ovo administration of DHEA on lipid metabolism and hepatic lipogenetic genes expression in broiler chickens during embryonic development . Lipids , 2007 . 42 ( 8 ): 749 - 757 . 17574488 DOI: 10.1007/s11745-007-3068-y http://doi.org/10.1007/s11745-007-3068-y . Effects of in ovo administration of DHEA on lipid metabolism and hepatic lipogenetic genes expression in broiler chickens during embryonic development X Zhao , Y Guo , S Guo , 等 . Effects of Clostridium butyricum and Enterococcus faecium on growth performance, lipid metabolism, and cecal microbiota of broiler chickens . Appl Microbiol Biotechnol , 2013 . 97 ( 14 ): 6477 - 6488 . 23666481 DOI: 10.1007/s00253-013-4970-2 http://doi.org/10.1007/s00253-013-4970-2 . Effects of Clostridium butyricum and Enterococcus faecium on growth performance, lipid metabolism, and cecal microbiota of broiler chickens We are grateful to Dr. Jun-liang DENG (Biotree Bio-technology Co., Ltd., Shanghai, China) for providing helps in metabolomics data analysis. Compliance with ethics guidelines Cun-xi NIE, Wen-ju ZHANG, Yong-qiang WANG, Yan-feng LIU, Wen-xia GE, and Jian-cheng LIU declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1400255 Project supported by the National Natural Science Foundation of China (No. 31360564) and the Graduate Research & Innovation Project of Xinjiang Uygur Autonomous Region of China (No. XJGRI2013059) 2014-09-24 2015-05-13 2015-02-08 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 16