Abstract

   Feeding sheep with high-concentrate diet (HCD) to shorten production
   cycle is a well-developed feeding strategy to increase lamb production.
   Here, metabolomics were performed to explore the mechanism that HCD
   changes lamb nutrition composition. Differential metabolites were
   enriched in primary bile acid biosynthesis. Significantly higher
   content of bile acids including taurodeoxycholic acid sodium salt
   (TDCA), taurochenodeoxycholic acid sodium salt (TCDCA) and taurocholic
   acid (TCA) was observed in lamb of HCD, while the content of
   lithocholic acid (LCA), cholic acid (CA), chenodeoxycholic acid (CDCA)
   and Chenodeoxycholic acid-3-beta-D-glucuronide (CDCA-3Gln) were higher
   in the controls. Furthermore, a significantly decreased content of
   fatty acids was observed in lamb of HCD group. Finally, primary
   skeletal cells treated with CA or TCA showed a significant decrease in
   contents of fatty acids, while TCA showed a stronger effect in
   decreasing fatty acid contents. Collectively, we suggest that HCD
   decreases lamb fatty acid contents by regulating bile acid composition.

   Keywords: High-concentrate diet, Metabolomics, Lamb, Bile acid, Fatty
   acid

Highlights

     * •
       High-concentrate diet affects the metabolites composition of
       mutton.
     * •
       Both CA and TCA inhibit the accumulation of fatty acids in skeletal
       muscle cells.
     * •
       High-concentrate diet decreases mutton fatty acid contents by
       regulating bile acid composition.

1. Introduction

   Currently, feeding sheep with high-concentrate diet has emerged as a
   sophisticated production technique in contrast to conventional grass
   feeding, facilitating an increase in the sales rate of livestock and
   thereby increasing overall production and profitability
   ([43]Perez-Trejo et al., 2022). High-concentrate diet is a kind of diet
   that contains a higher level of easily fermentable carbohydrates and
   starch, and a lower level of fiber. By increasing the nutrient level of
   diet, this production strategy not only can significantly improve
   slaughter performance but also change the meat quality of livestock
   ([44]Ma et al., 2023), which might be resulted from growth rate changes
   ([45]Koch et al., 2023). A major effect of high concentrate diet to
   meat is that it significantly decreases the slice shear force of meat
   ([46]Del Campo et al., 2021). While it has been reported that
   high-concentrate diet affects the sensory quality of mutton ([47]Gao et
   al., 2014). The compounds influencing meat flavor and taste, including
   lipids, hydrophilic metabolites, and volatile compounds, are highly
   associated with meat quality and customers' choice ([48]Wang et al.,
   2022).

   Bile acids are not only amphipathic molecules that facilitate
   digestion, but also important physiological agents. It remains unclear
   whether bile acids have influence on lamb. Bile acids,
   cholesterol-derived endogenous metabolites, have a vital role in
   maintaining energy homeostasis. The process of absorption and digestion
   of dietary lipids is facilitated by bile acids ([49]Zhong et al.,
   2022). Primary bile acids are those initially transformed from
   cholesterol in hepatocytes via two different metabolic pathways,
   including a classical pathway depending on cholesterol 7α-hydroxylase
   and an alternative pathway depending on sterol 27-hydroxylase ([50]Jia
   et al., 2021). These bile acids are then conjugated with taurine or
   glycine, secreted into the gallbladder, and released into the
   intestinal lumen after food ingestion. The intestinal microbiota
   facilitates the biotransformation of bile acids from primary to
   secondary within the intestine ([51]Yang, Yang, et al., 2023). The bile
   acids are then reabsorbed into enterohepatic circulation by the gut
   epithelium when they reach the distal part of the small intestine.
   During this process, bile acids can overflow into the systemic
   circulation and function as signaling molecules that influence almost
   all organs through the activation of dedicated bile acid receptors
   ([52]Perino & Schoonjans, 2022).

   Recently, metabolomics has been widely applied in analyzing the effects
   of pre-slaughter factors on meat quality. Metabolomics represents the
   downstream information of genomes, transcriptomes, and proteomes, which
   provides an overview of all small-molecule metabolites of living
   organisms ([53]Creydt & Fischer, 2022a). This promotes researchers to
   compare the meat samples processed with diverse treatments and identify
   quality markers through metabolomics ([54]Ramanathan et al., 2023), in
   which untargeted metabolomics using UHPLC-MS/MS platforms has become an
   increasingly important shotgun approach to analyze the chemistry of
   meat quality change ([55]Creydt & Fischer, 2022b). While, untargeted
   metabolomics is usually combined with targeted metabolomics in order to
   provide more detailed information of the meat under different feeding
   strategies. In this study, the differential metabolites between control
   and HCD lamb were significantly enriched in primary bile acid
   biosynthesis. Thus, we hypothesized that bile acids were involved in
   high concentrate diet caused lamb lipid contents change. Furthermore,
   we have verified and quantified the bile acids contents. Given the
   central role of bile acids in fatty acid biosynthesis, we have profiled
   fatty acids and validated the effect of bile acids by in vitro study.

2. Material and methods

2.1. Animals

   Ten male Hu Sheep were divided randomly into two groups at the age of
   day 60, including an HCD and control group in a commercial farm
   (Zhejiang Yihui Ecological Agriculture Co., Ltd., Zhejiang Province,
   China). All the sheep were fed with oat grass and peanut vine as
   roughage, while a pellet was used as concentrated feed with 22.24 %
   crude protein (Table S1). The diet for the sheep in the control group
   was comprised of 20 % of the pellet on dry matter basis, while the diet
   for the HCD group was comprised of 50 % pellet. Sheep were slaughtered
   at 9 months of age. The information of animal characteristics was shown
   in Table S2. Slaughtering was carried out in a commercial abattoir
   (Huzhou Changxing Muyuan Food Co., Ltd) by the slaughter house staff,
   the method of slaughtering followed the traditional procedures
   including stunning. The Institutional Animal Care and Use Committees at
   the Zhejiang Academy of Agricultural Sciences provided their approval
   to the experimental protocols used in animal research.

2.2. Sample preparation for UHPLC-MS/MS

   Longissimus dorsi samples from thirteenth rib were collected and
   separately grounded with liquid nitrogen followed by the reconstitution
   of the resulting homogenate in methanol (MeOH). After vortex mixing,
   the samples were subjected to incubation for 5 min followed by
   centrifugation (15,000g, 20 min, 4 °C), after which the supernatant was
   diluted to 53 % MeOH. After that, the samples were centrifuged for the
   duration of 20 min at 15000 g, 4 °C before being incorporated into the
   LC-MS/MS system.

   To examine bile acid, 100 mg of lamb sample were resuspended with
   liquid nitrogen followed by the incorporation of 0.9 mL of ddH[2]O.
   Following dilution, it was combined with a mixture of acetonitrile
   (ACN) and MeOH (8:2) containing mixed internal standards (IS). Samples
   were centrifuged (12,000 rpm, 20 min) and the supernatants were dried
   with a nitrogen blower followed by reconstitution with 100 μL of
   water/ACN (8:2) that contained formic acid (FA, 0.1 %). For fatty acid
   measurement, homogenization of 100 mg of the lamb samples was performed
   with 300 μL of mixed IS containing Isopropanol/ACN (1:1), and
   centrifuged for 10 min at 12,000 rpm.

2.3. UHPLC-MS/MS analysis

   UHPLC-MS/MS was performed using a Vanquish UHPLC system (Thermo Fisher,
   USA) coupled with an Orbitrap Q Exactive TMHF mass spectrometer (Thermo
   Fisher) in Novogene Co., Ltd. (Beijing, China). Samples were applied to
   a Hypesil Gold column and eluted with a linear gradient. FA (1 %) in
   the mixture of water and MeOH were utilized as eluents in the positive
   polarity mode while the negative polarity mode's eluents were based on
   ammonium acetate and MeOH. A Q Exactive TM HF mass spectrometer was
   used.

2.4. Data processing and metabolite identification

   Compound Discoverer 3.1 (CD3.1, ThermoFisher) was used to carry out
   peak picking, peak alignment, and quantification of individual
   metabolites in the UHPLC-MS/MS-generated raw data files. After that,
   the total spectral intensity was used to normalize the peak
   intensities. Using the normalized data, the molecular formula was
   predicted considering the molecular ion peaks along with fragment and
   additive ions. The accurate as well as relative quantitative results
   were acquired by matching the peaks with Cloud
   ([56]https://www.mzcloud.org/), mz Vault, and Mass List data bile
   acids. The statistical software R, Python, and CentOS were used to
   conduct the statistical studies. Annotation of metabolites was done
   with the KEGG ([57]https://www.genome.jp/kegg/pathway.html) databases.

2.5. The measurement of bile acids and fatty acids

   Six stable isotope-labeled standards as well as thirty-three of the
   bile acid standards, or fifty standards of fatty acids and five stable
   isotope-labeled standards were acquired from ZZ Standards Co., LTD.
   (Shanghai, China). LC-MS/MS was conducted by injecting 2 μL
   supernatant. For the purpose of separation, Waters ACQUITY UPLC BEH C18
   column (2.1 × 100 mm, 1.7 μm) was used which was kept at 50 °C. At
   0.30 mL/min flow rate, the mobile phase, a mixture of 0.1 % FA in water
   and ACN, was introduced for bile acid measurement, while 0.05 % FA in
   water and Isopropanol/ACN (1:1) was introduced for fatty acid
   measurement.

2.6. Cell isolation and culture

   Lamb longissimus dorsi was collected from Hu sheep, washed with PBS
   buffer containing antibiotic. The connective tissue and fat were
   removed. Then, the lamb sample was cut into small pieces. Subsequently,
   0.2 % collagenase IV was added and sample was digested in a water bath
   of 37 °C for 1 h. Then, 0.25 % trypsin was added to digest for 20 min.
   After digestion was terminated, the samples were resuspended, and
   filtered, centrifuged at 1000 r/min for 10 min. The primary cells of
   the lamb longissimus dorsi were isolated and purified by Percoll
   density gradient centrifugation after being suspended in complete
   culture medium for precipitation. Resuspend the cells and cultured in a
   37 °C and 5 % CO[2] incubator. The cells were incubated with either 2
   μΜ CA or 1 μΜ TCA for 2 days before fatty acid profile.

2.7. Data analysis

   Data are presented as means ± SEM unless otherwise indicated.
   Differences were determined with independent-samples t-tests using SPSS
   20.0 software (IBM Corp., USA.).

3. Results

3.1. Multivariate statistical analysis of lamb metabolites

   We analyzed the lamb samples by UHPLC-QTOF/MS in the positive (ESI+)
   and negative ion (ESI-) modes. The ESI- and ESI+ modes respectively
   detected a total of 797 and 519 metabolites. Then, the metabolites were
   annotated based on secondary mass spectrometry and database mapping
   (Fig. S1). PCA was performed to illustrate the differences between
   control and HCD samples. The analysis revealed that the first two main
   components described 68.81 % of the variability, corresponding to
   13.73 % and 55.08 % of the total variance in the ESI-mode (Fig. S2A).
   While 66.18 % variability was observed for the first two primary
   components corresponding to 47.47 %, and 18.71 % of the total variance
   in ESI+ mode (Fig. S2B).

   PLS-DA was then performed to determine the lamb metabolic profile.
   PLS-DA analysis showed that the 70.68 % variability demonstrated by the
   first two primary components, represented total variances of 31.01 %,
   and 39.67 % in the ESI- mode (Fig. S1 A), while 67.42 % variability of
   the top two primary components accounted for variances of 44.99 %, and
   22.43 % in ESI+ mode (Fig. S1B). A discernible metabolite profile was
   observed between HCD lamb and control, as evidenced by the separation
   and placement of samples in distinct quadrants. Due to the similar
   distribution of the score plot to that of PCA, and the results of
   validation tests of the PLS-DA model (Fig. S1C and D), we suggested
   that the validity of the profiling of metabolites distinguish the
   different groups. According to fold change >1.5 or < 0.67 and p<0.05,
   52 and 83 differentially abundant metabolites were determined using
   ESI- ([58]Fig. 1A, Table S3) and ESI+ ([59]Fig. 1B, Table S4) mode,
   respectively, including 88 overabundant metabolites in HCD lamb and 47
   excessively abundant metabolites in control lamb.

Fig. 1.

   [60]Fig. 1
   [61]Open in a new tab

   A volcano map showing ESI- (A) and ESI+ modes (B) of differential
   metabolites in lamb of high-concentrate diet.

3.2. Bioinformatics analysis of differentially abundant metabolites

   After differential metabolite identification, KEGG enrichment analysis
   was performed to reveal the effect of HCD on metabolic pathways. In
   ESI- mode, differentially abundant metabolites were significantly
   enriched in the biosynthesis of primary bile acid (map00120),
   metabolism of taurine and hypotaurine (map00430), and cholesterol
   metabolism (map04979)([62]Fig. 2A, Table S5). Differential metabolites
   in ESI+ mode were not significantly enriched, while Phenylalanine
   metabolism (map00360), Steroid hormone biosynthesis (map00140),
   Glycine, serine and threonine metabolism (map00260),
   Aldosterone-regulated sodium reabsorption (map04960) and ABC
   transporters (map02010) were among the top five enriched KEGG terms in
   positive mode ([63]Fig. 2B, Table S6).

Fig. 2.

   [64]Fig. 2
   [65]Open in a new tab

   KEGG pathway enrichment analysis of differentially abundant metabolites
   in high-concentrate diet group in the ESI- (A) and ESI+ modes (B).
   Pathways are organized based on their corresponding p-value.

3.3. Differential bile acids in HCD lamb longissimus dorsi

   In the KEGG terms that involved in bile acids, we found the content of
   taurochenodeoxycholic acid, taurine, TCA and L-glutamic acid
   significantly higher in HCD group, while the content of CA were
   significantly higher in control group (Table S3 and S4). Then, we
   reviewed the rest of differential bile acids and found the level of
   taurolithocholic acid sodium salt (TLCA) and taurochenodeoxycholic acid
   sodium salt (TCDCA) significantly higher in HCD lamb, while LCA and
   ursodeoxycholic acid (UDCA) were down-regulated (Table S3 and S4).
   These data indicated a diverse pattern of bile acids contents between
   HCD group and control group.

   Thus, we were promoted to profile the bile acids composition between
   control and HCD lamb by non-targeted metabolomics. A total of fifteen
   bile acids were identified, among which seven bile acids differed
   markedly between the groups, including TDCA, TCDCA, CDCA, CA,
   CDCA-3Gln, LCA and TCA. These differential bile acids can be classified
   into four primary and three secondary, or four conjugated and three
   unconjugated bile acids. Similar to the non-targeted metabolomics
   results, the content of TDCA, TCDCA, and TCA were considerably lower in
   the control lamb (p<0.05), while considerably lower levels of LCA, CA,
   CDCA, and CDCA-3Gln were observed in HCD lamb (p<0.05) ([66]Table 1).

Table 1.

   Bile acid profile of lamb longissimus dorsi between control and HCD
   groups ng/g.
   name Control HCD
   Taurodeoxycholic acid sodium salt (TDCA) 48.86 ± 12.61 159.23 ± 44.82*
   Hyodeoxycholic acid (HDCA) 4.90 ± 1.45 2.69 ± 1.00
   12-ketolithocholic acid (12- ketoLCA) 9.44 ± 1.98 7.98 ± 1.38
   Deoxycholic acid (DCA) 217.41 ± 111.99 9.96 ± 3.51
   Taurochenodeoxycholic acid sodium salt (TCDCA) 43.09 ± 17.60
   130.25 ± 23.83*
   Chenodeoxycholic acid (CDCA) 85.42 ± 27.12 1.62 ± 0.38*
   Cholic acid (CA) 921.43 ± 442.43 9.90 ± 3.66*
   Chenodeoxycholic acid-3-beta-D-glucuronide (CDCA-3Gln) 16.40 ± 3.55
   0.76 ± 0.05*
   Lithocholic acid (LCA) 19.67 ± 1.32 11.83 ± 1.34*
   Glycochenodeoxycholic acid sodium salt (GCDCA) 13.68 ± 9.02 3.19 ± 1.41
   Glycocholic acid hydrate (GCA) 48.96 ± 25.08 22.71 ± 6.03
   Taurolithocholic acid sodium salt (TLCA) 3.79 ± 0.75 4.80 ± 0.92
   Taurocholic acid (TCA) 166.93 ± 50.78 405.01 ± 96.99
   Tauro-alpha-Muricholic acid sodium salt (T-α-MCA) 3.35 ± 1.28
   1.90 ± 0.78
   Glycodeoxycholic acid (GDCA) 7.50 ± 3.39 1.60 ± 0.42
   [67]Open in a new tab

   Note: data are presented as MEAN ± SEM, n = 5, * means p < 0.05.

3.4. Differential fatty acids in the lamb longissimus dorsi between HCD and
control groups

   Overall, 48 fatty acids were found, with 28 of them showing
   significantly lower levels in the HCD group, including C15:0, C15:1,
   C15:1 T, C16:0, C16:1, C16:1 T, C17:0, C17:1, C17:1 T, C18:0,
   C18:1(n-9), C18:1(n-7), C18:1(n−12)T, C18:1(n-9)T, C18:1(n-7)T,
   C18:2(n-6), C18:2(n-6)T, C20:0, C22:1, C22:1 T, C20:3(n-3), C23:0,
   C22:2, C20:5, C22:5(n-3), C24:1 and C22:6 (p<0.05) ([68]Table 2). The
   rest of fatty acids showed no significant difference between two
   groups.

Table 2.

   Fatty acids profile of lamb longissimus dorsi between control and
   high-concentrate diet groups ng/g.
   Name Control HCD
   Decanoic acid (C10:0) 402.80 ± 89.96 238.20 ± 34.07
   Dodecanoic acid (C12:0) 391.66 ± 121.39 211.61 ± 22.78
   Tridecanoic acid (C13:0) 27.50 ± 1.82 22.71 ± 1.33
   Tetradecanoic acid (C14:0) 8004.59 ± 1876.64 4634.21 ± 456.74
   Myristoleic acid (C14:1) 326.35 ± 89.04 160.40 ± 16.46
   Myristelaidic acid (C14:1 T) 366.09 ± 164.38 58.29 ± 3.70
   Pentadecanoic acid (C15:0) 1215.07 ± 173.52 582.53 ± 72.93*
   cis-10-Pentadecenoic acid (C15:1) 31.00 ± 6.84 12.52 ± 1.83*
   trans-10-Pentadecenoic acid (C15:1 T) 46.91 ± 6.57 31.09 ± 1.42*
   Hexadecanoic acid (C16:0) 89,179.56 ± 7436.88 44,765.42 ± 4014.39*
   Palmitoleic acid (C16:1) 15,941.07 ± 3261.84 8094.44 ± 937.73*
   Palmitelaidic acid (C16:1 T) 6723.03 ± 343.78 4288.79 ± 669.86*
   Heptadecanoic acid (C17:0) 6988.69 ± 912.21 2404.29 ± 234.37*
   cis-10-Heptadecenoic acid (C17:1) 7535.43 ± 1506.77 3350.01 ± 538.05*
   trans-10-Heptadecenoic acid (C17:1 T) 450.26 ± 85.49 197.60 ± 27.05*
   Octadecanoic acid (C18:0) 52,259.17 ± 6226.20 25,032.25 ± 3907.08*
   Petroselinic acid (C18:1(n-12)) 85,121.34 ± 13,197.91
   42,799.03 ± 6315.05*
   Oleic acid (C18:1(n-9)) 91,720.86 ± 14,719.47 45,726.71 ± 6729.29*
   cis-Vaccenic acid (C18:1(n-7)) 4395.72 ± 102.29 2910.32 ± 180.07*
   Petroselaidic acid (C18:1(n-12)T) 12,661.20 ± 1543.11 2968.07 ± 473.28*
   Elaidic acid (C18:1(n-9)T) 12,084.54 ± 1613.81 2945.28 ± 446.32*
   trans-Vaccenic acid (C18:1(n-7)T) 169,563.70 ± 13,771.38
   55,071.07 ± 4168.27*
   Linoleic acid (C18:2(n-6)) 7491.71 ± 897.95 2388.03 ± 190.97*
   Linoelaidic acid (C18:2(n-6)T) 475.81 ± 91.16 227.99 ± 25.29*
   trans-7-Nonadecenoic acid (C19:1(n-12)T) 494.10 ± 130.67 279.34 ± 71.65
   trans-10-Nonadecenoic acid (C19:1(n-9)T) 2647.07 ± 588.09
   1726.54 ± 257.08
   Arachidic acid (C20:0) 7302.06 ± 1521.05 4020.02 ± 476.54*
   gamma-Linolenic acid (C18:3(n-6)) 2127.34 ± 493.63 2547.76 ± 510.08
   cis-11-Eicosenoic acid (C20:1) 448.17 ± 143.86 309.15 ± 49.03
   trans-11-Eicosenoic acid (C20:1 T) 6883.02 ± 1438.47 4077.39 ± 501.58
   alpha-Linolenic acid (C18:3(n-3)) 732.04 ± 242.46 385.40 ± 55.76
   Heneicosanoic acid (C21:0) 3113.61 ± 705.18 2475.00 ± 404.94
   cis-11,14-Eicosadienoic acid (C20:2) 1435.03 ± 249.36 1313.74 ± 215.41
   Docosanoic acid (C22:0) 528.19 ± 146.47 422.66 ± 81.14
   homo-gamma-Linolenic acid (C20:3(n-6)) 436.96 ± 110.13 566.78 ± 97.10
   Erucic acid (C22:1) 4204.35 ± 495.51 1699.15 ± 237.63*
   Brassidic acid (C22:1 T) 120,981.1 ± 10,988.27 55,670.06 ± 7142.89*
   cis-11,14,17-Eicosatrienoic acid (C20:3(n-3)) 136.17 ± 28.47
   60.23 ± 10.23*
   Arachidonic acid (C20:4) 279.89 ± 82.08 317.37 ± 63.20
   Tricosanoic acid (C23:0) 4612.67 ± 534.99 1148.38 ± 77.37*
   cis-13,16-Docosadienoic acid (C22:2) 6689.10 ± 1093.51 3510.85 ± 623.22
   *
   cis-5,8,11,14,17-Eicosapentaenoic acid (C20:5) 10,979.60 ± 947.77
   3018.93 ± 370.26*
   cis-7,10,13,16-Docosic acidtraenoic acid (C22:4) 2967.07 ± 434.73
   2157.89 ± 384.96
   cis-7,10,13,16,19-Docosapentaenoic acid (C22:5(n-3)) 154.84 ± 31.30
   70.81 ± 14.14*
   cis-4,7,10,13,16-Docosapentaenoic acid (C22:5(n-6)) 2931.79 ± 807.46
   3855.68 ± 811.28
   Tetracosanoic acid (C24:0) 271,356.42 ± 53,249.25
   154,247.47 ± 26,563.53
   Nervonic acid (C24:1) 3463.27 ± 775.98 1380.45 ± 148.42*
   cis-4,7,10,13,16,19-Docosahexaenoic acid (C22:6) 1397.83 ± 378.75
   432.01 ± 45.56*
   [69]Open in a new tab

   Note: data are presented as MEAN ± SEM, n = 5, * means p < 0.05.

3.5. Differential fatty acids in the longissimus dorsi cells following CA or
TCA treatment

   When compared with control group, we found the content of C11:0, C12:0,
   C13:0, C14:0, C14:1, C14:1 T, C15:0, C15:1, C16:0, C16:1, C16:1 T,
   C17:0, C17:1 T, C18:1(n-12), C18:1(n-9), C18:1(n-7), C18:1(n-12)T,
   C18:1(n-7)T, C18:2(n-6), C19:1(n-12)T, C19:1(n-9)T, C20:0, C20:1,
   C20:1 T, C21:0, C20:2, C22:1, C20:4, C23:0, C22:2, C20:5, C22:4,
   C22:5(n-3), C24:1, C22:6 significantly lower in CA treated cells, while
   the content of C10:0, C18:0, C18:1(n-9)T, C22:0, C22:1 T, C22:5(n-6)
   and C24:0 were significant higher in CA treatment group (p<0.05)
   ([70]Table 3).

Table 3.

   Fatty acids profile of sheep skeletal muscle cells treated with CA and
   TCA ng/g.
       Name          Control              CA               TCA
   C10:0        0.50 ± 0.01^c     1.02 ± 0.04^b     1.37 ± 0.05^a
   C11:0        3.15 ± 0.22^a     1.25 ± 0.06^b     2.55 ± 0.04^a
   C12:0        55.91 ± 1.18^a    46.61 ± 0.92^b    46.11 ± 0.13^b
   C13:0        16.54 ± 0.32^a    10.43 ± 0.26^b    11.87 ± 0.48^b
   C14:0        167.60 ± 0.90^a   136.00 ± 1.55^b   136.06 ± 1.84^b
   C14:1        28.93 ± 0.63^a    17.67 ± 0.37^b    17.34 ± 0.71^b
   C14:1 T      30.75 ± 0.86^a    19.32 ± 0.69^b    21.27 ± 0.88^b
   C15:0        187.09 ± 2.41^a   144.49 ± 1.99^b   113.82 ± 2.15^c
   C15:1        30.92 ± 0.56^a    26.75 ± 0.41^b    30.11 ± 0.41^a
   C16:0        3138.44 ± 7.81^a  2673.45 ± 17.02^b 2479.69 ± 8.78^c
   C16:1        314.88 ± 2.63^a   227.75 ± 2.13^b   209.42 ± 2.64^c
   C16:1 T      341.26 ± 2.63^a   276.81 ± 3.98^b   332.81 ± 1.82^a
   C17:0        117.49 ± 1.45^a   95.81 ± 1.40^b    84.13 ± 1.02^c
   C17:1        201.04 ± 1.05^a   198.43 ± 1.59^a   143.74 ± 2.31^b
   C17:1 T      178.04 ± 2.73^a   108.58 ± 4.17^b   96.50 ± 3.35^b
   C18:0        1978.59 ± 11.95^c 2662.67 ± 24.83^a 2236.42 ± 16.09^b
   C18:1(n-12)  3534.37 ± 17.66^a 3112.93 ± 28.06^b 2375.57 ± 14.70^c
   C18:1(n-9)   3222.30 ± 9.34^a  2935.29 ± 31.81^b 2177.09 ± 36.17^c
   C18:1(n-7)   64.53 ± 0.84^a    44.07 ± 1.83^b    42.81 ± 1.37^b
   C18:1(n-12)T 56.58 ± 0.41^a    46.56 ± 1.86^b    23.96 ± 1.63^c
   C18:1(n-9)T  161.81 ± 6.52^c   364.16 ± 9.27^a   222.38 ± 4.55^b
   C18:1(n-7)T  2667.76 ± 10.79^a 1807.18 ± 13.61^b 1251.21 ± 19.68^c
   C18:2(n-6)   445.06 ± 3.61^a   316.53 ± 7.60^b   282.39 ± 5.47^c
   C19:1(n-12)T 40.49 ± 1.20^a    24.21 ± 0.59^b    16.30 ± 0.36^c
   C19:1(n-9)T  235.23 ± 3.87^a   156.74 ± 2.06^b   163.27 ± 2.68^b
   C20:0        452.20 ± 3.01^a   236.39 ± 3.88^b   181.82 ± 2.39^c
   C18:3(n-6)   379.23 ± 1.95^a   387.12 ± 3.12^a   321.16 ± 4.29^b
   C20:1        221.64 ± 2.80^a   54.69 ± 1.98^c    125.86 ± 3.21^b
   C20:1 T      408.16 ± 3.15^a   258.51 ± 4.45^b   109.69 ± 3.56^c
   C18:3(n-3)   12.97 ± 0.26^a    12.89 ± 0.41^a    8.56 ± 0.27^b
   C21:0        457.68 ± 3.02^a   393.98 ± 6.79^b   251.41 ± 3.35^c
   C20:2        145.65 ± 1.93^a   130.16 ± 1.49^b   88.71 ± 1.50^c
   C22:0        103.63 ± 1.30^b   114.00 ± 1.63^a   82.64 ± 1.32^c
   C22:1        802.10 ± 5.28^a   719.07 ± 7.21^b   439.85 ± 6.95^c
   C22:1 T      5010.25 ± 19.43^b 5437.07 ± 7.08^a  3981.74 ± 8.15^c
   C20:3(n-3)   4.28 ± 0.12^b     3.85 ± 0.15^b     4.99 ± 0.13^a
   C20:4        200.49 ± 1.65^a   187.94 ± 1.26^b   132.58 ± 1.75^c
   C23:0        917.95 ± 4.25^a   878.10 ± 3.90^b   598.14 ± 4.88^c
   C22:2        1908.57 ± 6.02^a  1535.23 ± 6.83^b  1385.66 ± 7.71^c
   C20:5        385.78 ± 3.71^a   260.23 ± 3.90^b   238.30 ± 4.17^c
   C22:4        315.92 ± 3.84^a   259.76 ± 2.88^b   213.02 ± 2.66^c
   C22:5(n-3)   14.42 ± 0.32^a    12.95 ± 0.20^b    15.03 ± 0.38^a
   C22:5(n-6)   122.00 ± 3.46^b   136.54 ± 2.54^a   113.44 ± 2.28^b
   C24:0        2780.30 ± 7.22^b  3026.61 ± 16.40^a 2372.92 ± 19.05^c
   C24:1        1555.23 ± 12.18^a 1388.60 ± 17.62^b 830.48 ± 18.54^c
   C22:6        28.28 ± 1.52^a    16.43 ± 0.69^b    14.23 ± 0.39^c
   [71]Open in a new tab

   Note: data are presented as MEAN ± SEM, n = 3 repeats, * means
   p < 0.05.

   Comparing with control group, the contents of C11:0, C12:0, C13:0,
   C14:0, C14:1, C14:1 T, C15:0, C16:0, C16:1, C17:0, C17:1, C17:1 T,
   C18:1(n-12), C18:1(n-9), C18:1(n-7), C18:1(n-12)T, C18:1(n-7)T,
   C18:2(n-6), C19:1(n-12)T, C19:1(n-9)T, C20:0, C20:1, C20:1 T,
   C18:3(n-3), C21:0, C20:2, C22:0, C22:1, C22:1 T, C20:4, C23:0, C22:2,
   C20:5, C22:4, C24:0, C24:1, C22:6 were significantly lower in TCA
   treated cells, while the content of C10:0, C18:0, C18:1(n-9)T,
   C20:3(n-3) were significant higher (p<0.05) ([72]Table 3).

   When comparing with CA treated cells, the contents of C15:0, C16:0,
   C16:1, C17:0, C17:1, C18:0, C18:1(n-12), C18:1(n-9), C18:1(n-12)T,
   C18:1(n-9)T, C18:1(n-7)T, C18:2(n-6), C19:1(n-12)T, C20:0, C18:3(n-6),
   C20:1 T, C18:3(n-3), C21:0, C20:2, C22:0, C22:1, C22:1 T, C20:4, C23:0,
   C22:2, C20:5, C22:4, C22:5(n-6), C24:0, C24:1 and C22:6 were
   significantly lower in TCA treated cells, while the content of C10:0,
   C11:0, C15:1, C16:1 T, C20:1, C20:3(n-3) and C22:5(n-3) were
   significantly higher in TCA treated cells when comparing with CA
   treated cells (p<0.05) ([73]Table 3).

4. Discussion

   The intramuscular content of fatty acid and its composition are
   important indicators of meat quality, which also play essential roles
   in metabolic homeostasis ([74]Valdes-Hernandez et al., 2023). In this
   study, we have found that high-concentrate diet changed the metabolite
   composition of lamb. Bile acids played an essential role in regulating
   fatty acid composition. Both CA and TCA decrease the contents of
   various species of fatty acids. These results highlight an attractive
   characteristic of bile acid in regulating lamb fatty acid accumulation.

   Recently, bile acids have emerged as important regulators of various
   physiological and pathological processes. Diet nutrition is known to
   influence bile acid secretion, however, current understanding to the
   molecular species of bile acid remains unclear ([75]Yoshitsugu et al.,
   2019). In mice, it has been reported that high-fat diet changes gut
   microbiota composition and metabolism, which, in turns, influences bile
   acid composition ([76]Ocvirk & O'Keefe, 2021). In pig, it has been
   reported that dietary fatty acids changed the bile acid composition of
   liver, plasma, and colon ([77]Manjarin et al., 2022). Similar to
   previous studies, we found the composition of bile acid in lamb changed
   after fed with high concentrate diet, while Metabolomics analysis also
   showed that biosynthesis of primary bile acid, metabolism of taurine
   and hypotaurine, and cholesterol metabolism were key KEGG pathways that
   high concentrate diet influences lamb. Since cholesterol is a precursor
   of bile acids, these evidences indicate that bile acids are important
   regulator in lamb fed with high concentrate diet.

   Since FXR was reported low expression in skeletal muscle ([78]Perino &
   Schoonjans, 2022), TGR5 and S1PR2 are considered the key receptors that
   interacts with bile acids in skeletal muscle ([79]Kitada et al., 2016;
   [80]Maldonado et al., 2023). TGR5 is highly expressed in the muscle,
   adipose and intestine, which participates in the stimulation energy
   metabolism. Bile acids can induce type 2 iodothyronine deiodinase
   expression in skeletal muscle cell via a TGR5-dependent manner
   ([81]Watanabe et al., 2006). D2 then induces uncoupling protein
   expression by converting thyroxine to tri-iodothyronine, which finally
   dissipate the proton gradient in electron transport chain and influence
   energy homeostasis ([82]Fujiwara et al., 2023). Thus, activation of
   TGR5 by bile acids or its agonist inhibits fatty acid uptake and
   decreases lipid accumulation ([83]Wang et al., 2024). S1PR2 is the
   receptor for bile acids conjugated with taurine including TCA, TDCA and
   TUDCA ([84]Yang, Yu, et al., 2023). The conjugation of taurine with
   bile acids increase the polarity of bile acids, which facilitates the
   absorption of lipid and lipid-solved vitamins, and reduces the
   cytotoxicity of hydrophobic bile acids ([85]Vettorazzi et al., 2016).
   It has been reported that high-fat diet triggers the production bile
   acids conjugated taurine in the bile acid pool ([86]Devkota et al.,
   2012). Activation of S1PR2 by taurine-conjugated bile acids increases
   sphingosine kinase expression, which promotes lipid metabolism
   ([87]Studer et al., 2012). In this study, we found the content of TDCA,
   TCDCA, and TCA increased in HCD lamb, while the content of LCA, CA,
   CDCA, and CDCA-3Gln decreased in HCD lamb. Furthermore, the content of
   plenty species of fatty acids decreased. These evidences indicated that
   HCD decrease fatty acid accumulation in lamb by changing the
   composition of bile acid.

   Our in vitro study showed that both CA and TCA decreased the content of
   various species of fatty acids, in which more species of fatty acids
   showed relative lower contents in the cells treated with TCA. Taurine
   is not only a free osmolyte, but also a conjugated metabolite, which
   can modulate energy and lipid metabolism through regulating proteins
   involves mitochondrial biogenesis and respiratory function
   ([88]Murakami, 2015). Supplying taurine in diet increases fatty acid
   oxidation ([89]De Carvalho et al., 2021). Previous research studies
   have also found that Taurine-conjugated UDCA could reduce fat
   deposition in hepatocytes ([90]Cui et al., 2023). TUDCA increased
   oxidation of fatty acids via AMPK/HSL signaling pathway in brown fat
   tissue ([91]Dos Reis Araujo et al., 2022). A study on fish also showed
   that high lipid diet supplemented with TCA decreased lipid deposition
   in fish ([92]Xu et al., 2022). These data indicated that
   taurine-conjugated bile acids exhibited a stronger inhibitory effect to
   the accumulation of various species of fatty acid in lamb.

5. Conclusions

   Collectively, the high concentrate diet influences lamb metabolites.
   Our results revealed that high concentrate diet changed the bile acid
   composition by increasing the content of TCA, TCDCA, and TDCA, and
   decreasing the content of CA, LCA, CDCA, and CDCA-3Gln in lamb. This
   composition changes of bile acids resulted in the decrease of fatty
   acids accumulation in lamb longissimus dorsi. Our data also indicated
   that taurine-conjugated bile acids exhibited a stronger inhibitory
   effect to fatty acid accumulation in lamb.

CRediT authorship contribution statement

   Kaizhi Zheng: Writing – review & editing, Writing – original draft,
   Investigation. Liangyong Guo: Data curation, Conceptualization. Yang
   Cao: Data curation, Conceptualization. Yuyang Yin: Methodology,
   Investigation. Hui Gao: Methodology, Investigation. Xiaowei Zhang:
   Methodology, Investigation. Junfang Jiang: Investigation. Jinbing Li:
   Investigation. Xin Huang: Funding acquisition. Kui Li: Supervision.
   Sangang He: Project administration.

Declaration of competing interest

   The authors declare that they have no known competing financial
   interests or personal relationships that could have appeared to
   influence the work reported in this paper.

Acknowledgements