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Mulberry leaf supplementation inhibits skatole deposition by regulating gut microbiota and upregulating liver cytochrome P450 1A1 expression in finishing pigs
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Yuqing Suna, Xiaoming Menb, Tianbao Lina, Bo Dengb, Shi Zhonga, Jinxi Huoa, Kaipeng Qinb, Zhiqiang Lva, Ziwei Xub, *, Yougui Lia, *
Animal Nutrition | 2024, 16(1) : 34 - 44
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Animal Nutrition | 2024, 16(1): 34-44
• Original Research Article •
Mulberry leaf supplementation inhibits skatole deposition by regulating gut microbiota and upregulating liver cytochrome P450 1A1 expression in finishing pigs
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Yuqing Suna, Xiaoming Menb, Tianbao Lina, Bo Dengb, Shi Zhonga, Jinxi Huoa, Kaipeng Qinb, Zhiqiang Lva, Ziwei Xub, *, Yougui Lia, *
Affiliations
  • aInstitute of Sericulture and Tea, Zhejiang Academy of Agricultural Sciences, 298 Deshengzhong Road, Shangcheng, Hangzhou 310021, China
  • bInstitute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, 298 Deshengzhong Road, Shangcheng, Hangzhou 310021, China
Published: 2024-03-10 doi: 10.1016/j.aninu.2023.05.018
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Skatole, a strong fecal odor substance, is generated through microbial degradation of tryptophan in the animal hindgut. It easily accumulates in adipose tissue and affects meat quality. In this study, the effect of mulberry leaf supplementation on skatole in finishing pigs was studied. In a 35-day trial, 20 finishing pigs (barrows and gilts) were fed with a basal diet or basal diet with 6% mulberry leaves. Growth performance of the pigs (n = 10) was automatically recorded by a performance-testing feeder system and 8 pigs in each treatment were slaughtered and sampled for the remaining tests. Skatole and short-chain fatty acids were detected using HPLC and gas chromatography, respectively. Fecal microbiota were analyzed using 16S rRNA gene sequencing. The metabolomics analysis of feces and serum was performed with UHPLC-MS/MS. The major cytochrome P450 (CYP) enzymes that catalyze skatole degradation in the liver were tested by using RT-PCR and Western blot. Effects of major bioactive compounds in mulberry leaves on the CYP genes were verified in the hepatic cell line HepG2 in an in vitro test (n = 3). In finishing pigs, mulberry leaf supplementation had no significant effect on the average daily gain, average daily feed intake, and feed conversion ratio (P > 0.05), but reduced skatole levels in feces, serum, and backfat (P < 0.05), and increased acetic acid levels in feces (P = 0.027). Mulberry leaf supplementation decreased the relative abundance of the skatole-producing bacteria Megasphaera and Olsenella (P < 0.05). Indole-3-acetic acid, the intermediate that is essential for skatole production, was significantly reduced in feces by mulberry leaf supplementation (P < 0.05) and was positively correlated with skatole content in feces (P = 0.004). In pigs treated with mulberry leaves, liver CYP1A1 expression was increased (P < 0.05) and was negatively correlated with skatole content in backfat (P = 0.045). The in vitro test demonstrated that mulberry leaf polyphenols and polysaccharides could directly stimulate CYP1A1 expression in hepatic cells. These findings suggest that mulberry leaf supplementation reduces skatole production and deposition in finishing pigs by regulating the gut microbiota and promoting skatole degradation in liver.

Pig  /  Skatole  /  Mulberry leaf  /  Microbiota  /  Cytochrome P450
Yuqing Sun, Xiaoming Men, Tianbao Lin, Bo Deng, Shi Zhong, Jinxi Huo, Kaipeng Qin, Zhiqiang Lv, Ziwei Xu, Yougui Li. Mulberry leaf supplementation inhibits skatole deposition by regulating gut microbiota and upregulating liver cytochrome P450 1A1 expression in finishing pigs[J]. Animal Nutrition, 2024 , 16 (1) : 34 -44 . DOI: 10.1016/j.aninu.2023.05.018
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1.　Introduction
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Skatole (3-methylindole) is a fecal-odor-causing substance that is produced by the microbial degradation of tryptophan in the animal gut. It is the main substance causing the unpleasant odor of pork and one of the main odor substances emitted by swine manure during pig farming (Ma et al., 2021). Skatole can be perceived by humans at very low concentrations. For example, skatole concentrations of as low as 26 ng/g in fat are easily detected by the human nose (Annor-Frempong et al., 1997), and the olfactory threshold of skatole in air is reported to be less than 0.0004 μg/m3 (Rosenfeld et al., 2007). Skatole produced in the gut is partly excreted through feces and partly absorbed by the gut. The absorbed skatole is transported to the liver through the portal vein, then rapidly degraded by cytochrome P450 (CYP) enzymes and excreted through urine (Lanthier et al., 2007). Skatole that is not degraded in the liver is further transported to the peripheral blood and deposited in the body. Skatole accumulates easily in adipose tissue due to its lipophilic characteristics, thus causing a reduction in pork quality (Meinert et al., 2017; Morlein et al., 2012; Rowe et al., 2014). Therefore, decreasing the production and deposition of skatole in pigs is of great importance for the environment and pork quality.
Mulberry, an important cash crop in China, has been used as silkworm feed and as a traditional Chinese medicine since ancient times. Mulberry leaves contain numerous active components, such as polyphenols (PP), polysaccharides (PS), alkaloids (AL), flavonoids, and steroids, and have been reported to have various physiological activities, including antidiabetic, antioxidant, anti-hyperlipidemic, and antiatherosclerotic activities (Chan et al., 2016). The application of mulberry leaves in pig diets has also been reported. We previously reported that the inclusion of mulberry leaves in feed could improve meat quality by decreasing serum triglyceride and cholesterol and increasing inosinic acid and intramuscular fat content in muscle in finishing pigs (Li et al., 2012). Other studies reported that in finishing pigs, the mulberry leaf diet improves antioxidative capacity; inhibits lipid oxidation; increases polyunsaturated fatty acid content in muscle (Liu et al., 2021); and decreases the shear force, cooking loss, and drip loss of muscle (Zeng et al., 2019). Some compounds in mulberry leaves are also beneficial to pigs. For example, mulberry leaf PS can improve the ecology of the gut microbiota, reduce diarrhea rate, and improve growth performance in early-weanling pigs (Zhao et al., 2015). Mulberry leaf flavonoids can improve growth performance, meat tenderness, and polyunsaturated fatty acid content in finishing pigs (Liu et al., 2022). Song et al. (2023) recently found that mulberry leaf extract could reduce odorous substances in feces of weaned piglets. Although the application of mulberry leaves in pig feed has been reported, the effects of mulberry leaves on skatole deposition in finishing pigs have not been reported. In the present study, we explored the effects of mulberry leaves on skatole in finishing pigs and investigated their underlying mechanism by using 16S rRNA sequencing, metabolomics analysis, gene expression analysis, and correlation analysis. We aim to explore new ways to reduce skatole deposition in finishing pigs and to expand the functional application of mulberry leaves.
2.　Materials and methods
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2.1.　 Animal ethics statement
All the experimental procedures applied in this study were in accordance with the guidelines of Farm Animal Welfare Requirements: Pigs (T/CAS 235-2014) promulgated by China Association for Standardization and approved by the Ethics Committee of the Zhejiang Academy of Agricultural Sciences (authorization code number: 2022ZAASLA77). All animal experiments were compliant with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Percie du Sert et al., 2020).
2.2.　 Preparation of mulberry leaves and mulberry leaf extracts
Mulberry leaf powder was supplied by Zhejiang Cathaya International Co., Ltd. (Hangzhou, China) and was prepared as follows: mulberry leaves were dried naturally, crushed through an 80-mesh screen, and stored in a dry environment at room temperature before use. Some of the mulberry leaf powder was extracted to prepare PP, PS, and AL for the cell experiment according to previous methods (Ma et al., 2019; Wu et al., 2013) with some modifications. Firstly, mulberry leaf powder was boiled with 1:50 (wt/vol) ultrapure water, and the supernatant was filtered and concentrated with a rotary evaporator. Ethanol was slowly added to the concentrate to a final concentration of 75%. The mixture was then centrifuged at 12,000 × g at room temperature for 10 min. The supernatant was concentrated with a rotary evaporator, and then dried through lyophilization to obtain mulberry leaf PP. The precipitate was dissolved in hot water, and protein was removed by using isoamyl alcohol:chloroform solution (1:4, vol/vol). Then, the redissolved solution was dried via lyophilization to obtain mulberry leaf PS. For the extraction of total AL, mulberry leaf powder was extracted with 80% ethanol at 1:50 (wt/vol) and 60 °C for 2 h. The supernatant was filtered, and ethanol was removed by using a rotary evaporator. The supernatant was collected through centrifugation and then adsorbed with cation exchange resins for 24 h. After being rinsed with ultrapure water, the resins were eluted with 0.5 mol/L ammonia, and the eluent was collected for the removal of ammonia by using a rotary evaporator. The concentrated eluent was dried through lyophilization to obtain mulberry leaf AL. The total PP content in PP extract was 65.5% as detected by using the Folin–Ciocalteau method (Chamorro et al., 2012). The total PS content in the PS extract was 62.7% as detected by using the phenol–sulfuric acid method (He et al., 2017). The total AL content in the AL extract was 76.6% as detected via the Reinecke salt colorimetry method (Li et al., 2008).
2.3.　 Experimental animals, diets, and design
The pigs used in this experiment were a crossbreed with approximately 30% Chinese native pig genotypes bred by our team. A total of 20 healthy pigs (10 barrows and 10 gilts) aged at 150 ± 10 days with an average body weight (BW) of 75.66 ± 7.89 kg were selected. Pigs in each gender were blocked by BW into 2 blocks with similar BW, and pigs in each block were randomly assigned to 2 dietary treatment groups in a completely randomized block design resulting in 10 animals (5 barrows and 5 gilts) per treatment. All pigs were raised in the same environmentally controlled fattening house. Pigs in the same treatment were kept in 1 pen equipped with an Osborne FIRE performance testing feeder system (Osborne, USA) that could automatically record the BW and feed intake of every single pig. Pigs were fed with a basal diet (CON group) or basal diet with 6% mulberry leaves (MB group) for 35 days. The diets were formulated in accordance with the nutrient requirements of swine (meat-type pig) recommended by the National Standards of the People's Republic of China (GB/T 39235-2020). The composition and nutritional levels of the experimental diets are shown in Tables 1 and 2. The test methods of nutrient levels of the diets are supplied in the Supplementary materials. The pigs were allowed to eat and drink freely during the experiment and were vaccinated in accordance with the requirements of the farm. The BW and feed intake of pigs were automatically recorded by using the Osborne FIRE performance testing feeder system. The average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated (n = 10 per treatment).
2.4.　 Sample collection and processing
At the end of the experiment, pigs were slaughtered humanely after electrical stunning in a commercial slaughterhouse. In order to save the cost of subsequent tests, 8 pigs (4 barrows and 4 gilts) in each treatment group were randomly selected for sample collection under the premise that the sample size was sufficient for statistical analysis. The samplers were blinded to the animal experimental design. The blood, rectal feces, liver, backfat, and longissimus dorsi muscle of the selected pigs were sampled. Blood was kept at room temperature for 3 h to allow clotting, and then centrifuged at 3500 × g at 4 °C for 10 min to obtain serum. The serum samples were stored at −80 °C. Part of longissimus dorsi muscle samples were measured for meat color (CIE L*a*b*), drip loss, mill loss, cooking loss, and intramuscular fat content according to the method in Technical Specification for Determination of Pork Quality (NY/T 821-2019) promulgated by the Ministry of Agriculture of the People's Republic of China. Other samples were frozen in liquid nitrogen immediately, and then stored at −80 °C. For all samples, the testers were blinded to the experimental design.
2.5.　 Detection of biochemical indices in serum and muscle
Total protein (TP), albumin (ALB), total cholesterol (TC), triglyceride (TG), nonesterified fatty acid (NEFA), total antioxidant capacity (TAOC), and malondialdehyde (MDA) in serum or muscle (n = 8 per treatment) were tested respectively by total protein quantitative assay kit (A045-2-2), albumin assay kit (A028-2-1), total cholesterol assay kit (A111-1-1), triglyceride assay kit (A110-1-1), nonesterified free fatty acids assay kit (A042-2-1), total antioxidant capacity assay kit (A015-1-2), and malondialdehyde assay kit (A003-1-2) purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) in accordance with the manufacturer's instructions.
2.6.　 Detection of skatole
Skatole in feces: approximately 0.3 g of fecal sample (n = 8 per treatment) was weighed into a 2-mL centrifuge tube, then 1.5 mL of methanol and 2 steel balls were added. Subsequently, the mixture was homogenized at 60 Hz for 60 s and centrifuged at 13,500 × g at room temperature for 5 min. The supernatant was filtered through a 0.45-μm filter and transferred to a headspace vial for testing.
Skatole in serum: aliquots of 1 mL of serum (n = 8 per treatment) were added into 2-mL centrifuge tubes, mixed with 1 mL of diethylether, rigorously vortexed for 1 min, and centrifuged at 13,500 × g at room temperature for 5 min. The diethylether phase was separated and dried under a gentle stream of nitrogen at 30 °C, redissolved in 500 μL of methanol, and rigorously vortexed. The samples were further purified by a Microcon 30 kDa centrifugal filter device (Millipore, USA) via centrifugation at 13,500 × g at room temperature for 10 min. A 400 μL aliquot of the extract was diluted with 400 μL of acetonitrile:water solution (60:40, vol/vol). The diluted extract was filtered through a 0.45-μm filter and transferred to a headspace vial for testing.
Skatole in muscle: longissimus dorsi muscle (n = 8 per treatment) was vacuum freeze-dried and ground into powder. Approximately 0.3 g of muscle powder was weighed into a 2-mL centrifuge tube then 1.5 mL of methanol and 2 steel balls were added, then homogenized at 60 Hz for 60 s, and centrifuged at 13,500 × g at room temperature for 5 min. The supernatant was filtered through a 0.45-μm filter and transferred to a headspace vial for testing.
Skatole in backfat: approximately 1 g of backfat (n = 8 per treatment) was weighed into a 2-mL centrifuge tube and microwaved on medium–high for 3 min twice. The melted fat was pipetted into another 2-mL centrifuge tube and weighed, and the tube was swirled for 30 s after the addition of 0.5 mL of hexane. A 1.2 mL aliquot of acetonitrile:water solution (75:25, vol/vol) was added. Then, the mixture was vortexed for 30 s and centrifuged at 13,500 × g at room temperature for 5 min. Approximately 0.5 to 1.0 mL of the lower aqueous phase was pipetted into a headspace vial for testing.
An ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm) column (Agilent, USA) was used for HPLC under the following conditions: column temperature of 40 °C, injection volume of 10 μL, mobile phase of acetonitrile:water (60:40, vol/vol), flow rate of 0.8 mL/min, and FLD detector excitation wavelength of 270 nm and emission wavelength of 360 nm.
2.7.　 Detection of short-chain fatty acids (SCFA) in feces
Acetic, propionic, butyric, isobutyric, and isovaleric acids in feces (n = 8 per treatment) were tested by using gas chromatography. Approximately 2 g of fecal sample was weighed into a 15-mL centrifuge tube, then 10 mL of ultrapure water was added, and the mixture was vortexed. Then the mixture was centrifuged at 12,000 × g at room temperature for 5 min, and the supernatant was separated. An aliquot (2 mL) of the supernatant was mixed with 0.2 mL of 50% sulfuric acid and 2 mL of diethylether in a 10-mL centrifuge tube. The mixture was centrifuged at 12,000 × g at room temperature for 5 min. Then, the supernatant was separated for testing. SCFA were tested with a gas chromatograph (Agilent model 7890, Palo Alto, CA, USA) coupled with a flame ionization detector and capillary Nukol column (30 m × 0.25 mm × 0.25 μm, Supelko, Bellefonte, PA, USA). The chromatographic conditions were as follows: injector temperature of 270 °C, detector temperature of 280 °C, injection volume of 2 μL, splitless mode, and carrier gas of nitrogen at 2.0 mL/min. The column oven was programmed as follows: 100 °C for 1 min, followed by heating to 150 °C at speed of 5 °C/min and holding for 5 min.
2.8.　 Gut microbiota analysis by 16S rRNA gene sequencing
The microbial genomic DNA of rectal feces (n = 8 per treatment) was isolated and purified by using a QIAamp DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany). The V3–V4 hypervariable regions of the 16S rRNA genes were PCR-amplified by using primers B341F and B806R (B341F: CCTACGGGNGGCWGCAG, B806R: GGACTACHVGGGTATCTAAT). The resulting PCR products were gel purified, quantified, pooled, and sequenced. Sequencing libraries were generated by using a NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, USA). The library was sequenced on an Illumina PE250 platform. The resulting sequences were clustered into operational taxonomic units (OTU) by using UPARSE software at 97% sequence similarity. The taxonomy of each 16S rRNA gene sequence was determined by the RDP Classifier (http://rdp.cme.msu.edu/) with confidence greater than 70%. Data processing and analysis were performed by using the Omicsmart cloud platform (Guangzhou Gene Denovo Biotechnology Co., Ltd., Guangzhou, China).
2.9.　 Nontargeted metabolomics analysis of feces and serum
For feces (n = 8 per treatment), 100 mg of a sample was resuspended with 500 μL of 80% methanol (prechilled at 4 °C) by well vortexing. For serum (n = 8 per treatment), 100 μL of a sample and 400 μL of prechilled 80% methanol were mixed in a 1.5-mL centrifuge tube by well vortexing. The following steps were the same for fecal and serum samples. The samples were incubated on ice for 5 min then centrifuged at 15,000 × g and 4 °C for 20 min. The supernatant was transferred into a new tube and diluted with distilled water to the final methanol concentration of 53%, then centrifuged at 15,000 × g and 4 °C for 20 min. The supernatant was then ready for testing by the UHPLC–MS/MS system.
UHPLC–MS/MS analyses were performed by using a Vanquish UHPLC system (Thermo Fisher Scientific, Germany) coupled with a Hypesil Gold column (100 × 2.1 mm, 1.9 μm) and an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Germany) in Gene Denovo Co., Ltd. (Guangzhou, China). The samples were loaded by using a 17-min linear gradient at a flow rate of 0.2 mL/min. For the positive polarity mode, the eluents were 0.1% formic acid in water (eluent A) and methanol (eluent B). For the negative polarity mode, the eluents were 5 mM ammonium acetate (eluent A) and methanol (eluent B). The elution process was as follows: 2% B, 0–1.5 min; 2%–100% B, 1.5–12.0 min; 100% B, 12.0–14.0 min; 100%–2% B, 14.0–14.1 min; 2% B, 14.1–17 min. The ESI parameters were as follows: spray voltage of 3.2 kV, sheath gas flow rate of 40 arb, aux gas flow rate of 10 arb, and capillary temperature of 320 °C. The MS/MS secondary scans were data-dependent scans, and the scan range was M/Z 100–1500. The raw data files generated by UHPLC–MS/MS were processed by using Compound Discoverer 3.1 (CD3.1, Thermo Fisher Scientific, Germany). Peak intensities were normalized to the total spectral intensity, and peaks were matched with the mzCloud, mzVault, and MassList databases to obtain the accurate qualitative and relative quantitative results.
2.10.　 In vitro cell test
The hepatic cell line HepG2 was obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (Gibco, USA) at 37 °C and 5% CO2. The cells were incubated in 6-well plates with mulberry leaf PP, PS, or AL (100 or 200 μg/L) for 24 h with 3 replicates per treatment. Then, the culture medium was removed, and the cells were stored at −80 °C before testing.
2.11.　 Quantitative real-time polymerase chain reaction (qRT-PCR)
The gene expression levels of CYP genes were analyzed by using qRT-PCR as previously reported (Sun et al., 2021). The total RNA from pig liver (n = 8 per group) or HepG2 cells (n = 3 per group) was isolated by using a universal mRNA purification kit (TaKaRa, Japan) and reverse transcribed into cDNA by using a reverse transcription reagent kit with gDNA Eraser (TaKaRa, Japan). The qRT-PCR was performed by using the SYBR Fast qPCR Mix (TaKaRa, Japan) and the StepOne Plus real-time PCR system (Life Technologies, Carlsbad, CA, USA). The amplification conditions were denaturation at 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The gene-specific primers used in qRT-PCR are shown in Tables 3 and 4. The expression level of each target gene was normalized to the housekeeping gene 18S rRNA (for pig liver) or GAPDH (for HepG2 cells) by using the 2−△△Ct method.
2.12.　 Western blot analysis
The TP of liver were extracted with the Minute Total Protein Extraction Kit for Animal Cultured Cells/Tissues (Invent, USA), and protein concentrations were determined by using a BCA protein assay kit (Beyotime, China). Protein extracts were adjusted to the same protein concentration. As a total of 16 samples could not be all loaded into 1 protein gel and 3 replicates for Western blot trial are usually conducted in most studies, the protein extracts from 8 pigs under 1 treatment were mixed into 3 samples by mixing every 2 or 3 extracts into 1 sample. The mixed samples were then denatured with loading buffer at 95 °C for 5 min. Protein samples (n = 3) were loaded into each well of a 4%–12% SDS polyacrylamide gel (Invent, USA) then transferred to a nitrocellulose membrane (Invent, USA) by using an IBlot Dry transfer system (Life Technologies, USA). After blocking with SuperBlock T20 blocking buffer (37516, Thermo Fisher Scientific, USA) for 1 h at room temperature, the membranes were incubated with the corresponding primary antibodies at room temperature for 1 h. The membranes were washed 3 times with 1× TBST buffer (20 mmol/L Tris–HCl [pH 7.6], 8 g/L NaCl, and 0.1% Tween-20) for 5 min each time then incubated with the secondary antibody at room temperature for 1 h. The primary antibodies were rabbit CYP1A1 (1324-1-AP, Proteintech, China) and β-actin (ab8227, Abcam, UK). The secondary antibody was Alexa Fluor 790 goat antirabbit IgG H&L (ab175781, Abcam, UK). The membranes were imaged after another 3 washes by using a ChemiDoc touch imaging system (BIO-RAD, USA). Protein expression levels were detected by using ImageLab software.
2.13.　 Data analysis
Data were expressed as means ± SD or medians with interquartile ranges. Differences in bacterial abundance were analyzed by using Welch's test. Microbial Tax4Fun function predictions were analyzed via the Wilcoxon rank sum test. For phylotype analysis, the alpha diversity of the microbiome was calculated on the basis of OTU level by using mothur (version 1.30.1). Principal coordinate analysis (PCoA), the nonmetric multidimensional scaling (NMDS) analysis of the microbiome, and the partial least squares discriminant analysis (PLS-DA) of the metabolome were performed and visualized by R, and significant differences were evaluated by conducting Adonis analysis. P-values were adjusted for multiple comparisons by using the Benjamini-Hochberg correction. The false discovery rate and significance were set at q < 0.05. Other statistical analyses were performed by using Student's t-test with SPSS, version 16 (SPSS Inc., Chicago, IL, USA). The Pearson correlation coefficient was used for correlation analysis. The P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely significant.
3.　Results
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3.1.　 Growth performance, meat quality traits and biochemical indices in serum and muscle
For growth performance, no significant difference in initial BW, final BW, ADG, ADFI, and FCR (P > 0.05) between CON and MB groups were observed (Table 5), and none of these results showed differences between the two groups when analyzed separately by sex (Table S1). For meat quality traits of longissimus dorsi muscle, the meat color L* was decreased (P = 0.018) in MB group compared to CON group, while the meat color a*, meat color b*, drip loss, mill loss, cooking loss and intramuscular fat did not show significant differences (P > 0.05) between the two groups (Table S2). Biochemical indices, such as TP, ALB, TC, TG, TAOC and MDA in serum, and TP, TC, TG, TAOC, MDA and NEFA in muscle, also did not show significant differences (P > 0.05) between the two treatment groups (Fig. S1).
3.2.　 Skatole content in feces, serum, muscle, and backfat
As shown in Table 5, the mulberry leaf treatment significantly reduced the skatole content in feces (P = 0.011), serum (P = 0.018), and backfat (P = 0.010) but had no significant effect on skatole content in muscle (P > 0.05). The skatole contents followed the order of feces > backfat > muscle > serum. When analyzed separately by sex, none of these results showed differences between the two groups (Table S1).
3.3.　 SCFA content in feces
SCFA have been reported to regulate the production of skatole in the gut. Therefore, the SCFA content in feces was measured. As shown in Table 5, mulberry leaf treatment significantly increased the acetic acid content in feces (P = 0.027) but did not affect the contents of propionic, butyric, isobutyric, and isovaleric acids in feces (P > 0.05).
3.4.　 Gut microbial community
Given that skatole is produced by microbes, the gut microbial community in rectal feces was analyzed by using 16S rRNA gene sequencing to reveal the responses of gut microbes in finishing pigs to mulberry leaves. Fig. 1A illustrates that the Chao1 and Shannon indices in the MB group were significantly higher (P < 0.05) than those in the CON group. These results suggested that the richness and diversity of microbiota in the MB group was increased. PCoA and NMDS analyses at the OTU level (Fig. 1 B) revealed that the gut microbial community of the MB group was separate from that of the CON group, indicating that mulberry leaf supplementation changed the gut microbial structure. Fig. 1C illustrates the genera that were significantly downregulated by mulberry leaf supplementation. The correlations of these genera with skatole and acetic acid in feces were also investigated. Nine genera were significantly downregulated by mulberry leaf supplementation. Two of these genera, namely, Olsenella and Megasphaera, were skatole-producing bacteria that have been previously reported. Olsenella showed a significant positive correlation with skatole in feces. Taxonomy analysis demonstrated that Olsenella belonged to family Atopobiaceae in phylum Actinobacteria, and Megasphaera belonged to family Veillonellaceae in phylum Firmicutes. Although Megasphaera was not significantly correlated with skatole in feces, Mitsuokella and Anaerovibrio, two genera that also belonged to family Veillonellaceae, showed a significantly positive correlation with skatole in feces. Moreover, Mitsuokella and Anaerovibrio were significantly and negatively correlated with acetic acid in feces (P < 0.01), but further research is needed to reveal their role in skatole production. Coprococcus_3, a genus belonging to family Lachnospiraceae in phylum Firmicutes, also showed a significant positive correlation with skatole in feces. Fig. 1D displays the genera that were significantly upregulated by mulberry leaf supplementation. Six genera were significantly upregulated by mulberry leaf supplementation. Among these genera, Eubacterium_ruminantium_group, a genus belonging to family Lachnospiraceae, showed a significantly negative correlation with skatole in feces. Tax4Fun function prediction analysis indicated that the relative abundance of the OTU related to carbohydrate metabolism had significantly increased, whereas that related to amino acid metabolism had significantly decreased by mulberry leaf supplementation. Other three predicted functions, such as metabolism of cofactors and vitamins, transcription, and substance dependence, also changed, but the relative abundance of OTU related to these functions were much lower (less than 0.10%).
3.5.　 Metabolomics analysis of feces
Changes in microbiota are also reflected by changes in metabolites. Feces were subjected to metabolomics analysis to explore the effect of mulberry leaves on the metabolism of microbiota. Fig. 2A shows that in PLS-DA, the samples in the MB group were separate from those in the CON group, indicating the existence of differences between the compositions of the fecal metabolites in the two groups. Compared with the CON group, 91 metabolites were downregulated and 81 metabolites were upregulated in the MB group (Fig. 2B). The KEGG pathway enrichment of these different metabolites showed that the purine metabolism pathway and drug metabolism–cytochrome P450 pathway had significantly changed (Fig. 2C). A heat map of the differential metabolites in the two pathways is provided in Fig. 2D. Tryptophan degradation metabolites, such as indole and indole derivatives, were compared between the two groups to understand the anabolic processes of skatole. As shown in Fig. 2E, indole-3-acetic acid (IAA), the precursor compound of skatole (Jensen et al., 1995a), had significantly decreased under mulberry leaf treatment (P < 0.05), whereas 10 other metabolites were not significantly affected (P > 0.05). Moreover, IAA showed a significantly positive correlation with skatole content in feces (P = 0.004; Fig. 2F).
3.6.　 Metabolomics analysis of serum
Metabolomics analysis was performed on serum to further investigate the effect of mulberry leaf treatment on metabolites in the blood. Similar to that in the fecal metabolomics analysis, in PLS-DA, the samples of the MB group were distinct from those of the CON group, indicating variations in the composition of serum metabolites between the two groups (Fig. 3A). A total of 48 metabolites in the MB group were significantly changed compared to those in the CON group, of which 10 were downregulated and 38 were upregulated (Fig. 3B). The KEGG pathway enrichment analysis of the differential metabolites (Fig. 3C) showed that the phenylalanine, tyrosine, and tryptophan biosynthesis; protein digestion and absorption; cyanoamino acid metabolism; phenylalanine metabolism; and aromatic compound degradation pathways had significantly changed (P < 0.05). A heat map of the differential metabolites in these pathways is provided in Fig. 3D. Indole and indole derivatives with levels that significantly differed between groups are presented in Fig. 3E. DL-tryptophan, indole, and indole-3-lactic acid in the serum of the MB group had significantly increased compared with those in the CON group (P < 0.05). Their correlation with skatole content in serum was analyzed to explore the relationship between these indole derivatives and skatole. Fig. 3F shows that DL-tryptophan, indole, and indole-3-lactic acid were all negatively correlated with skatole content in serum (P < 0.05).
3.7.　 Expression of CYP in the liver and hepatic cell line HepG2
The gene expression of major CYP enzymes that were demonstrated to catalyze skatole degradation in the liver was examined to determine whether mulberry leaves have an effect on skatole degradation in finishing pigs. As shown in Fig. 4A, the mRNA expression of CYP1A1 significantly increased in the MB group (P < 0.05), whereas that of CYP1A2, CYP2E1, and CYP2A did not significantly change (P > 0.05). CYP can be inhibited by androstenone in intact males, therefore leading to a higher skatole concentration in intact males than in females. In our study, the male pigs were castrated, and gene expression of these CYP did not show differences between sexes (Table S1).
Correlation analysis (Fig. 4B) revealed that the gene expression of CYP1A1 was negatively correlated with skatole concentration in fat (P = 0.045) but was not significantly correlated with skatole in serum (P > 0.05). The upregulation of CYP1A1 in pigs under mulberry leaf treatment was also verified at the protein level through Western blot analysis (Fig. 4C). The effects of major bioactive compounds in mulberry leaves on CYP genes were tested in vitro by using the hepatic cell line HepG2. As shown in Fig. 4D, PP and PS upregulated CYP1A1 mRNA expression in HepG2 cells in a dose-dependent manner (P < 0.01), and the regulatory effect of PP was considerably stronger than that of PS. By contrast, both doses of AL downregulated CYP1A1 expression (P < 0.01). The expression levels of CYP1A2, CYP2E1, and CYP2A were not significantly affected by treatment with PP, PS, or AL (P > 0.05).
4.　Discussion
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We previously investigated the effects of basal diets containing 10% or 20% mulberry leaves on the growth performance, fat metabolism, and meat quality of finishing pigs in a 65-day trial. We found that in finishing pigs, the 20% mulberry leaf diet reduced ADG and increased FCR, whereas the 10% mulberry leaf diet did not affect ADG and FCR (Li et al., 2012). The 10% and 20% mulberry leaf diets decreased leaf lard percentage and backfat thickness and increased intermuscular fat content (Li et al., 2012). Liu et al. (2021) reported that 3%, 6%, and 9% mulberry leaf diets had no adverse effects on the growth performance of finishing pigs, whereas a 12% mulberry leaf diet decreased growth performance in a 50-day feed experiment. In addition, the serum antioxidant properties were improved linearly with an increase in the amount of mulberry leaves included in the diet (Liu et al., 2021). In an 85-day trial on finishing pigs, a 15% mulberry leaf diet decreased ADG and increased FCR but improved meat quality by changing myofiber characteristics, enhancing antioxidative capacity, and increasing intramuscular fat content (Zeng et al., 2019). In this study, feeding 6% mulberry leaf in the diet for 35 days had no significant effect on growth performance, but significantly decreased the meat color L* value of longissimus dorsi muscle in finishing pigs. The meat color L* value represents the brightness of the meat. Pale, soft and exudative (PSE) meat has high L* value and low a* value. Our results suggested that mulberry leaf in the diet improved meat color to some extent. Mulberry leaf inclusion did not affect drip loss, mill loss, cooking loss or intramuscular fat of longissimus dorsi muscle. The antioxidative capacity of serum and muscle were also unaffected by a 6% mulberry leaf diet. This may be due to the relatively low content of mulberry leaves in the diet and the shorter feeding time in this study compared to previous studies.
We observed a significant reduction in skatole content in the feces, serum, and backfat of mulberry leaf-treated pigs. Skatole is produced from tryptophan by microbes in the hindgut (Ma et al., 2021). Undigested carbohydrates, such as fibers, PS, and oligosaccharides, have been proven to regulate gut microbiota and reduce skatole production (Bekkelund et al., 2022; Jensen et al., 1995b; Lösel et al., 2006; Vhile et al., 2012). Undigested carbohydrates are thought to inhibit skatole production in two ways: First, skatole-producing bacteria preferentially utilize undigested carbohydrates, thereby reducing the utilization of tryptophan (Jensen et al., 1995b). Second, the increase in SCFA in the hindgut as a result of undigested carbohydrate fermentation reduces the pH of the gut and therefore leads to the bacterial metabolism of tryptophan into indole instead of skatole (Doerner et al., 2009; Vhile et al., 2012). Mulberry leaves are rich in dietary fiber and PS and may reduce skatole production by regulating the microbiota. In this study, mulberry leaf treatment changed the richness, diversity, and structure of the microbiota and decreased the abundance of the skatole-producing bacteria Olsenella and Megasphaera. To our knowledge, only a few skatole-producing bacteria have been identified thus far. Lactobacillus, Clostridium, Olsenella, and Megasphaera are the major known skatole-producing genera (Ma et al., 2021). In genus Olsenella, Olsenella uli DSM 7084 and Olsenella scatoligenes SK9K4 are two known skatole-producing strains that were first isolated from human gingival crevices and pig feces, respectively (Li et al., 2015; Olsen et al., 1991). Megasphaera is a genus belonging to family Veillonellaceae. The strain Megasphaera sp. TrE9262, which was first isolated from sheep, has been demonstrated to produce skatole (Attwood et al., 2006). However, not all bacteria that belong to the genus Megasphaera produce skatole. For example, Megasphaera sp. strain DJF_B143, an isolate from the pig hindgut, cannot produce skatole (Li et al., 2016). This situation may account for the lack of the positive correlation between Megasphaera and skatole content in feces. We found that Mitsuokella and Anaerovibrio, another two genera belonging to family Veillonellaceae, were significantly and positively correlated with skatole in feces. Moreover, they decreased in the MB group and were negatively correlated with acetic acid content in feces. However, further research is needed to reveal their role in skatole production. Although we found that Coprococcus 3, a butyrate-producing genus, was also positively correlated with skatole in feces, evidence or literature linking it to skatole production is unavailable. The upregulated genera Eubacterium ruminantium group, Anaeroplasma, Butyrivibrio, and Ruminococcus 1 are all SCFA-producing fibrolytic bacteria (Koike and Kobayashi, 2009; Liang et al., 2021; Luo et al., 2019; Niu et al., 2015) and may partially account for the increase in acetic acid in feces. The enhanced carbohydrate metabolism and weakened amino acid metabolism found by bacterial function prediction analysis also reflected in the change in gut bacteria. In general, this change was unfavorable to skatole production. Our results are consistent with the recent study of Song et al. (2023), who reported that 0.1% mulberry leaf extract supplementation could increase the number of carbohydrate degrading bacteria Lactobacillus and Bifidobacterium and reduce the number of nitrogen compound degrading bacteria Escherichia coli, thus increasing lactate in feces and reducing the formation of NH3–N, biogenic amines, and skatole in feces of weaned piglets.
Skatole-producing bacteria convert tryptophan into skatole through a two-step mechanism. During the first step, tryptophan is deaminated into IAA, and during the second step, IAA is further decarboxylated into skatole (Attwood et al., 2006; Doerner et al., 2009; Li et al., 2015). IAA is the essential intermediate for skatole production. In our study, the IAA level in feces from mulberry leaf-treated pigs significantly decreased. Furthermore, fecal IAA level was significantly and positively correlated with fecal skatole level. This correlation suggested that tryptophan deamination, the first step of skatole production, was suppressed by mulberry leaf supplementation. Tryptophan has been proven to be mostly converted into IAA or indole by bacteria (Doerner et al., 2009; Jensen et al., 1995a; Roager and Licht, 2018). Small amounts of other tryptophan metabolites, such as indole-3-lactic acid, may be also detected (Jensen et al., 1995a, b; Roager and Licht, 2018). Low pH benefits indole production, whereas high pH benefits IAA production (Doerner et al., 2009; Vhile et al., 2012). We observed a significant increase in indole in serum and an insignificant increase in indole in feces in pigs treated with mulberry leaves. Indole-3-lactic acid also increased in the serum of pigs treated with mulberry leaves. The increase in indole and indole-3-lactic acid implied that the conversion of tryptophan into IAA in pigs treated with mulberry leaves was reduced. Moreover, the levels of tryptophan in serum were increased in pigs treated with mulberry leaves. As mentioned earlier, skatole-producing bacteria preferentially utilize undigested carbohydrates instead of tryptophan (Jensen et al., 1995b). Therefore, we inferred that the increase in tryptophan in the serum of pigs treated with mulberry leaves may be due to the reduction in tryptophan utilization by bacteria and the consequent increase in tryptophan absorption by the large intestine. The negative correlation of skatole in serum with tryptophan, indole, and indole-3-lactic acid in the serum can be easily understood on the basis of the above analysis.
Skatole deposition in pigs is not only related to skatole production in the gut lumen by bacteria but also to the catabolism of skatole in vivo (Lanthier et al., 2007). The liver is the main metabolic organ for skatole degradation, and CYP is the key enzyme family catalyzing skatole production in the liver (Lanthier et al., 2007; Wiercinska et al., 2012). CYP isoforms, such as CYP1A, CYP2A, and CYP2E1, have been reported to be responsible for the degradation of skatole (Burkina et al., 2019; Liu et al., 2020; Wiercinska et al., 2012). Factors affecting these CYP enzymes can therefore affect skatole accumulation in the body.
In this study, we detected the gene expression levels of four well-known skatole-degrading CYP enzymes, namely, CYP1A1, CYP1A2, CYP2E1, and CYP2A, in the liver of finishing pigs. We found that the expression of CYP1A1 was significantly upregulated by mulberry leaf treatment. Moreover, CYP1A1 expression level was negatively correlated with skatole content in backfat but was not correlated with skatole content in serum. This result implied that CYP1A1 activation affected the deposition, but not absorption, of skatole. Our results suggested that mulberry leaves may promote skatole degradation by stimulating CYP1A1 expression in the liver of finishing pigs. It is known that boars have higher skatole levels than barrows and gilts because the androstenone secreted by the testicles inhibits the activities of CYP (Lösel et al., 2006; Wesoly et al., 2015; Zamaratskaia et al., 2012). In the present study, barrows and gilts were used as experimental animals because intact male pigs are not used for meat production in China. As castration of boars in some countries is prohibited due to the protection of animal welfare, effects of mulberry leaves on boars need to be studied in the future.
The in vitro test on the hepatic cell line HepG2 further demonstrated that mulberry leaf components, PP, and PS can directly simulate CYP1A1 expression. Considering that PS are large compounds that are probably not absorbed in entirety by the gut, we speculate that PP are likely the substances that effectively activate CYP1A1 in vivo.
5.　Conclusions
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In a 35-day trial on finishing pigs, the 6% mulberry leaf diet did not affect performance but reduced skatole levels in feces, serum, and backfat. Mulberry leaves may reduce skatole production in the gut by decreasing the abundance of the skatole producing bacteria Olsenella and Megasphaera and reducing the conversion of tryptophan into IAA, the precursor of skatole. Moreover, mulberry leaves may promote skatole degradation by stimulating the expression of CYP1A1 in the liver. PP in mulberry leaves may be the active compounds with stimulatory effects on CYP1A1. The above findings suggest that mulberry leaves could reduce odor emission in pig farming and improve pork quality.
References
Less
Annor-Frempong IE, Nute GR, Whittington FW, Wood JD. The problem of taint in pork: detection thresholds and odor profiles of androstenone and skatole in a model system. Meat Sci 1997;46:45-55.
Attwood G, Li D, Pacheco D, Tavendale M. Production of indolic compounds by rumen bacteria isolated from grazing ruminants. J Appl Microbiol 2006;100:1261-71.
Bekkelund A, Kjos NP, Øverland M. Effects of dried chicory and Jerusalem artichoke on skatole-producing microbial populations of entire male pigs. Livest Sci 2022;261:104957.
Burkina V, Zlabek V, Rasmussen MK, Zamaratskaia G. End-product inhibition of skatole-metabolising enzymes cyp1a, cyp2a19 and cyp2e1 in porcine and piscine hepatic microsomes. Toxicol Lett 2019;303:67-71.
Chamorro S, Viveros A, Alvarez I, Vega E, Brenes A. Changes in polyphenol and polysaccharide content of grape seed extract and grape pomace after enzymatic treatment. Food Chem 2012;133(2):308-14.
Chan EW, Lye PY, Wong SK. Phytochemistry, pharmacology, and clinical trials of Morus alba. Chin J Nat Med 2016;14(1):17-30.
Doerner KC, Cook KL, Mason BP. 3-Methylindole production is regulated in Clostridium scatologenes ATCC 25775. Lett Appl Microbiol 2009;48:125-213.
He M, Zeng J, Zhai L, Liu Y, Wu H, Zhang R, et al. Effect of in vitro simulated gastrointestinal digestion on polyphenol and polysaccharide content and their biological activities among 22 fruit juices. Food Res Int 2017;102:156-62.
Jensen MT, Cox RP, Jensen BB. 3-Methylindole (skatole) and indole production by mixed populations of pig fecal bacteria. Appl Environ Microbiol 1995a;61(8):3180-4.
Jensen MT, Cox RP, Jensen BB. Microbial production of skatole in the hind gut of pigs given different diets and its relation to skatole deposition in backfat. Anim Sci 1995b;61(2):293-304.
Koike S, Kobayashi Y. Fibrolytic rumen bacteria: their ecology and functions. Asia Austral J Anim 2009;22(1):131-8.
Lanthier F, Lou Y, Squires EJ. Skatole metabolism in the intact pre-pubescent male pig: the relationship between hepatic enzyme activity and skatole concentrations in plasma and fat. Livest Sci 2007;106:145-53.
Li F, Qiu YY, Qian WC, Lou LJ, Zeng S, Jiang HD. Determination and investigation of total alkaloids and 1-deoxynojirimycin in folium mori. Chin Pharmaceut J 2008;43(3):176-9 [In Chinese].
Li Y, Zhang L, Zhong S, Zhang X, Li Q, Lv Z, et al. Effects of dietary mulberry leaf on growth performance, fat metabolism and meat quality of finishing pigs. Chin J Anim Nutr 2012;24(9):1805-11 [In Chinese].
Li X, Jensen RL, Hojberg O, Canibe N, Jensen BB. Olsenella scatoligenes sp. nov., a 3-methylindole-(skatole) and 4-methylphenol-(p-cresol) producing bacterium isolated from pig faeces. Int J Syst Evol Microbiol 2015;65:1227-33.
Li X, Marshall I, Schreiber L, Højberg O, Canibe N, Jensen BB. Draft genome sequence of megasphaera sp. strain djf_b143, an isolate from pig hindgut unable to produce skatole. Genome Announc 2016;4(1):e00007-e00016.
Liang D, Zhang L, Chen H, Zhang H, Dai X. Potato resistant starch inhibits dietinduced obesity by modifying the composition of intestinal microbiota and their metabolites in obese mice. Int J Biol Macromol 2021;180:458-69.
Liu H, Zhao X, Yang G, Liu J, Zhu X. Effects of dietary stachyose levels on caecal skatole concentration, hepatic cytochrome p450 mrna expressions and enzymatic activities in broilers. Br J Nutr 2020;124(10):1013-20.
Liu Y, Li Y, Xiao Y, Peng Y, He J, Li F, et al. Mulberry leaf powder regulates the antioxidative capacity and lipid metabolism in finishing pigs. Anim Nutr 2021;7(2):421-9.
Liu Y, Xiao Y, Xie J, Peng Y, Li F, Chen C. Dietary supplementation with flavonoids from mulberry leaves improves growth performance and meat quality, and alters lipid metabolism of skeletal muscle in a Chinese hybrid pig. Anim Feed Sci Technol 2022;285:115211.
Lösel D, Lacorn M, Büttner D, Claus R. Flavor improvement in pork from barrows and gilts via inhibition of intestinal skatole formation with resistant potato starch. J Agric Food Chem 2006;54(16):5990-5.
Luo Q, Cheng D, Huang C, Li Y, Lao C, Xia Y, et al. Improvement of colonic immune function with soy isoflavones in high-fat diet-induced obese rats. Molecules 2019;24(6):1139.
Ma Q, Jiang JG, Yuan X, Qiu K, Zhu W. Comparative antitumor and antiinflammatory effects of flavonoids, saponins, polysaccharides, essential oil, coumarin and alkaloids from Cirsium japonicum DC. Food Chem Toxicol 2019;125:422-9.
Ma Q, Meng N, Li Y, Wang J. Occurrence, impacts, and microbial transformation of 3-methylindole (skatole): a critical review. J Hazard Mater 2021;416:126181.
Meinert L, Lund B, Bejerholm C, Aaslyng MD. Distribution of skatole and androstenone in the pig carcass correlated to sensory characteristics. Meat Sci 2017;127:51-6.
Morlein D, Lungershausen M, Steinke K, Sharifi AR, Knorr C. A single nucleotide polymorphism in the CYP2E1 gene promoter affects skatole content in backfat of boars of two commercial Duroc-sired crossbred populations. Meat Sci 2012;92(4):739-44.
Niu Q, Li P, Hao S, Zhang Y, Kim SW, Li H, et al. Dynamic distribution of the gut microbiota and the relationship with apparent crude fiber digestibility and growth stages in pigs. Sci Rep-UK 2015;5:9938.
Olsen I, Johnson JL, Moore LVH, Moore WEC. Lactobacillus uli sp. nov. and Lactobacillus rimae sp. nov. from the human gingival crevice and emended descriptions of Lactobacillus minutus and Streptococcus parvulus. Int J Syst Evol Microbiol 1991;41:261-6.
Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. PLoS Biol 2020;18:e3000410.
Roager HM, Licht TR. Microbial tryptophan catabolites in health and disease. Nat Commun 2018;9(1):3294.
Rosenfeld PE, Clark JJJ, Hensley AR, Suffet IH. The use of an odour wheel classification for the evaluation of human health risk criteria for compost facilities. Water Sci Technol 2007;55:345-57.
Rowe SJ, Karacaören B, de Koning DJ, Lukic B, Hastings-Clark N, Velander I, et al. Analysis of the genetics of boar taint reveals both single SNPs and regional effects. BMC Genom 2014;15(1):424.
Song M, Wang C, Cui Y, Deng D, Yu M, Liu Z, et al. Effects of mulberry leaf extract on number of main microbes and production of odorous substances in feces of weaned piglets. Chin J Anim Nutr 2023;35(2):823-33 [In Chinese].
Sun Y, Huo J, Zhong S, Zhu J, Li Y, Li X. Chemical structure and anti-inflammatory activity of a branched polysaccharide isolated from Phellinus baumii. Carbohydr Polym 2021;268:118214.
Vhile SG, Kjos NP, Sørum H, Øverland M. Feeding Jerusalem artichoke reduced skatole level and changed intestinal microbiota in the gut of entire male pigs. Animal 2012;6(5):807-12.
Wesoly R, Jungbluth I, Stefanski V, Weiler U. Pre-slaughter conditions influence skatole and androstenone in adipose tissue of boars. Meat Sci 2015;99:60-7.
Wiercinska P, Lou Y, Squires EJ. The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism. Animal 2012;6(5):834-45.
Wu CH, Chen SC, Ou TT, Chyau CC, Chang YC, Wang CJ. Mulberry leaf polyphenol extracts reduced hepatic lipid accumulation involving regulation of adenosine monophosphate activated protein kinase and lipogenic enzymes. J Funct Foods 2013;5(4):1620-32.
Zamaratskaia G, Stefanovic S, Lundstrom K, Doran O. Expression of the hepatic skatole- and androstenone-metabolising enzymes in entire male pigs of two live weights. Livest Sci 2012;145(1-3):124-30.
Zeng Z, Jiang JJ, Yu Jie, Mao XB, Yu Bing, Chen DW. Effect of dietary supplementation with mulberry (morus alba L.) leaves on the growth performance, meat quality and antioxidative capacity of finishing pigs. J Integr Agric 2019;18(1):143-51.
Zhao X, Li L, Luo Q, Ye M, Luo G, Kuang Z. Effects of mulberry (Morus alba L.) leaf polysaccharides on growth performance, diarrhea, blood parameters, and gut microbiota of early-weanling pigs. Livest Sci 2015;177:88-94.
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Year 2024 volume 16 Issue 1
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doi: 10.1016/j.aninu.2023.05.018
  • Receive Date：2022-09-29
  • Online Date：2026-01-28
  • Published：2024-03-10
Article Data
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History
  • Received：2022-09-29
  • Revised：2023-04-04
  • Accepted：2023-05-27
Affiliations
    aInstitute of Sericulture and Tea, Zhejiang Academy of Agricultural Sciences, 298 Deshengzhong Road, Shangcheng, Hangzhou 310021, China
    bInstitute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, 298 Deshengzhong Road, Shangcheng, Hangzhou 310021, China

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Corresponding authors. E-mail addresses: (Z. Xu),
(Y. Li).
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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