Mulberry leaf supplementation inhibits skatole deposition by regulating gut microbiota and upregulating liver cytochrome P450 1A1 expression in finishing pigs

Abstract

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.

Keywords: Cytochrome P450; Microbiota; Mulberry leaf; Pig; Skatole.

Fig. 1

Effects of mulberry leaf diet on the gut microbiota of finishing pigs. (A) Chao1 and Shannon indices. (B) PCoA and NMDS analyses. (C) Relative abundance, correlation analysis, and taxonomy of genera that were downregulated by the mulberry leaf diet. (D) Relative abundance, correlation analysis, and taxonomy of genera that were upregulated by the mulberry leaf diet. (E) Tax4Fun functional prediction analysis of bacterial operational taxonomic units (OTU). PCoA = principal coordinate analysis; NMDS = nonmetric multidimensional scaling; CON = basal diet; MB = mulberry leaf diet. n = 8 for all analyses. The asterisk indicates a significant difference from the basal diet group, ∗P < 0.05, ∗∗P < 0.01.

Fig. 2

Fecal metabolomics analysis of finishing pigs. (A) Partial least squares discriminant analysis (PLS-DA), (B) volcano plot of differential metabolites, (C) KEEG enrichment of differential metabolites, and (D) heatmap of differential metabolites in the changed pathways. (E) Peak intensities of metabolites related to indole and indole derivatives. (F) Correlation analysis of indole-3-acetic acid in feces and skatole in feces. CON = basal diet; MB = mulberry leaf diet. n = 8 for all analyses. The asterisk indicates a significant difference from the basal diet group, ∗P < 0.05.

Fig. 3

Metabolomics analysis of the serum of finishing pigs. (A) Partial least squares discriminant analysis (PLS-DA), (B) differential metabolite numbers, (C) KEEG enrichment of differential metabolites, and (D) differential metabolites in the changed pathways. (E) Peak intensities of significantly changed metabolites related to indole and indole derivatives. (F) Analysis of the correlation of DL-tryptophan, indole, and indole-3-lactic acid in serum with skatole in serum. CON = basal diet; MB = mulberry leaf diet. n = 8 for all analyses. The asterisk indicates a significant difference from the basal diet group, ∗P < 0.05.

Fig. 4

Cytochrome P450 expression levels in the liver of finishing pigs and the hepatic cell line HepG2. (A) mRNA expression levels of CYP1A1, CYP1A2, CYP2E1, and CYP2A in the liver of finishing pigs (n = 8). (B) Analysis of the correlation of CYP1A1 gene expression in the liver with skatole in fat and skatole in serum, respectively. (C) Western blot analysis of CYP1A1 protein level in the liver of finishing pigs (n = 3). (D) In vitro test of the effects of polyphenols (PP), polysaccharides (PS), and alkaloids (AL) extracted from mulberry leaves on the mRNA expression levels of CYP1A1, CYP1A2, CYP2E1, and CYP2A in the hepatic cell line HepG2 (n = 3). CON = basal diet; MB = mulberry leaf diet. The asterisk indicates a significant difference from the basal diet group or control cells, ∗P < 0.05, ∗∗P < 0.01.