Fecal bacteria and metabolite responses to dietary lysozyme in a sow model from late gestation until lactation

Abstract

Lysozyme (LZM) is a natural anti-bacterial protein that is found in the saliva, tears and milk of all mammals including humans. Its anti-bacterial properties result from the ability to cleave bacterial cell walls, causing bacterial death. The current study was conducted to investigate the effects of dietary LZM on fecal microbial composition and variation in metabolites in sow. The addition of LZM decreased the fecal short-chain fatty acids (SCFAs). Zonulin and endotoxin in the serum, and feces, were decreased with lysozyme supplementation. Furthermore, fecal concentrations of lipocalin-2 and the pro-inflammatory cytokine TNF-α were also decreased while the anti-inflammatory cytokine IL-10 was increased by lysozyme supplementation. 16S rRNA gene sequencing of the V3-V4 region suggested that fecal microbial levels changed at different taxonomic levels with the addition of LZM. Representative changes included the reduction of diversity between sows, decreased Bacteroidetes, Actinobacteria, Tenericutes and Spirochaetes during lactation as well as an increase in Lactobacillus. These findings suggest that dietary lysozyme supplementation from late gestation to lactation promote microbial changes, which would potentially be the mechanisms by which maternal metabolites and inflammatory status was altered after LZM supplementation.

Figure 1

Effects of feeding sows diets supplemented with lysozyme on the serum concentrations of zonulin (A1–3) and endotoxin (B1–3) and fecal concentrations of endotoxin (C1–3). Data are presented as means ± SEM (n = 8). Con. = control, LZM 150 = control diet + lysozyme 150 mg/kg, LZM 300 = control diet + lysozyme 300 mg/kg. A, B, significant effect of treatment (values with different uppercase letters are significantly different, P < 0.01).

Figure 2

Effects of feeding sows diets supplemented with lysozyme on the serum concentrations of zonulin (A1–3) and endotoxin (B1–3) and fecal concentrations of endotoxin. Data are presented as means ± SEM (n = 8). TNF-α = tumor necrosis factor-α, IL-6 = interleukin-6, IL-10 = interleukin-10. Con. = control, LZM 150 = control diet + lysozyme 150 mg/kg, LZM 300 = control diet + lysozyme 300 mg/kg. A, B, significant effect of treatment (values with different uppercase letters are significantly different, P < 0.01).

Figure 3

Comparison of the gut microbiota composition among treatments. Principal coordinate analysis to visualize the weighted UniFrac distances of fecal samples from individual sow. Con.d1 = control at day 1 of lactation, Con.d7 = control at day 7 of lactation, Con.d7 = control at day 21 of lactation, LZM 150 d1 = control diet + lysozyme 150 mg/kg at day 1 of lactation, LZM 150 d7 = control diet + lysozyme 150 mg/kg at day 7 of lactation, LZM 150 d21 = control diet + lysozyme 150 mg/kg at day 21 of lactation, LZM 300 d1 = control diet + lysozyme 300 mg/kg at day 1 of lactation, LZM 300 d7 = control diet + lysozyme 300 mg/kg at day 7 of lactation, LZM 300 d21 = control diet + lysozyme 300 mg/kg at day 21 of lactation.

Figure 4

Changes in the six distinct bacterial phyla in sow gut of feeding sows diets supplemented with lysozyme. (A) Bacteroidetes, (B) Actinobacteria, (C) Tenericutes, (D) Spirochaetes, (E) Proteobacteria, and (F) Euryarchaeota. Data are presented as means ± SEM (n = 6). Con. = control, LZM 150 = control diet + lysozyme 150 mg/kg, LZM 300 = control diet + lysozyme 300 mg/kg. A, B, significant effect of treatment (values with different uppercase letters are significantly different, P < 0.01), and a, b significant effect of treatment (values with different lowercase letters are significantly different, P < 0.05).

Figure 5

LEfSe analysis of the gut microbiota composition on d 21 of lactation of feeding sows diets supplemented with lysozyme. (A) Cladogram using LEfSe method indicating the phylogenetic distribution of gut microbiota in sows supplemented with lysozyme. Each successive circle represents a phylogenetic level. (B) Histogram of the LDA scores reveals the most differentially abundant taxa among different treatments. Con. = control, LZM 150 = control diet + lysozyme 150 mg/kg, LZM 300 = control diet + lysozyme 300 mg/kg.

Figure 6

Heatmap of the spearman r correlations between the gut microbiota (phylum level) significantly modified by metabolic parameters of sows. Data are presented as means ± SEM (n = 6). *P < 0.05; **P < 0.01 (following the Spearman correlation analysis). S.zon. = serum_zonulin, S.end. = serum endotoxin, F.end = fecal endotoxin, F.lip. = fecal lipocalin-2, F.TNF = fecal TNF-α, F.IL6 = fecal IL-6, F.IL10 = fecal IL-10, Acet. = acetate, Prop. = propionate, Buty. = butyrate, SCFAs is the sum of acetate, propionate, and butyrate.