A Multi-Enzyme Complex That Mitigates Hepatotoxicity, Improves Egg Production and Quality, and Enhances Gut and Liver Health in Laying Hens Exposed to Trace Aflatoxin B1

Abstract

Aflatoxin B₁ is a prevalent secondary hazardous metabolite generated by fungus present in feed ingredients and the surrounding environment: enzymes are currently being recognized as an efficient and promising approach to reducing the associated risks. The objective of this study was to assess the effects of varying doses of enzyme complexes on several parameters in laying hens that were exposed to aflatoxin. During an 8-week experiment, a total of 288 Yukou Jingfen No.6 laying hens were placed into four groups. These groups included a group treated with toxins (CON group) and groups supplemented with compound enzyme complexes at doses of 250 g/t (E1 group), 500 g/t (E2 group), and 1000 g/t (E3 group). The E2 and E3 groups exhibited a statistically significant 2.6% increase in egg production rate compared to the CON group (p < 0.05). In addition, the E2 group showed significant improvements in both the feed-to-egg ratio and egg weight (p < 0.05). In addition, the E2 and E3 groups showed improved hutch unit and egg white height compared to the control group (p < 0.05). The E2 and E3 groups showed a substantial rise in liver health indicators, namely serum alanine transaminase (ALT) and alkaline phosphatase (ALP) activity. On the other hand, malondialdehyde (MDA) was lowered, and total superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC) were raised. These findings were statistically significant (p < 0.05). The E2 and E3 groups showed notable enhancements in intestinal morphology, as evidenced by a rise in villus height and a decrease in crypt depth in all segments of the intestine (p < 0.05). Furthermore, analysis of 16S rRNA sequencing revealed that these participants had a higher prevalence and variety of microorganisms in their gut microbiota. More precisely, there was a significant rise in the abundance of Bacteroidota and a decline in Firmicutes at the level of the phylum. In general, the inclusion of the enzyme complex had advantageous impacts on performance, egg quality, intestinal morphology, intestinal barrier function, and intestinal flora in laying hens. Our results indicate that toxin-degrading enzymes, when used as feed additives, play a significant role in mitigating AFB₁ contamination in diets and improving the production performance of laying hens.

Keywords: AFB1; gut health; gut microbiota; liver damage; liver metabolites; production.

Figure 1

The effects of enzyme complexes on liver tissue morphology and functional abnormalities induced by AFB₁ in laying hens: (a–d) The actives of albumin (n = 8), ALP, ALT, and AST in serum; values are means ± SEM (n = 6). (e) Liver tissue HE results (100× and 400×). Partial fragmentation of the nucleus (black arrow), blood infiltration within the sinusoidal spaces (red arrow). The results are represented as mean ± SEM; ^{a, b} represent significant differences between groups (p < 0.05).

Figure 2

Effects of AFB₁, AFB₁+ enzyme complexes treatments on the oxidation and antioxidant levels of MDA, SOD, and T-AOC in the liver (a–c); values are means ± SEM (n = 6). Relative mRNA expression of SOD1 and Nrf2 (d,e); values are means ± SEM (n = 3). ^a–c Means with different letters within the same column are significantly different (p < 0.05).

Figure 3

Effects of enzyme complex levels on the mRNA expression levels of tight junctions in jejunum of laying hens feeding in AFB₁ and complex enzymes at different concentrations: (a) zonula occludens 1 (ZO-1); (b) mucin 2 (MUC2) (n = 4). ^a–c Results are expressed as mean ± SEM. Means with different letters within the same column are significantly different (p < 0.05).

Figure 4

Changes in the gut microbiota of laying hens feeding in AFB₁ and complex enzymes at different concentrations: (a) OTUS analysis. (b) $\beta$ diversity was estimated using PCoA. (c) Comparative analysis was performed, to examine the relative abundance of bacteria at the phylum level. The Chao (d) and Simpson index (e) were used to estimate sample richness and diversity, respectively. (f) The distribution of bacterial abundance at the phylum level. (g) Cladogram from LEfSe multi−level species difference discriminant analysis (LDA > 3), with different color nodes representing those communities significantly enriched in the corresponding phylum, showing significant differences between groups. (h) The relative abundance of bacteria communities was also evaluated between the E1 and CON at the genus level, as well as between E2 and CON at the genus level. (j) E3 and CON at genus (i). (k) The heat map illustrates the Spearman correlation between changes in the fecal microbial population and both production performance and egg quality. (l) The heat map shows the Spearman correlation between the changes in the fecal microbial population and small intestinal morphology. ^a–c Results were expressed as mean ± SEM. Means with different letters within the same column were significantly different (p < 0.05). * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.

Figure 5

The liver metabo in the comparison groups: (a) PLS−DA based on positive ion table. (b) Difference Venn diagram of the relative comparison groups. The differential metabolites identified were categorized into 78 distinct biochemical groups. (c) Superclass. (d) Class. (e) KEGG pathways enriched for the 78 differentially expressed metabolites both in the relative comparison group. Volcano plot showing the different fecal metabolites between E1 and CON (f), between E2 and CON (g), and between E3 and CON (h). KEGG enrichment analysis between E1 and CON (i), between E2 and CON (j), and between E3 and CON (k). (l) A Spearman correlation analysis was performed, to assess the associations between hepatic metabolites and markers of liver injury and antioxidant capacity. (m) Spearman correlation analysis between hepatic metabolites and gut microbiota. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.