Effects of cysteine addition to low-fishmeal diets on the growth, anti-oxidative stress, intestine immunity, and Streptococcus agalactiae resistance in juvenile golden pompano (Trachinotus ovatus)

Abstract

As the precursor of taurine, cysteine serves physiological functions, such as anti-oxidative stress and immune improvement. Investigation of cysteine and its derivatives has made positive progress in avian and mammalian species, yet the study and application of cysteine in aquatic animals are relatively rare. Therefore, we evaluated the effects of supplementing a low-fishmeal diet with various levels of cysteine on the growth, antioxidant capacity, intestine immunity, and resistance against Streptococcus agalactiae of the juvenile golden pompano (Trachinotus ovatus). According to our study, exogenous supplementation with 0.6-1.2% cysteine greatly increased the final body weight (FBW) and specific growth rate (SGR) of golden pompano compared to the control group. Under the present conditions, the optimum dietary cysteine supplementation level for golden pompano was 0.91% based on the polynomial regression analysis of SGR. Meanwhile, we found that the Nrf2/Keap1/HO-1 signaling pathway was notably upregulated with the increase of exogenous cysteine, which increased antioxidant enzyme activity in serum and gene expression in the intestine and reduced the level of reactive oxygen species (ROS) in the serum of golden pompano. In addition, morphological analysis of the midgut demonstrated that exogenous cysteine improved muscle thickness and villi length, which suggested that the physical barrier of the intestine was greatly strengthened by cysteine. Moreover, cysteine increased the diversity and relative abundance of the intestinal flora of golden pompano. Cysteine suppressed intestinal NF-κB/IKK/IκB signaling and pro-inflammatory cytokine mRNA levels. Conversely, intestinal anti-inflammatory cytokine gene expression and serum immune parameters were upregulated with the supplementary volume of cysteine and improved intestine immunity. Further, exogenous cysteine supplementation greatly reduced the mortality rate of golden pompano challenged with S. agalactiae. In general, our findings provide more valuable information and new insights into the rational use of cysteine in the culture of healthy aquatic animals.

Keywords: Streptococcus agalactiae; Trachinotus ovatus; antioxidant capacity; cysteine; growth; intestine immunity.

Figure 1

Estimation of the optimal dietary cysteine supplementation level for T. ovatus by means of polynomial regression analysis using the SGR.

Figure 2

Effect of antioxidant capability such as T-AOC (A), CAT (B), SOD (C), GSH-PX (D), MDA (E) and ROS (F) in the serum of T. ovatus fed diets with different dose cysteine supplementation after 8 weeks. Mean values (n = 9) within values in the picture above with different superscripts are significantly different (P< 0.05).

Figure 3

Effect of immunological parameters such as LZM (A), IgA (B), IgG (C), IgM (D), C3 (E), C4 (F) in the serum of T. ovatus fed diets with different dose cysteine supplementation after 8 weeks. Mean values (n = 9) within values in the picture above with different superscripts are significantly different (P< 0.05).

Figure 4

Effects of dietary cysteine on mid-gut morphology of T. ovatus. (A) 0% cysteine; (B): 0.40% cysteine; (C): 0.80% cysteine; (D): 1.20% cysteine; (E): 1.60% cysteine. Scale bar: 200 μm. The villus length (F), muscular thickness (G), and goblet cells quantity (H) of mid-gut in T. ovatus. data are presented as mean ± SD (n = 9). Asterisks *, **, ***, and **** indicate statistically significant difference between treated group and control group at P< 0.05, P< 0.01 P< 0.001, and P< 0.0001, respectively.

Figure 5

Cysteine supplementation regulated gut microbiota composition and abundance. Profile of the intestinal microbiota (n = 3). The basic structure of the gut microbiota was determined by Venn diagram (A), Rarefaction curves (B), Rank-Abundance curves (C), species accumulation curves (D), relative abundance histograms of phylum (E) and genus (F), Mcrobial community barplot with cluster tree (G), and heat map comparison (H) were depicted. The diversity and richness of the communities were compared by alpha diversity indices, including Chao 1 index (I), Shannon index (J), and Simpson index (K). The community diversity and richness were compared by Principal Component Analysis (L), UniFrac-based principal co-ordinates analysis (M), and UniFrac-based non-metric multi-dimensional scaling analysis (N) were performed for β-diversity analysis. Communities or species with significantly different effects on sample delineation were identified by UniFrac-based cluster tree (O), Evolutionary branching plots (P), and Linear discriminant analysis (Q). "n.s."indicate No significant difference. Asterisks *, **, and **** indicate statistically significant difference between treated group and control group at P < 0.05, P < 0.01, and P < 0.0001, respectively.

Figure 6

The expression profiles of antioxidant genes (A) and signaling pathway (B) in the intestine of T. ovatus fed diets with different dose cysteine supplementation after 8 weeks. Mean values (n = 9) within values in the picture above with different superscripts are significantly different (P< 0.05).

Figure 7

The expression profiles of inflammatory genes (A) and signaling pathway (B) in the intestine of T. ovatus fed diets with different dose cysteine supplementation after 8 weeks. Mean values (n = 9) within values in the picture above with different superscripts are significantly different (P< 0.05).

Figure 8

The Kaplan-Meier survival analysis of T. ovatus after S. agalactiae infection. Asterisks *, **, and *** indicate statistically significant difference between the two groups at P < 0.05, P < 0.01, and P < 0.001, respectively.