Metabolomics Analysis of Effects of Commercial Soy-based Protein Products in Red Drum (Sciaenops ocellatus)

Abstract

We investigated the metabolic effects of four different commercial soy-based protein products on red drum fish (Sciaenops ocellatus) using nuclear magnetic resonance (NMR) spectroscopy-based metabolomics along with unsupervised principal component analysis (PCA) to evaluate metabolic profiles in liver, muscle, and plasma tissues. Specifically, during a 12 week feeding trial, juvenile red drum maintained in an indoor recirculating aquaculture system were fed four different commercially available soy formulations, containing the same amount of crude protein, and two reference diets as performance controls: a 60% soybean meal diet that had been used in a previous trial in our lab and a natural diet. Red drum liver, muscle, and plasma tissues were sampled at multiple time points to provide a more accurate snapshot of specific metabolic states during the grow-out. PCA score plots derived from NMR spectroscopy data sets showed significant differences between fish fed the natural diet and the soy-based diets, in both liver and muscle tissues. While red drum tolerated the inclusion of soy with good feed conversion ratios, a comparison to fish fed the natural diet revealed that the soy-fed fish in this study displayed a distinct metabolic signature characterized by increased protein and lipid catabolism, suggesting an energetic imbalance. Furthermore, among the soy-based formulations, one diet showed a more pronounced catabolic signature.

Keywords: NMR; Sciaenops ocellatus; aquaculture; metabolomics; red drum; soy.

Figure 1

Unsupervised PCA score plots derived from ¹H NOESY 1D NMR spectra from red drum liver tissue (independent models). A) Natural diet; B) diet #1 (SBM60); C) diet #2; D) diet #3; E) diet #4; F) diet #5. Sampled time points were T₀ (n = 63) for sampling at the end of the conditioning period, T₂ to T₅ and T₉ to T₁₂ (n = 12/time point) for sampling at weeks 2 to week 5 and week 9 to week 12, respectively. Error bars represent the mean ± 1 SEM.

Figure 2

Unsupervised PCA score plots (independent models) from (A) liver tissue and (B) muscle tissue for the five soy-based experimental diets (diet #1 to diet #5) at the end-point (T_end) of the metabolic trajectory including the natural diet (N). For comparison, recalculated PCA score plots for (C) liver and (D) muscle exclude the natural diet (N). Error bars represent the mean ± 1 SEM. Sample numbers are: diet #1 (n = 44, liver; n = 47, muscle), diet #2 (n = 44, liver; n = 46, muscle), diet #3 (n = 46, liver; n = 48, muscle), diet #4 (n = 47, liver; n = 48, muscle), diet #5 (n = 47, liver; n = 48, muscle), natural diet (n = 47, liver; n = 48, muscle).

Figure 3

(A) Liver end-point (T_end) PC1 loading plot (95^th percentile) for the five experimental diets (diet #1–#5) compared with the natural diet. (B) Expansion of the PC1 loading plot in (A). (C) Liver end-point (T_end) PC1 loading plot (95^th percentile) for the five experimental diets (diet #1 to #5) excluding the natural diet. (D) Expansion of the PC1 loading plot in (C). Assignment information is shown in Tables 1 and 2. Glycero-PC, glycerophosphocholine; glycerol 3-P, glycerol 3-phosphate. (A and B) Loadings with a negative sign indicate metabolites that are present at lower levels in the soy-based diets and higher in the natural diet and vice versa. (C and D) Loadings with a negative sign indicate metabolites that are present at higher levels in diets #1, #3, #4 and #5 and lower in diet #2 and vice versa. The horizontal axis represents NMR chemical shift in ppm.

Figure 4

Profile of significantly altered metabolites identified in soy-based diets compared to natural diet for red drum (A) liver tissue and (B) muscle tissue at T_end. Glycero-PC, glycerophosphocholine; glycerol 3-P, glycerol 3-phosphate; TMAO, trimethylamine-N-oxide. The histograms represent fold change, which is calculated by the average bin intensities of metabolite in soy-based diet samples (average value for diets #2 to #5) divided by the average bin intensities of natural diet samples. Error bars represent mean ± SD (n = 5).

Figure 5

(A) Muscle end-point (T_end) PC1 loading plot (95^th percentile) for the five experimental diets (diets #1 – diet #5) compared with the natural diet. (B) Expansion of the PC1 loading plot in (A). (C) Muscle end-point (T_end) PC1 loading plot (95^th percentile) for the five experimental diets (diet #1 to #5) excluding the natural diet. (D). Expansion of the PC1 loading plot in (C). Assignment information is shown in Tables 1 and 2. TMAO, trimethylamine-N-oxide. (A and B) Loadings with a negative sign indicate metabolites that are present at lower levels in the soy-based diets and higher in the natural diet and vice versa. (C and D) Loadings with a negative sign indicate metabolites that are present at higher levels in diets #1, #3, #4 and #5 and lower in diet #2 and vice versa. The horizontal axis represents NMR chemical shift in ppm.

Figure 6

Levels of A) glycerol 3-phosphate and B) O-phosphocholine in soy-based diets from red drum liver tissue at T_end. The natural diet was not included. The histograms represent bin intensities. Error bars represent mean ± 1 SEM. *** indicate statistical significance level for P < 0.001 (one-way ANOVA with Tukey post-hoc test; n = 44 to 47 samples for each dietary treatment as indicated).

Figure 7

Levels of A) betaine, B) choline, C) glycine and D) serine in soy-based diets (diet #1 to #5) from red drum muscle tissue at T_end. The natural diet was not included. The histograms represent bin intensities. Error bars represent mean ± 1 SEM. Statistical significance levels for diet #2 are illustrated as * for P < 0.05, ** for P < 0.01, and *** for P < 0.001 (one-way ANOVA with Tukey post-hoc test; n = 46 to 48 samples for each dietary treatment as indicated).

Figure 8

Metabolomics pathway analysis overview for A) liver tissue and B) muscle tissue indicating the metabolic pathways that are mostly affected by the different dietary regimes (natural diet and soy-based formulations). The diameter of the circles (nodes) indicates the pathway impact; the color of the nodes is graded from white to red with increasing P-value derived from enrichment analysis.

Figure 9

Overview of the general metabolic network related to energy metabolism affected by the different diets. Red arrows indicate the general pathways that appear to be upregulated in red drum fed any of the experimental soy-based diets compared with the natural diet.