Metabolomics revealed diurnal heat stress and zinc supplementation-induced changes in amino acid, lipid, and microbial metabolism

Abstract

Heat stress (HS) dramatically disrupts the events in energy and nutrient metabolism, many of which requires zinc (Zn) as a cofactor. In this study, metabolic effects of HS and Zn supplementation were evaluated by examining growth performance, blood chemistry, and metabolomes of crossbred gilts fed with ZnNeg (no Zn supplementation), ZnIO (120 ppm ZnSO4), or ZnAA (60 ppm ZnSO4 + 60 ppm zinc amino acid complex) diets under diurnal HS or thermal-neutral (TN) condition. The results showed that growth performance was reduced by HS but not by Zn supplementation. Among measured serum biochemicals, HS was found to increase creatinine but decrease blood urea nitrogen (BUN) level. Metabolomic analysis indicated that HS greatly affected diverse metabolites associated with amino acid, lipid, and microbial metabolism, including urea cycle metabolites, essential amino acids, phospholipids, medium-chain dicarboxylic acids, fatty acid amides, and secondary bile acids. More importantly, many changes in these metabolite markers were correlated with both acute and adaptive responses to HS. Relative to HS-induced metabolic effects, Zn supplementation-associated effects were much more limited. A prominent observation was that ZnIO diet, potentially through its influences on microbial metabolism, yielded different responses to HS compared with two other diets, which included higher levels of short-chain fatty acids (SCFAs) in cecal fluid and higher levels of lysine in the liver and feces. Overall, comprehensive metabolomic analysis identified novel metabolite markers associated with HS and Zn supplementation, which could guide further investigation on the mechanisms of these metabolic effects.

Keywords: Heat stress; metabolism; metabolomics; zinc supplementation.

Figure 1

Effects of HS and Zn supplementation on physiological responses and growth performance of growing pigs. (A) Timeline of 21‐day feeding experiment. Pigs had ad libitum access to water and diet and were fed with one of assigned diets (ZnNeg, ZnIO, or ZnAA) through entire feeding experiment. (B) Designed daily temperature settings in TN and HS treatments. (C–D) Average RT and RR of pigs after 2 h of HS. T is the room temperature when RT and RR were measured. (E–G) ADG, ADFI, and G:F ratio of pigs under HS and TN conditions. Significant differences between TN and HS are labeled as ***P < 0.001.

Figure 2

Effects of HS and Zn supplementation on blood chemistry of growing pigs. (A) Serum glucose, (B) Serum triglycerides, (C) Serum cholesterol, (D) Serum BUN, (E) Serum creatinine, (F) Serum Zn.

Figure 3

Metabolomic analysis of amino‐containing metabolites in serum during 1‐week HS and Zn supplementation. Amino‐containing metabolites were derivatized by DC prior to LC‐MS analysis. (A) The scores plot from PCA analysis on serum metabolome. The t[1] and t[2] values of each data point are the average scores of eight samples in principal components 1 and 2 of the model, respectively. These eight samples were harvested from the same treatment group on specified dates (marked in the plot) between day 0 and 7. (B) The S‐loadings plot on the ions contributing to the separation of HS and TN samples in an OPLS‐DA model. Major contributing metabolites are labeled. (C) The heat map on serum amino‐containing metabolites during 1‐week HS and Zn supplementation. The metabolites were grouped by HCA. Concentrations of each metabolite at different time points and in different sample groups were compared by its Z scores and are presented according to inlaid color keys. Significant differences between day 0 and day 1 in HS groups are determined by the Student's t‐test and labeled as *P < 0.05, **P < 0.01, and ***P < 0.001. (D) The concentrations of total AAs in HS and TN groups. (E–N) The concentrations of lysine, tryptophan glycine, serine, hydroxyproline, proline, arginine, citrulline, ornithine, and ammonia in serum, respectively.

Figure 4

Metabolomic analysis of serum lipid profiles during 1‐week HS and Zn supplementation. (A) The scores plot from PCA analysis on serum metabolome. The t[1] and t[2] values of each data point are the average scores of eight samples in principal components 1 and 2 of the model, respectively. These eight samples were harvested from the same treatment group on specified dates (marked in the plot) between day 0 and 7. (B) The S‐loadings plot on the ions contributing to the separation of HS and TN samples in an OPLS‐DA model. Major contributing PCs are labeled. (C) Structure and MSMS spectrum of PC (15:0/18:2). (D) Structure and MSMS spectrum of PC (17:0/18:2). (E) The heat map on lipid species during 1‐week HS and Zn supplementation. Lipid species were grouped by HCA. Relative abundances of each lipid species at different time points and in different sample groups were compared by its Z scores and are presented according to inlaid color keys.

Figure 5

LC‐MS‐based metabolomic analysis of hepatic extract, cecal fluid, feces extract, and urine samples from pigs under HS and Zn supplementation. (A) Scores plot of a PCA model on hepatic extracts. The distribution of HS‐ZnIO samples in the model is illustrated by a rectangle. (B) Scores plot of a PCA model on cecal fluid samples. The distribution of HS‐ZnIO samples in the model is illustrated by a rectangle. (C) Scores plot of a PCA model on feces extract samples. (D) Scores plot of a PCA model on urine samples.

Figure 6

Metabolite markers of HS and Zn supplementation from LC‐MS‐based metabolomic analysis of hepatic extracts. The concentrations of FAAs and relative abundances of phospholipids in the liver are converted to the Z scores and presented in the heat map according to inlaid color keys. All enlisted metabolites have been confirmed by authentic standards or MSMS fragmentation.

Figure 7

Metabolite markers of HS and Zn supplementation from LC‐MS‐based metabolomic analysis of cecal fluid. The markers labeled with * were confirmed with authentic standards. Putative identities of other markers were based on database search. (A) The heat map of cecal metabolite markers. The metabolites are grouped by HCA. Relative abundances of each metabolite across sample groups are converted to the Z scores and presented in the heat map according to inlaid color keys. (B) The concentration of acetic acid in cecal fluid. (C) The concentration of propionic acid in cecal fluid. (D) The concentration of butyric acid in cecal fluid.

Figure 8

Summary of major HS‐induced metabolic changes. (A) HS‐induced changes in the metabolites related to nitrogen and amino acid metabolism. (B) HS‐induced changes in fatty acids and phospholipids.