Investigating the Short-Term Effects of Cold Stress on Metabolite Responses and Metabolic Pathways in Inner-Mongolia Sanhe Cattle

Abstract

Inner-Mongolia Sanhe cattle are well-adapted to low-temperature conditions, but the metabolic mechanisms underlying their climatic resilience are still unknown. Based on the ¹H Nuclear Magnetic Resonance platform, 41 metabolites were identified and quantified in the serum of 10 heifers under thermal neutrality (5 °C), and subsequent exposure to hyper-cold temperature (-32 °C) for 3 h. Subsequently, 28 metabolites were pre-filtrated, and they provided better performance in multivariate analysis than that of using 41 metabolites. This indicated the need for pre-filtering of the metabolome data in a paired experimental design. In response to the cold exposure challenge, 19 metabolites associated with cold stress response were identified, mainly enriched in "aminoacyl-tRNA biosynthesis" and "valine, leucine, and isoleucine degradation". A further integration of metabolome and gene expression highlighted the functional roles of the DLD (dihydrolipoamide dehydrogenase), WARS (tryptophanyl-tRNA synthetase), and RARS (arginyl-tRNA synthetase) genes in metabolic pathways of valine and leucine. Furthermore, the essential regulations of SLC30A6 (solute carrier family 30 (zinc transporter), member 6) in metabolic transportation for propionate, acetate, valine, and leucine under severe cold exposure were observed. Our findings presented a comprehensive characterization of the serum metabolome of Inner-Mongolia Sanhe cattle, and contributed to a better understanding of the crucial roles of regulations in metabolites and metabolic pathways during cold stress events in cattle.

Keywords: Sanhe cattle; climatic resilience; cold stress; metabolomics.

Figure 1

The analytical workflow performed in this study.

Figure 2

Sanhe cattle during severe cold stress exposure. (a) Young bull, and (b) Heifer.

Figure 3

The serum metabolic profiling of Sanhe cattle. (a) The ¹H NMR spectra (0–10.0 ppm) of serum from Sanhe cattle. The numbers represent signals of equivalent hydrogen, and the DSS is 0 ppm for the chemical shift of the whole spectrum. (b) Forty-one unique metabolites were commonly identified and quantified in the serum metabolic profiling of Sanhe cattle.

Figure 4

The pre-selection of metabolic profiling. (a) The changes of 3-hydroxyisobutyrate in 10 animals exposed to severe cold. (b) The changes of arginine in 10 animals exposed to severe cold. (c) Twenty-eight metabolites changed in the same trend after cold stress. (d) Score plots of PCA with 28 metabolites (R²X = 0.636). (e) Score plots of PCA with 41 metabolites (R²X = 0.616).

Figure 5

PLS-DA analyses of metabolic profiling with 28 and 41 metabolites. (a) Score plots of PLS-DA with 28 metabolites (R²X = 0.603). (b) Score plots of PLS-DA with 41 metabolites (R²X = 0.383). (c) Cross-validation results of PLS-DA with 28 metabolites (components = 5, R²Y = 0.95, Q²Y = 0.62). (d) Cross-validation results of PLS-DA with 41 metabolites (components = 5, R²Y = 0.94, Q²Y = 0.49). (e) 1000 random permutation tests in PLS-DA with 28 metabolites (p = 0.007). (f) 1000 random permutation tests in PLS-DA with 41 metabolites (p = 0.087). *: p-Value < 0.05.

Figure 6

Correlation-based networks of 28 metabolites in Sanhe cattle. (a) The correlation among metabolites that were common in the serum of Sanhe cattle before and after severe cold stress. (b) The correlation among metabolites that were only found in the serum of Sanhe cattle before severe cold stress. (c) The correlation among metabolites that were only found in the serum of Sanhe cattle after severe cold stress. The solid line is significantly positive correlated, while the dotted line is significantly negative correlated. The purple circles are the metabolites with a VIP value > 1 in PLS-DA, and the green flags are the metabolites with a p-value < 0.05 in the paired t-test.

Figure 7

Networks between metabolites and genes involved in metabolic pathways. (a) Overview for metabolite set enrichment analysis of metabolic response to cold stress in Sanhe cattle. (b) Relevant metabolic pathways involved in the response to cold stress in Sanhe cattle. Triangles represent metabolites, ovals represent genes, and rectangles are the metabolic pathways. (c) The changes of gene expressions involved in metabolic pathways. *: p-Value < 0.05; ns: no significance; SEPHS1: selenophosphate synthetase 1; ECHDC1: enoyl coenzyme A hydratase domain containing 1; DLD: dihydrolipoamide dehydrogenase; RARS: arginyl-tRNA synthetase; WARS: tryptophanyl-tRNA synthetase.

Figure 8

Networks between metabolites and genes involved in transportation pathways and protein biosynthesis. (a) Transportation pathways. (b) Protein biosynthesis. Triangles represent metabolites, ovals represent genes, and rectangles represent the metabolic pathways. (c) The changes of gene expressions involved in transportation pathways. (d) The changes of gene expressions involved in protein biosynthesis. *: p-Value < 0.05; **: p-Value < 0.01; ns: no significance; SLC30A6: solute carrier family 30 (zinc transporter), member 6; LPL: lipoprotein lipase; LIPA: lipase A, lysosomal acid, cholesterol esterase; EIF2S3: eukaryotic translation initiation factor 2, subunit 3 gamma; FXYD3: FXYD domain containing ion transport regulator 3; NPC1: Niemann-Pick disease, type C1; EIF2B1: eukaryotic translation initiation factor 2B, subunit 1 alpha; MRPL30: mitochondrial ribosomal protein L30; RARS: arginyl-tRNA synthetase; SRP72: signal recognition particle 72 kDa; RPL22L1: ribosomal protein L22-like 1; RPS18: ribosomal protein S18; WARS: tryptophanyl-tRNA synthetase; RPLP2: ribosomal protein, large, P2.

Figure 9

Proposed mechanisms of cold stress response to related functional pathways. Red indicates upregulated metabolites, and blue represents downregulated metabolites, however, blue italics indicates downregulated genes. RPL22L1: ribosomal protein L22-like 1; SRP72: signal recognition particle 72 kDa; EIF2B1: eukaryotic translation initiation factor 2B, subunit 1 alpha; MRPL30: mitochondrial ribosomal protein L30; RPS18: ribosomal protein S18; RARS: arginyl-tRNA synthetase; RPLP2: ribosomal protein, large, P2; WARS: tryptophanyl-tRNA synthetase; SLC30A6: solute carrier family 30 (zinc transporter), member 6; EIF2S3: eukaryotic translation initiation factor 2, subunit 3 gamma; LPL: lipoprotein lipase; LIPA: lipase A, lysosomal acid, cholesterol esterase; FXYD3: FXYD domain containing ion transport regulator 3; NPC1: Niemann-Pick disease, type C1; DLD: dihydrolipoamide dehydrogenase; ECHDC1: enoyl coenzyme A hydratase domain containing 1.