Characterization of genes and pathways that respond to heat stress in Holstein calves through transcriptome analysis

Abstract

This study aimed to investigate the genes and pathways that respond to heat stress in Holstein bull calves exposed to severe ranges of temperature and humidity. A total of ten animals from 4 to 6 months of age were subjected to heat stress at 37 °C and 90 % humidity for 12 h. Skin and rectal temperatures were measured before and after heat stress; while no correlation was found between them before heat stress, a moderate correlation was detected after heat stress, confirming rectal temperature to be a better barometer for monitoring heat stress. RNAseq analysis identified 8567 genes to be differentially regulated, out of which 465 genes were significantly upregulated (≥2-fold, P < 0.05) and 49 genes were significantly downregulated (≤2-fold, P < 0.05) in response to heat stress. Significant terms and pathways enriched in response to heat stress included chaperones, cochaperones, cellular response to heat stress, phosphorylation, kinase activation, immune response, apoptosis, Toll-like receptor signaling pathway, Pi3K/AKT activation, protein processing in endoplasmic reticulum, interferon signaling, pathways in cancer, estrogen signaling pathway, and MAPK signaling pathway. The differentially expressed genes were validated by quantitative real-time PCR analysis, which confirmed the tendency of the expression. The genes and pathways identified in this analysis extend our understanding of transcriptional response to heat stress and their likely functioning in adapting the animal to hyperthermic stress. The identified genes could be used as candidate genes for association studies to select and breed animals with improved heat tolerance.

Keywords: Differentially expressed genes; Heat stress; KEGG; RNAseq; Transcriptome.

Fig. 1

Measurements of skin and rectal temperatures for Holstein calves (a, b) during the course of the experiment; c, d correlation between measurements of skin and rectal temperature before heat stress (09:00 hours) and after heat stress (19:02 hours)

Fig. 2

Volcano plot showing differentially expressed genes between AHS and BHS; the dots in red are genes that were considered significant FC ≤ or ≥2-fold and P value <0.05 (color figure online)

Fig. 3

Hierarchical clustering of all the expressed genes before (BHS) and after (AHS) heat stress. Red corresponds to downregulated gene product, and green corresponds to upregulated gene product (color figure online)

Fig. 4

Expression levels of selected genes from RNAseq analysis (blue bar) and their validation by qRT-PCR (red bar). R ² is the correlation of expression levels between the two methods (color figure online)

Fig. 5

Summary of GO terms for biological process (BP), molecular function (MF), and cellular component (CC) ontologies for upregulated and downregulated gene products in response to heat stress. Chart labels are GO terms, and the percentage indicates the total percentage of the DEGs involved in that process

Fig. 6

Gene interaction network of DEGs analyzed using STRING protein database. Nodes are the genes, and the edges are the interaction between the nodes. The color of the edge indicates the type of interaction

Fig. 7

A network map of pathways significantly enriched after heat stress. The nodes are the pathways, and edges connect the genes involved in the pathway