Comparison of the transcriptome in circulating leukocytes in early lactation between primiparous and multiparous cows provides evidence for age-related changes

Abstract

Background: Previous studies have identified many immune pathways which are consistently altered in humans and model organisms as they age. Dairy cows are often culled at quite young ages due to an inability to cope adequately with metabolic and infectious diseases, resulting in reduced milk production and infertility. Improved longevity is therefore a desirable trait which would benefit both farmers and their cows. This study analysed the transcriptome derived from RNA-seq data of leukocytes obtained from Holstein cows in early lactation with respect to lactation number.

Results: Samples were divided into three lactation groups for analysis: i) primiparous (PP, n = 53), ii) multiparous in lactations 2-3 (MP 2-3, n = 121), and iii) MP in lactations 4-7 (MP > 3, n = 55). Leukocyte expression was compared between PP vs MP > 3 cows with MP 2-3 as background using DESeq2 followed by weighted gene co-expression network analysis (WGCNA). Seven modules were significantly correlated (r ≥ 0.25) to the trait lactation number. Genes from the modules which were more highly expressed in either the PP or MP > 3 cows were pooled, and the gene lists subjected to David functional annotation cluster analysis. The top three clusters from modules more highly expressed in the PP cows all involved regulation of gene transcription, particularly zinc fingers. Another cluster included genes encoding enzymes in the mitochondrial beta-oxidation pathway. Top clusters up-regulated in MP > 3 cows included the terms Glycolysis/Gluconeogenesis, C-type lectin, and Immunity. Differentially expressed candidate genes for ageing previously identified in the human blood transcriptome up-regulated in PP cows were mainly associated with T-cell function (CCR7, CD27, IL7R, CAMK4, CD28), mitochondrial ribosomal proteins (MRPS27, MRPS9, MRPS31), and DNA replication and repair (WRN). Those up-regulated in MP > 3 cows encoded immune defence proteins (LYZ, CTSZ, SREBF1, GRN, ANXA5, ADARB1).

Conclusions: Genes and pathways associated with lactation number in cows were identified for the first time to date, and we found that many were comparable to those known to be associated with ageing in humans and model organisms. We also detected changes in energy utilization and immune responses in leukocytes from older cows.

Keywords: Ageing; Cow; Leukocytes; Longevity; Multiparous; Primiparous.

Fig. 1

A Cluster dendrogram of 13,769 genes (individual black lines at top) clustered by their topological overlap dissimilarity scores. The multi-coloured panel next to “Dynamic Tree Cut” shows 42 identified modules using the Dynamic Tree Cut algorithm. The second multi-coloured panel shows 32 larger modules identified after highly correlated smaller modules were merged together (agreement of 0.8). B Number of genes per module identified using WGCNA. C Module-trait correlations according to the parity of the cow: i) primiparous (PP, n = 53), ii) multiparous in lactations 2–3 (MP 2–3, n = 121), and iii) MP in lactations 4–7 (MP > 3, n = 55). Red, positive correlation, blue, negative correlation. Numbers indicate the r value: significance was taken at P = ± 0.25

Fig. 2

GO enrichment analysis of significantly correlated modules (FDR < =0.1). A Positively correlated in PP cows: violet, darkred, midnightblue, orange, yellow; B negatively correlated in PP cows: black, turquoise

Fig. 3

David functional annotation Cluster 4 contained many genes (shown in blue) in the Glycolysis/Gluconeogenesis KEGG pathway which were more highly expressed in MP > 3 vs PP cows (figure drawn by the authors). This would potentially maximise the use of glucose as a precursor of acetyl CoA for use in the TCA cycle

Fig. 4

David functional annotation Cluster 6 contained genes involved in immunity which were more highly expressed in MP > 3 vs PP cows. This included genes encoding proteins for pattern recognition receptors (PRR), the bovine MHC complex, the complement system and several with antimicrobial activity. NRROS and TNFAIP8L2 can both act as negative regulators of TLR signalling mediated via inhibition of NF-kappa-B activation