Expression profiles of pro-inflammatory cytokine genes in milk somatic cells at different stages of the first lactation in Holstein dairy cattle

Milk somatic cells produce numerous soluble proteins like cytokines that play important roles in the immunity of the mammary gland. This study aimed to investigate the expression profiles of bovine pro-inflammatory cytokine genes including IL-2, IL-6, IL-8, TNF-α, IFN-γ, and GM-CSF in the somatic cells of milk in healthy Holstein cows at different lactation stages in their first lactation cycle. For this purpose, milk samples were collected from eighteen dairy cows at the early, middle, and late lactation stages. Total RNA was extracted from the somatic cells of milk and then the first strand of cDNA was synthesized. Real-time PCR was performed for the bovine pro-inflammatory cytokine genes. As reference genes, the β-actin and GAPDH genes were used to normalize the data. The real-time PCR data were analyzed with the REST and SAS programs. According to the results, the six-cytokine genes were expressed in the milk somatic cells of healthy cows in different lactation stages. The results showed that the expressions of almost all cytokine genes (except for the TNF-α gene) were significantly higher in animals at the middle compare to the early lactation stage. However, the expression of cytokine genes also showed a trend to be higher at the late lactation stage compared to early lactation. Still, these differences were only significant for mRNA levels of TNF-α and GM-CFS genes. Furthermore, the expression differences of cytokine genes were not significant in cows at the late relative to animals at the middle lactation stage. In the entire lactation cycle, the mRNA transcription levels of IL-6 and IL-2 were observed at high and low concentrations compare to other cytokine genes, respectively. The highest stability was shown for IL-6 throughout the three lactation stages, while the lowest stability was found for the expression of TNF-α. The correlation between the gene expression levels was almost not significant for most of the studied genes in different stages of lactation, however, a significant correlation was found between IL-8 and GM-CSF in the entire, early and late stages of lactation.