Immune response of the mammary gland during different stages of lactation cycle in high versus low yielding Karan Fries crossbred cows

To investigate the magnitude of innate immunity of mammary glands during different stages of the lactation cycle, milk samples were collected from both high (n=10) and low (n=10) yielding Karan Fries (KF) crossbred cows during early, mid and late lactation stages. Milk somatic cells released during different stages of the lactation cycle were measured microscopically. In vitro immune activity of each milk leukocytes (viz. neutrophils, lymphocytes and macrophages) were also evaluated after isolating each of the cells from the total milk somatic cell pellet. Relative mRNA expression profiles of TNF-α, IL-8 and TLR-4 by RT polymerase chain reaction were also studied in total milk somatic cells during various stages of the lactation cycle in high yielding cows, whereas low yielding KF cows were taken as control. The results were analyzed and significance was tested by employing two way ANOVA, and the relative expression ratio of the target genes of high yielding cows was tested and analyzed for significance by Relative Expression Software Tool REST version 2009. Irrespective of the stage of lactation, high yielding animals were having higher (P<0.01) somatic cell counts (SCC) compared to low yielders. Milk SCC was found to be significantly higher (P<0.01) during mid-lactation compared to other stages in high yielding cows. In vitro immune response of milk leukocytes were lower in high yielders (P<0.01) compared to low yielders, irrespective of stage of lactation. In vitro immune response of all the milk leukocytes was found to be lower (P<0.01) in high yielding cows compared to low yielding cows. In vitro phagocytic index (PI) of milk neutrophils was higher (P<0.01) during mid lactation periods in both the groups of cows. PI of milk macrophages was found to be higher (P<0.01) during early lactation period in both high and low yielding cows. LPS induced milk lymphocyte blastogenic response was found to be higher (P<0.01) in early lactation period of low yielders. Abundance of IL-8, TNF-α and TLR-4 transcripts were up-regulated (P<0.01) during mid lactation of high yielders as compared to control cows (low yielding cows). Abundance of TNF-α and TLR-4 transcripts were down-regulated (P<0.01) during late lactation in high yielding cows. This study indicated that innate immune responses of milk somatic cells in KF cows are modulated by both lactation stage and milk yield. It was also found that mammary glands of high yielding cows have better magnitude of innate immune response during mid lactation stage compared to early and late lactation in terms of milk SCC and in vitro immune response of isolated milk leukocytes.