Leukocytes, microRNA, and complement activity in raw, heat-treated, and frozen colostrum and their dynamics as colostrum transitions to mature milk in dairy cows

The objectives of this study were to evaluate the abundance and viability of leukocytes, the abundance of microRNA, and the activity of the complement pathway in (1) colostrum following heat-treatment or freezing, and (2) colostrum, transition milk, and mature milk. In experiment 1, composite colostrum samples were harvested from individual cows (n = 14) on a commercial dairy farm in NY and split into 3 aliquots using single-use colostrum bags. One aliquot was immediately cooled on ice following harvest (RAW) and stored at 4°C overnight, one was heat-treated for 60 min at 60°C (HT) before being cooled on ice and stored at 4°C overnight, and one was frozen at -20°C overnight (FR). The following morning, all samples were warmed to 40°C before further processing. In experiment 2, cows were sampled in a longitudinal study where composite samples were collected from colostrum (first milking, n = 23), transition milk (3 to 4 d postpartum, n = 13), and mature milk (6 to 7 d postpartum, n = 13). In both experiments colostrum was harvested from the first milking within 8 h of calving and samples were processed within 14 h of collection. Colostral leukocytes were isolated before viability was determined by trypan blue exclusion and manual differential cell counts were performed. Extracellular vesicles were isolated from whey by ultracentrifugation to isolate and quantify microRNA. Activity of the alternative complement pathway was determined in casein-depleted whey by semi-solid phase hemolysis assay. Somatic cell counts were determined for all raw samples. Macrophages and neutrophils made up the greatest proportion of leukocytes in colostrum followed by lymphocytes. Lymphocyte proportion increased as colostrum transitioned to mature milk, but overall somatic cell numbers declined concurrently. Viable cells were not isolated from HT or FR samples. Abundance of microRNA isolated from transition and mature milk was decreased compared with colostrum, did not differ between HT and RAW, but was increased in FR compared with RAW. Alternative complement pathway activity was decreased in HT, but not FR compared with RAW, and was not measurable in transition or mature milk. Postharvest heat-treatment and freezing of colostrum eliminated viable colostral leukocytes and affected microRNA abundance and complement activity. Leukocyte proportions, microRNA abundance, and complement activity changed as colostrum transitioned to mature milk. Although there were clear changes in the colostral components under study in relation to treatment and transition to mature milk, the biological significance of the described treatment effects and temporal changes were not investigated here.