Waste milk consumption in Holstein calves: effect on systemic adaptive immunity

Waste milk (WM) is widely used as the main liquid diet in calf-rearing systems worldwide. However, it contains a high microbial load and residues of drugs, particularly antimicrobials and anti-inflammatory agents, which may impair the development of the adaptive immune response and, consequently, resilience in the short and long term. This study aimed to evaluate the effects of WM on humoral and cellular adaptive immune parameters against pathogenic and probiotic bacteria in suckling Holstein calves.Animals were allocated into three experimental groups: saleable milk (SM = 10), pasteurized waste milk (PWM = 10), and raw waste milk (WM = 10), and were evaluated on days 7, 21, 35, 49, and 63 of life. Clinical scoring was performed, and body weight was estimated using thoracic girth measurements. Blood samples were collected for mononuclear cell count and isolation using density gradient centrifugation, followed by cell culture to assess proliferation through 5-bromo-2'-deoxyuridine (BrdU) incorporation. Cytokine levels were quantified in the culture supernatant using ELISA; total serum protein (TSP) and Brix index were measured using refractometry; and IgG concentrations were quantified by ELISA.For cellular immunity assessments, PBMC cultures were performed in quintuplicates under stimulation with Concanavalin A (ConA), Escherichia coli, Lactobacillus johnsonii, Lactobacillus reuteri, and Bifidobacterium longum, in addition to unstimulated controls. Statistical analysis was conducted using SAS software. Most continuous variables were analyzed using the General Linear Model (GLM), revealing significant differences across experimental groups. Health assessments indicated a higher incidence of neonatal diseases, particularly diarrhea, in calves fed WM, as evidenced by the lower fecal dry matter content. A trend toward lower respiratory disease incidence was observed in the SM group. A significant interaction between treatment and development was found for weekly weight gain during the first month of life. Calves in the PWM and WM groups showed higher counts of monocytes, lymphocytes, and total mononuclear cells. PBMCs from SM and PWM calves demonstrated greater proliferative responses to stimuli such as ConA, E. coli, and L. johnsonii. Moreover, PBMCs from SM and PWM calves produced higher levels of interleukin-4 (IL-4), while WM was associated with increased production of IL-17, particularly after stimulation with E. coli and ConA. IL-10 production was more pronounced in the SM group. Regarding the humoral response, baseline IgG levels were similar among groups before treatment. However, IgG concentrations increased progressively and more intensely in the WM group, followed by PWM, while SM calves maintained lower IgG levels throughout the suckling period. Indirect biomarkers of humoral immunity, such as total serum protein and the Brix index, mirrored the IgG dynamics. In conclusion, different types of liquid diets modulate both cellular and humoral adaptive immune responses. Calves receiving raw waste milk exhibited a more reactive and pro-inflammatory immune profile. Pasteurization of waste milk (PWM) partially mitigated the antigenic burden's effects on the adaptive immune system, while saleable milk (SM) was associated with a less reactive and more immunomodulatory profile.