Effects of a metabolizable protein supply on metabolic function of CD4+ T lymphocytes in mid-lactation dairy cows

The objective of the present experiment was to determine the effects of MP supply on metabolic function of nonactivated (NA) and activated (AC) CD4⁺ T lymphocytes in mid-lactation dairy cows. Thirty-two multiparous Holstein cows with a mean ± SD lactation of 3.28 ± 0.99, milk yield 51.6 ± 6.1 kg/d, and milk protein 3.38% ± 0.47% were enrolled at 86 ± 18 DIM in a barn equipped with 16 bin feeders. Cows and bins (16 cows/8 bins per treatment) were randomly assigned to treatments. All cows were fed the same herd diet (MP = 3,006 g/d) during a 2-wk adaptation period before enrollment and a 1-wk covariate period following enrollment to allow acclimation to the bins and collect baseline measurements. Following the covariate period, cows received 1 of 2 treatments for 12 wk: diets formulated to be either adequate in MP supply (MPA; MP = 3,111 g/d) or MP inadequate (MPI; MP = 2,983 g/d). The difference in MP supply was achieved by including porcine blood meal in the MPA, whereas it was omitted in the MPI treatment. Blood was collected during wk 11 at 173 ± 18 DIM to isolate peripheral blood mononuclear cells followed by magnetic isolation of CD4⁺ T lymphocytes using bovine-specific monoclonal antibodies. Isolated CD4⁺ T lymphocytes from each cow were split into 2 tubes and randomly assigned to incubate in an assay medium (250 µL) or with a combination of phorbol 12-myristate 13-acetate (100 µL, 20 ng/mL) and ionomycin (10 µL, 1 µg/mL) for 2 h at 37°C to evaluate metabolic function under metabolically NA and AC states, respectively, using an extracellular flux analyzer. Mitochondrial and glycolytic functional kinetics were recorded for NA and AC CD4⁺ T lymphocytes based on real-time measurement of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and proton efflux rate (PER) under basal conditions and in response to complex V inhibitor (oligomycin A), a protonophore uncoupler (BAM 15), and complex I and complex III inhibitors (rotenone and antimycin A). Cows fed the MPI diet exhibited lower OCR leading to a reduced maximal respiration compared with MPA-fed cows. Feeding MPI diet decreased maximal respiration in both metabolically NA and AC CD4⁺ T lymphocytes. Feeding MPI diet decreased sparing respiratory capacity in both metabolically NA and AC CD4⁺ T lymphocytes. During the T-cell persistence assay, an interaction between diet and time within the assay was observed for ECAR and PER, indicating that CD4⁺ T cells from cows fed MPI showed a progressive decrease in aerobic glycolysis over the 1.50 to 73.0 min measurement period, whereas CD4⁺ T cells from cows fed MPA maintained higher glycolytic activity. Mitochondrial, glycolytic, and total ATP production rates were unaffected by MP supply; however, glycolytic ATP production rates were greater in AC than NA CD4⁺ T lymphocytes. These results indicate that suboptimal supply of MP reduced the metabolic fitness and persistence of CD4⁺ T lymphocytes, which might compromise immune responses during nutrient imbalance, such as early lactation or health challenges.