Repeated inflammatory challenges alter inflammatory and metabolic responses in lactating dairy cows under negative energy balance.

During the periparturient period, dairy cows enter a state of negative energy balance (NEB), a metabolic state characterized by increased lipid mobilization and physiological adaptations. Additionally, this period is frequently accompanied by inflammatory and endotoxemic insults associated with common periparturient disorders. Importantly, cows affected by an adverse health event during the first weeks of lactation face increased odds of developing a second disease within weeks of the first, compounding the risk of morbidity, mortality, and early removal from the herd. While the metabolic effects of NEB are well recognized, the impact of repeated inflammatory challenges during this state remains poorly understood. The objective of this study was to characterize the metabolic and inflammatory responses of dairy cows in NEB to a repeated endotoxemic challenge. Thirty-two multiparous mid-lactation Holstein cows underwent a 4-period experimental protocol. During Period 0 (P0; 0-7 d), baseline data were collected under ad libitum feeding. In Period 1 (P1; 8-11 d), cows were subjected to feed restriction to induce NEB (-14 Mcal/d), and on d 10, cows received an i.v. infusion of either sterile saline (SAL; n = 16) or LPS (1 µg/kg BW; n = 16). A recovery period (REC; 12-18 d) served as a washout with ad libitum feeding. In Period 2 (P2; 19-22 d), the feed restriction protocol was repeated, and cows were assigned within their original groups to receive a second infusion of SAL or LPS on d 21, resulting in 4 treatment groups: S-SAL, S-LPS, L-SAL, and L-LPS (n = 8 per group). Feed restriction reduced milk yield, plasma glucose, and BHB, while increasing plasma NEFA. During P1, LPS reduce BHB compared with SAL. LPS also increased body temperature (+1.0°C over baseline in the first 6 h), circulating cytokines, and acute-phase proteins. In addition, LPS induced metabolic acidosis and hypocalcemia compared with SAL. During P2, L-LPS showed higher body temperature than S-LPS (+1.82 vs. +1.0°C, over baseline at 6 h post-infusion, respectively). Compared with S-LPS, L-LPS elicited a greater increase in heart rate. Furthermore, L-LPS induced an earlier increase and faster resolution of lipopolysaccharide-binding protein compared with S-LPS, while the haptoglobin response was attenuated by 31% in L-LPS. Additionally, S-LPS displayed lower concentrations of IP-10, MCP-1, and MIP-1β compared with L-LPS. Hypocalcemia and acidosis were milder in L-LPS compared with S-LPS. Together, these findings indicate that in the context of NEB, repeated endotoxemic events in dairy cows induce an altered inflammatory and metabolic phenotype characterized by selective immune training and attenuated metabolic disturbances, which may contribute to altered disease susceptibility during early lactation.