Effect of rumen-protected standardized dry grape extract supplementation on antioxidant and immune-related gene expression in leukocytes of mid-lactating Fleckvieh cows exposed to natural heat stress.

Heat stress (HS) disrupts oxidative homeostasis and impairs immune function in dairy cows. In this context, plant polyphenols, when included in the diet, may modulate antioxidant defenses and immune response, including the antioxidant ability of leukocytes. This study evaluated whether supplementation with rumen-protected standardized dry grape extract (Nor-Grape BP-O; Nor-Feed) modulates antioxidant and immune-related gene expression in circulating leukocytes of mid-lactating Fleckvieh cows under natural HS. Thirty cows were randomly assigned to a control diet (CTR; n = 15) or to the same diet supplemented with 470 mg/cow per day of Nor-Grape BP-O (NG-BPO; n = 15) and monitored for 35 d during summer. Blood samples were collected at 0, 17, and 35 d for leukocyte isolation and quantitative PCR analysis. Relative gene expression was calculated using LinReg software and then transformed into ln fold change (lnFC) relative to 0 d. Data were subjected to PROC GLIMMIX procedure of SAS version 9.4 (SAS Institute). Overall, NG-BPO cows showed higher lnFC expression of glutathione peroxidase 1 (GPX1) and glutathione-disulfide reductase (GSR) than CTR cows. The lnFC superoxide dismutase 1 (SOD1) expression tended to be greater in NG-BPO cows compared with CTR cows, whereas superoxide dismutase 2 (SOD2) expression was unaffected by the supplementation. Among immune-related genes, interleukin-1β (IL1B) had an overall greater lnFC expression in NG-BPO cows, particularly at d 35, when a heat wave occurred, compared with CTR cows. A similar pattern was observed for lnFC interleukin-10 (IL10) expression, which was greater at 35 d in NG-BPO cows compared with CTR cows. In contrast, toll-like receptor 2 (TLR2), myeloid differentiation primary response 88 (MYD88), interleukin-1 receptor (IL1R), and glucose transporter 3 (GLUT3) were not affected by supplementation. The early upregulation of GPX1 and GSR at 17 d suggests a transitory activation of oxidative detoxification pathway driven by polyphenols, followed by downregulation at 35 d. Concomitantly, the increased IL10 and IL1B expression at 35 d in NG-BPO cows may suggest an enhanced capacity to regulate inflammatory signaling and maintain immune homeostasis, by preventing the negative effect of excessive immune activation. Overall, rumen-protected standardized dry grape extract supplementation was associated with a positive modulation of leukocyte genes involved in antioxidant defense system and regulation of inflammation, providing new molecular insight supporting previously reported improvements in physiological and immunometabolic responses during HS.