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The interactive effect of dietary fat supplementation and milk yield level on dairy cows performance under heat stress has not been thoroughly investigated. The purpose of this study was to evaluate the effect of production level, the source of fat supplements and their interaction on dairy cows performance under heat stress. In this study, 64 Holstein multiparous cows were divided into 2 groups and received one of two rations having either calcium salts of fatty acids (Ca-FA) or high-palmitic acid (PA) supplements (2.8% of DM; dry matter). After completing the experiment and based on maturity-equivalent milk, cows were divided into two groups of high-yielding (14,633 kg) and medium-yielding (11,616 kg). Average temperature humidity index (THI) was 71 during the trial period. Apparent digestibility of dry matter (p = 0.04), organic matter (p = 0.05), and neutral detergent fiber (NDF; p = 0.04) for cows fed Ca-FA were greater than cows fed PA. The milk fat content in high-producing cows was 0.3% greater than medium-producing cows (p = 0.03). The milk protein content in cows fed Ca-FA was greater than cows fed PA (p < 0.01). High-producing cows had greater serum cholesterol (p = 0.02) than medium-producing cows. The cows fed PA tended to have a greater BUN than cows fed Ca-FA (p = 0.06). Alanine aminotransferase and aspartate aminotransferase tended to be increased by PA, which indicates that cows in PA treatment may have experienced more adverse effect on the liver function than cows on Ca-FA. Therefore, under heat stress and in 90 d trial, milk production level does not affect the cows’ response to PA or Ca-FA. Although cows fed Ca-FA received lower energy than those fed PA, they compensated for this shortage likely with increasing the digestibility and produced a similar amount of milk.

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