The calculation of the relative bioavailability (RBV) of rumen-protected AA supplements using the plasma free AA dose-response technique currently relies on blood samples obtained 2, 4, 6, and 8 h after the 0500 h feeding during the last 3 d of each period in Latin square experiments with cows fed every 8 h (0500, 1300, and 2100 h). The objective of this study was to determine if this current blood sampling protocol captures the changes that may occur in plasma Met concentrations within a 24-h day to adequately determine the RBV of Met from Smartamine M (SM). Five multiparous lactating Holstein cows were used in a 5 × 5 Latin square design with 7-d periods. Treatments were (1) control (abomasal infusion of tap water), (2) 12 g/d of abomasally infused dl-Met, (3) 24 g/d of abomasally infused dl-Met, (4) 15 g/d of fed Met (20 g/d of SM), and (5) 30 g/d of fed Met (40 g/d of SM). Blood samples were collected via jugular catheters every 2 h after the 0500 h feeding starting on d 5 and ending on d 7 of each period. Plasma Met analysis was conducted using gas chromatography after chloroformate derivatization. Plasma Met concentration was averaged across days for 2-8 h after the 0500 h feeding, 2-8 h after the 1300 h feeding, 2-8 h after the 2100 h feeding, and 2-24 h after the 0500 h feeding. In addition, plasma Met concentration was regressed on 0, 12, and 24 g of infused dl-Met and 0, 15, and 30 g of fed Met. The calculated RBV of Met from SM averaged 83.8%, 83.6%, 87.4%, and 83.0% for the 2-8 h, 10-16 h, 18-24 h, and 2-24 h sampling periods, respectively. The similarity in the estimations of RBV for the 2-8 h and 2-24 h sampling periods indicates that our original blood sampling protocol seems reliable for determining the RBV of ruminally protected Met products.
