*4.1. Animals*

Wagyu steers (aged 29–30 months, *n* = 4) and Holstein steers (aged 21–22 months, *n* = 4) were used in this study. They received a concentrate (78% total digestible nutrients and 13% crude protein) and orchard grass hay (56% total digestible nutrients and 8% crude protein) ad libitum from 10 months of age until they were slaughtered. Longissimus muscle (sirloin) samples were collected at slaughter. The sirloin samples (1.5 kg) were collected between the third and fourth lumbar vertebrae from the left side of the carcass. Samples were vacuum-packed and wet-aged at 4 ◦C for 7 days and then stored at −80 ◦C for later metabolome analysis. All animals received humane care as outlined in the Guide for the Care and Use of Experimental Animals (No.1631B004, National Agriculture and Food Research Organization).
