*4.1. Sample Collection*

A single 10 mL blood sample was taken from 708 clinical healthy cows, located on 13 farms in south-eastern Australia between September 2017 and July 2019. All cows had been calved 30 days

or less at the time of sampling. Cows on all farms except Farm 1 were Australian Holstein-Friesians, while cows on Farm 1 were crossbred animals (including Holstein–Friesian, Jersey, and Australian Red breeds). All farms operated a feeding system reliant on grazed pasture plus other forages, and concentrates fed in the bail at milking time.

Blood samples were collected from the coccygeal vein into 10 mL serum clot activator vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Samples were allowed to clot at room temperature, before being centrifuged at 1000 g for 20 min at 20 ◦C. Sera were divided into two aliquots. The first aliquot was refrigerated at 4 ◦C then shipped on ice to a commercial laboratory for BHBA analysis. The second aliquot was stored at –20 ◦C until processing for NMR spectroscopy.

#### *4.2. Reference BHBA Measurements*

Serum BHBA concentrations were determined using a colorimetric enzymatic kinetic assay [51]. All assays were performed by Regional Laboratory Services (Benalla, Victoria, Australia) using a Kone 20 XT clinical chemistry analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The uncertainty of measurement (at a 95% confidence level) was ± 0.060 mmol/L at 0.85 mmol/L.
