Milkoscan 203

Cows were milked twice daily at approximately 0700 and 1530 h. Weekly milk production was derived from individual milk yields (kg) recorded at each milking (Dairymaster). Milk fat, protein, and lactose contents were determined once weekly from a consecutive p.m. and a.m. milk sample for each cow and tested using infrared spectrophotometry (Milkoscan 203, Foss Electric) . Milk solids (kg of fat + protein; MSo) yield per cow and solids-corrected milk (SCM; Tyrrell and Reid, 1965) (link) were also calculated. For the purpose of this experiment, only milk production data relating to the weeks of the DMI measurement periods are presented. Full-lactation milk production results can be found in McClearn et al. (2019) . Cows were weighed in the week before, during the week of, and in the week after the DMI measurement period upon exit from the milking parlor using an electronic scale (Tru-Test Ltd.). Body condition score was assessed, in a similar manner, by the same individual throughout the study on a scale of 1 to 5 in increments of 0.25 (where 1 = emaciated and 5 = extremely fat) as outlined by Edmonson et al. (1989) (link).

Cows were milked twice daily at 0700 and 1530 h. Individual yields (kg/cow) were recorded at each milking (Dairymaster, Causeway, Ireland). Milk composition was measured weekly from one successive evening and morning milking. The concentrations of protein, fat, and lactose were determined using a Milkoscan 203 (Foss Electric, Hillerød, Denmark) .
Body weight and BCS were measured on a weekly basis. All cows were weighed using an electronic portable weighing scale and Winweigh software package (Tru-Test Limited, Auckland, New Zealand). Body condition score was recorded weekly and was scored by an experienced independent observer using a 1 to 5 scale (1 = emaciated and 5 = extremely fat) with 0.25 increments (Edmonson et al., 1989) .

Duplicate bulk tank milk samples and duplicate milk line samples were analyzed daily for fat, protein and lactose composition and SCC using a Milkoscan 203 (Foss Electric, Hillerød, Denmark) .
Duplicate bulk storage tank milk samples were submitted for wet chemistry analysis of nitrogen fractions after storage for 0, 48 and 96 h. The percentage total protein, NPN, and noncasein nitrogen content of the milk samples were determined using the Kjeldahl method [methods 20-3 (IDF, 2004b ), 20-4 (IDF, 2001 ), and 29-1 (IDF, 2004a) , respectively] using a Tecator Digestor Auto and Kjeltec 8400 distiller (Foss Electric).
The protein composition of duplicate bulk storage tank milk samples was quantified daily (0, 24, 48, 72, and 96 h) by HPLC using the method described by Mounsey and O'Kennedy (2009) . Briefly, 200 μL of milk was diluted in 3,800 μL of dissociating buffer (7 M urea and 20 mM Bis-Tris propane, pH 7.5), to which 5 μL of 2-mercaptoethanol was added, before filtering through a 0.22-μm filter. Separation of the milk protein fractions was achieved in reverse-phase mode, using an Agilent Poroshell 300SB C18 column (2.1 × 75 mm; Agilent Technologies, Santa Clara, CA). The HPLC equipment consisted of an Agilent 1200s with quaternary pump and multi-wavelength detector. Gradient elution and peak detection were performed according to the method of Mounsey and O'Kennedy (2009) .