Effect of Metabolizable Protein Supply on Milk Performance, Ruminal Fermentation, Apparent Total-Tract Digestibility, Energy and Nitrogen Utilization, and Enteric Methane Production of Ayrshire and Holstein Cows

**Background:** Holstein cows comprise approximately 94% of the dairy cow population in North America, and nutrient requirement models (CNCPS, NRC) have been developed primarily based on Holstein characteristics. Ayrshire cows represent only 1.5% and 0.3% of dairy cows in Canada and the USA, respectively, and differ phenotypically and genetically from Holsteins (smaller body weight, lower milk yield but higher milk fat and protein concentrations). Whether existing ration formulation models accurately predict the metabolizable protein (MP) requirements of Ayrshire cows is unknown. Additionally, potential differences between breeds in digestive physiology, ruminal microbial populations, and methane emissions have not been well characterized. This study aimed to evaluate the effects of increasing MP supply (85%, 100%, and 115% of requirement) on milk performance, ruminal fermentation, nutrient digestibility, energy and nitrogen utilization, and enteric methane production in Ayrshire versus Holstein lactating dairy cows.

**Methods:** Eighteen multiparous lactating cows (9 Ayrshire, 9 Holstein; 6 of each breed ruminally cannulated) were used in a replicated 3 × 3 Latin square design with 35-day periods. Diets were formulated using the CNCPS model (version 6.55) to supply 85%, 100%, or 115% of daily MP requirement, with breed-specific diets to account for differences in body weight, dry matter intake (DMI), milk production, and milk composition. Forage-to-concentrate ratios were 67:33 for Holsteins and 62:38 for Ayrshires. The 35-day periods included adaptation (d 1–14), methane measurement in respiration chambers (d 15–23), ruminal fluid sampling (d 25–26), and total collection of feces and urine for digestibility and nitrogen balance (d 27–33). Ruminal pH was measured continuously using indwelling probes. Methane concentration was measured continuously over 5 days using respiration chambers (4.09 m × 2.95 m × 2.84 m) with inline mass flowmeters and SERVOPRO 4100 analyzers. Data were analyzed using the MIXED procedure of SAS with breed, MP supply, breed × MP interaction, and period as fixed effects.

**Key Results:** Dry matter intake was 20% lower in Ayrshire vs. Holstein cows (20.5 vs. 25.8 kg/d, p < 0.01), but did not differ when expressed as % BW (average 3.66%). Milk yield was 25% lower in Ayrshire cows (32.8 vs. 43.7 kg/d, p < 0.01), with a significant breed × MP supply interaction (p = 0.05): milk yield increased linearly in Ayrshire cows with increasing MP, while a quadratic response was observed in Holsteins (no further increase from 100% to 115% MP). Energy-corrected milk (ECM) yield was lower in Ayrshire cows (36.0 vs. 45.3 kg/d, p < 0.01). Milk fat concentration (4.06% vs. 3.73%, p = 0.06) and milk protein concentration (3.39% vs. 3.05%, p < 0.01) were higher in Ayrshire cows. Feed efficiency (ECM/DMI) averaged 1.75 kg/kg and did not differ between breeds (p = 0.97). Nitrogen use efficiency averaged 33.7 g milk N/100 g N intake and did not differ between breeds (p = 0.53). Apparent total-tract digestibility of DM (70.3% vs. 68.2%, p = 0.04), OM (72.0% vs. 69.9%, p = 0.03), CP (67.8% vs. 65.1%, p = 0.01), NDF (43.3% vs. 38.8%, p = 0.05), and GE (70.0% vs. 67.7%, p = 0.02) were greater in Ayrshire cows. Daily methane emissions were 20% lower in Ayrshire cows (386 vs. 484 g/d, p = 0.06), but methane yield (18.9 g/kg DMI, p = 0.93) and intensity (10.8 g/kg ECM, p = 0.94) did not differ between breeds. Increasing MP supply linearly increased ECM yield (p < 0.01), feed efficiency (p < 0.01), and milk protein yield (quadratic, p = 0.01). Nitrogen use efficiency decreased linearly from 36.2 to 31.0 g milk N/100 g N intake (p < 0.01) as MP supply increased. Urinary N excretion increased linearly from 22.5% to 32.1% of N intake (p < 0.01). Methane yield and intensity were unaffected by MP supply.

**Clinical Implications:** This study demonstrates that Ayrshire and Holstein cows respond similarly to increasing MP supply, with no meaningful differences in feed efficiency, nitrogen use efficiency, or methane emission intensity between breeds. The CNCPS model appears to adequately predict MP requirements for both breeds, though the significant breed × MP interaction for milk yield suggests Ayrshire cows may have slightly higher MP requirements for milk production than predicted. Feeding MP above 100% of requirement does not improve milk production but significantly increases urinary nitrogen excretion (by up to 9.6 percentage units of N intake), worsening the environmental footprint of milk production. Methane emissions are primarily driven by DMI rather than breed or MP supply, and methane intensity is not affected by dietary protein manipulation. These findings support the use of existing ration formulation models for Ayrshire cows and emphasize that precision feeding to meet—but not exceed—MP requirements optimizes production while minimizing nitrogen losses to the environment.