Methane (CH₄) production of ruminants typically increases with increased dry matter intake (DMI). However, few studies have observed the effects of feeding multiple times a day and its effects on diurnal variation in CH₄ production and energy balance in late-lactation dairy cattle. A study using headbox-style indirect calorimetry and 12 multiparous (225 ± 16.2 d in milk; mean ± SD) lactating Jersey cows was conducted to determine the effects of feeding twice daily on diurnal variation in CH₄ production and total energy balance. A crossover design with 14-d periods (10 d of adaption and 4 d of collection) was used to compare 2 treatments. Treatments consisted of either once a day feeding (1×; 100% of feed given at 1000 h) or twice a day feeding (2×; 50% of feed given at 1000 h and the final 50% at 2000 h) with a common diet fed in both treatments. Dry matter intake was not different between treatments, with a mean of 16.9 ± 0.88 kg/d. Once a day feeding tended to have greater milk yield compared with twice a day feeding (21.2 vs. 20.4 ± 1.59 kg/d, respectively). Milk fat and milk protein percentage were not different, with means of 6.18 ± 0.20% and 3.98 ± 0.08%, respectively. Total CH₄ production did not differ between treatments, with a mean of 402.1 ± 20.8 L/d. Similarly, CH₄ per unit of milk yield and DMI was not different between treatments, with means of 20.5 ± 1.81 and 23.8 ± 1.21 L/kg, respectively. Feeding frequency did not affect diurnal variation of hourly CH₄ production, with a mean of 17.1 ± 0.74 L/h. A trend was observed for a treatment × hour interaction. Methane production per hour increased after the second feeding for cattle fed twice versus once daily. Gross energy, digestible energy, metabolizable energy, and balance (milk plus tissue) per kilogram of DMI did not differ by feeding frequency, with means of 4.41 ± 0.01, 3.05 ± 0.03, 2.63 ± 0.03, and 1.32 ± 0.08 Mcal/kg of DM, respectively. Metabolizable energy for maintenance was 146 kcal/kg of metabolic body weight, with an efficiency of converting metabolizable energy to net energy balance (milk plus tissue) of 76%. Nitrogen balance did not differ among treatments, with a mean balance of 17.3 ± 13.0 g/d. Therefore, total CH₄ production and energy maintenance were not affected by feeding frequency. However, CH₄ was variable throughout the day, and caution should be exercised when collecting CH₄ samples at a limited number of time points because this may under- or overestimate total production.

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