The impact of sward type and inorganic nitrogen application rate on the rumen metabolome of dairy cows as determined by nuclear magnetic resonance

This study examined the effects of pasture species and inorganic nitrogen application rate on the rumen fluid metabolome of spring-calving dairy cows. Twelve rumen-cannulated Holstein Friesian cows were randomly assigned to 1 of 4 treatments in a 2 × 2 incomplete crossover design. The dietary treatments consisted of: (1) perennial ryegrass (PRG) receiving 25 kg inorganic N/ha per cut; (2) PRG receiving 50 kg inorganic N/ha per cut; (3) PRG-white clover (WC) receiving 0 kg inorganic N/ha per cut; and (4) PRG-WC receiving 25 kg inorganic N/ha per cut. Milk and rumen fluid samples were collected, milk samples were analyzed for composition using MilkoScan, whereas rumen fluid metabolome was analyzed using proton nuclear magnetic resonance spectroscopy. Metabolomic profiles were analyzed using a mixed-effects model and multivariate analysis demonstrated that pasture species had a greater effect on the rumen fluid metabolome than inorganic nitrogen application rate within treatments. Specifically, cows fed PRG-WC diets exhibited significantly higher concentrations of VFA, including isobutyrate, isovalerate, and valerate, indicating enhanced rumen fermentation. Additionally, concentration of alcohols, particularly isopropanol and methanol, were also higher in cows fed PRG-WC compared with cows fed PRG which could be due to presence of bioactive compounds in WC. Conversely, cows fed PRG diets had increased levels of nucleosides and nucleotides, likely due to controlled microbial growth, increased turnover, and nucleic acid release compared with PRG-WC. Other metabolites that were affected by treatment were fumarate and dimethylamine. Both of which have been previously reported to have an effect on methane emission. Fumarate is associated with reduced methane emission, while dimethylamine is associated with an increase in methane emission. Furthermore, correlation analysis demonstrated that milk fat percentage was correlated with choline (r = 0.58), glucose (r = 0.58), maltose (r = 0.54), glutamate (r = -0.54), and nicotinate (r = -0.55). Milk urea was positively correlated with VFA, p-cresol (r = 0.67) and phenyl acetate (r = 0.75). These findings emphasize the strong relationship between experimental treatment and rumen metabolic processes, highlighting the potential benefits of incorporating WC into pasture systems to enhance rumen fermentation efficiency.