Background: Heat stress (HS) becomes a serious threat to dairy cow industry in certain circumstances, Ncarbamylglutamate (NCG) is a novel feed additive that may be used to alleviate HS. However, the effects and mechanisms of NCG on dairy cows under HS are still unknown. A total of 48 Holstein dairy cows with similar days in milk (154.37±13.56 d), parity (1-3), and body condition score (BCS) were randomly divided into 4 groups of 12 animals each. Under HS, the cows were fed a TMR diet supplemented with 0 (control), 15, 20, 25 g of NCG/d per cow for 60 days. Milk production performance was recorded and serum parameters were examined. Meanwhile, metabolomics study of plasma based on liquid chromatography-mass spectrometry (LC-MS) was also applied to investigate metabolites and pathways in response to NCG supplementation.Results: Milk yield (MY) was increased but milk urine nitrogen (MUN) was reduced by the NCG treatment.Addition of 15 g of NCG/d increased the milk protein percentage (MPP) compared with the control. No effect of NCG was observed on dry matter intake (DMI), lactose percentage (LP), milk fat percentage (MFP) and somatic cell count (SCC). Serum glucose (GLU) levels in cows fed with 15, 20, 25 g of NCG/d were increased by 14.35%, 19.34% and 18.63% in comparison with the control. Meanwhile, the concentrations of blood urea nitrogen (BUN) and blood ammonia (BA) were decreased with the addition of NCG. Results for antioxidant capacity and immune function showed bene cial effects of NCG, such as the increases in total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), IgG, IgM and IgA. Clear separations of plasma metabolic pro les between control and NCG groups were observed in the score plots. Sixteen different metabolites involved in metabolisms of amino acids, ketone bodies, butanoate and energy, as well as gut microbiome-derived metabolism were regulated by NCG supplementation.Conclusions: This study found that NCG treatment improved antioxidant capacity, immune function, production performance, and metabolic pro le of dairy cows under HS and provided new evidence for the better utilization of NCG.

Background: Heat stress (HS) becomes a serious threat to dairy cow industry in certain circumstances, N-carbamylglutamate (NCG) is a novel feed additive that may be used to alleviate HS. However, the effects and mechanisms of NCG on dairy cows under HS are still unknown. A total of 48 Holstein dairy cows with similar days in milk (154.37±13.56 d), parity (1-3), and body condition score (BCS) were randomly divided into 4 groups of 12 animals each. Under HS, the cows were fed a TMR diet supplemented with 0 (control), 15, 20, 25 g of NCG/d per cow for 60 days. Milk production performance was recorded and serum parameters were examined. Meanwhile, metabolomics study of plasma based on liquid chromatography–mass spectrometry (LC-MS) was also applied to investigate metabolites and pathways in response to NCG supplementation.Results: Milk yield (MY) was increased but milk urine nitrogen (MUN) was reduced by the NCG treatment. Addition of 15 g of NCG/d increased the milk protein percentage (MPP) compared with the control. No effect of NCG was observed on dry matter intake (DMI), lactose percentage (LP), milk fat percentage (MFP) and somatic cell count (SCC). Serum glucose (GLU) levels in cows fed with 15, 20, 25 g of NCG/d were increased by 14.35%, 19.34% and 18.63% in comparison with the control. Meanwhile, the concentrations of blood urea nitrogen (BUN) and blood ammonia (BA) were decreased with the addition of NCG. Results for antioxidant capacity and immune function showed beneficial effects of NCG, such as the increases in total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), IgG, IgM and IgA. Clear separations of plasma metabolic profiles between control and NCG groups were observed in the score plots. Sixteen different metabolites involved in metabolisms of amino acids, ketone bodies, butanoate and energy, as well as gut microbiome-derived metabolism were regulated by NCG supplementation.Conclusions: This study found that NCG treatment improved antioxidant capacity, immune function, production performance, and metabolic profile of dairy cows under HS and provided new evidence for the better utilization of NCG.

The objective of the present study was to investigate the effects of N-carbamylglutamate (NCG) supplementation on metabolic profile and microbiota in ruminal content and feces of lactating dairy cows under heat stress (HS). Forty-eight lactating Holstein cows (154 ± 13.6 days in milk) were assigned randomly to four treatments (n = 12), to receive 0, 15, 20, or 25 g/day of commercial NCG (proportion: 97.7%) for the period of 60 days. The recorded ambient temperature–humidity index (THI) suggested that the cows were exposed to HS for almost the entire experimental period (average THI: 80.6). Samples of ruminal content and feces were collected at the end of the trial (day 60) to determine the biological effects of NCG supplementation on metabolome and microbiota using mass spectrometry-based metabolomics and 16S rRNA gene sequencing techniques, respectively. Results showed that NCG supplementation enhanced the levels of ruminal microbial protein, total volatile fatty acids (VFAs), and the molar proportion of propionate in the rumen, but lowered the ruminal pH, ammonia nitrogen (NH3-N), and the ratio of acetate to propionate. NCG at doses of 20 and 25 g/day reduced the community richness and diversity of ruminal microbiota with the decrease of Shannon and Simpson diversity. Compositions of ruminal and fecal microbiotas were altered by NCG, and the PICRUSt results revealed that metabolic pathways of the bacteria, such as amino acid metabolism, energy metabolism, and pyruvate metabolism, were enriched in NCG groups. Distinct changes in the metabolomic profile of ruminal fluid were observed between the control and NCG groups. Changes of 26 metabolites mainly involved in arginine metabolism, glutamate metabolism, and nitrogen metabolism were observed associated with NCG supplementation. These results provided new insights into the effects of NCG on metabolomic profile and microbiota in ruminal content and feces, and the optimal dose of NCG supplemented to dairy cows was 20 g/hd/day, which contributed to understanding the effects of NCG on HS in lactating dairy cows.