Metabolomics combined with 16S rDNA revealed the effects of low protein diet on rumen microbiome structure, nitrogen utilization rate and differential metabolites in feces and urine of dairy cows

Abstract

Background

The use of low-protein diets on the basis of amino acid balance is conducive to optimizing feed formulations, reducing production costs, and improving economic efficiency. Aims of the study: Research objective: This experiment aims to conduct a feeding test by selecting 40 Holstein dairy cows and randomly dividing them into 2 groups, with 20 cows in each group and 20 replicates in each group, and one cow in each replicate. The effects of reducing dietary protein levels, adding RP-Lys and RP-Met on rumen microbial diversity, nitrogen metabolism and different metabolites in feces and urine of dairy cows were investigated by using 16S rDNA and metabolomics. To provide a certain theoretical basis for guiding dairy cows to save protein feed production. Results: Low protein amino acid balanced diet had no significant effect on rumen fermentation parameters such as rumen pH, NH₃-N and TVFA. At the genus level, the relative abundance of Ruminococcus in LP group [LP, low-protein diet with 16.0 % of crude protein +50 g/cow/d rumen-protected lysine (RP-Lys) + 30 g/cow/d rumen-protected methionine (RP-Met)] was significantly decreased. In the Linear discriminant analysis Effect Size (LEfSe) analysis, a total of 54 microorganisms with significant differences in relative abundance between groups were screened from phylum to genus. The abundance of 35 species such as Ruminococcus and Ruminobacillus in HP group (HP = high-protein group, regular diet with 17.5 % of crude protein) was significantly higher than that in LP group, while the abundance of 19 species such as Vibrio butyrate and Helicobacter was significantly lower than that in LP group (P < 0.05). The cows in LP group had higher dry matter intake (DMI) than the HP group after 90d. The dietary nitrogen intake in LP group was markedly lower than that in HP group (P < 0.05). Compared to HP group, the total nitrogen excretion was significantly lower in LP group (P < 0.01). The LP group treatment increased the nitrogen utilization efficiency (P < 0.05) when compared with HP group. Through metabolomics analysis, 620 differential metabolites were screened out from fecal samples, of which 329 were significantly up-regulated, such as uracil, uridine and uric acid, and 291 were significantly down-regulated, such as cholesterol, xanthine and N-acetyl serotonin. 502 differential metabolites were screened out from urine samples, of which 268 were significantly up-regulated, such as creatine, tryptamine and phenylacetylglycine (PAGly), and 234 were significantly down-regulated, such as phenol, L-proline (L-Pro) and L-tyrosine (L-Tyr). KEGG pathway enrichment analysis showed that LP group had significant differences in protein digestion and absorption, nucleotide metabolism, amino acid biosynthesis and metabolism. Conclusion: Adding protective rumen lysine and methionine to low-protein diets has no significant effect on rumen fermentation parameters such as pH, NH3-N, acetic acid and propionic acid, and has positive significance for maintaining the structural stability of rumen microbiota. It can reduce nitrogen excretion and improve nitrogen utilization efficiency, which is in line with the current development concept of green breeding and provides a scientific basis for reducing breeding pollution and optimizing resource utilization. And by regulating the metabolism of L-try and L-ala, it affects protein digestion and absorption, energy supply and rumen microbiota, laying a theoretical foundation for further improving the nutritional supply system of ruminants and enhancing production performance, and has extremely high practical guiding value.