To investigate the effect of 5-aminolevulinic acid (5-ALA) on in vitro rumen gas production, fermentation characteristics, and bacterial community profiles, five levels of 5-ALA (0, 100, 500, 1000, and 5000 mg/kg DM) were supplemented into a total mixed ration (concentrate/forage = 40:60) as substrate in an in vitro experiment. Results showed that as the supplementation level of 5-ALA increased, asymptotic gas production (b) decreased linearly and quadratically (p < 0.01) while the dry matter degradation rate increased quadratically (p < 0.01). Meanwhile, the propionate concentration of 72 h incubation fluid increased linearly (p = 0.03) and pH value increased linearly and quadratically (p < 0.01), while the concentrations of butyrate, isobutyrate, valerate, isovalerate, and NH₃-N and the ratio of acetate/propionate (A/P) decreased linearly and quadratically (p < 0.05). There was no significant difference in any alpha diversity indices of bacterial communities among the various 5-ALA levels (p < 0.05). PCoA and PERMANOVA analysis revealed that the bacterial profiles showed a statistical difference between the treatment 5-ALA at 1000 mg/kg DM and the other levels except for 5000 mg/kg DM (p < 0.05). Taxonomic classification revealed a total of 18 and 173 bacterial taxa at the phylum and genus level with relative abundances higher than 0.01% in at least half of the samples, respectively. LEfse analysis revealed that 19 bacterial taxa were affected by 5-ALA levels. Correlation analysis showed that Actinobacteriota was positively correlated with the gas production parameter b, the ratio of A/P, and the concentration of butyrate, isovalerate, and NH₃-N (p < 0.05) and negatively correlated with pH (p < 0.05). WPS-2 exhibited a negative correlation with the gas production parameter b, the ratio of A/P, and the concentration of butyrate, valerate, isobutyrate, isovalerate, and NH₃-N (p < 0.05), along with a weaker positive correlation with pH (p = 0.04). The Bacteroidales BS11 gut group was negatively correlated with the concentration of propionate but positively correlated with gas production parameter b and the concentration of butyrate and NH₃-N (p < 0.05). The Lachnospiraceae NK3A20 group was found to have a positive correlation with gas production parameter b, the ratio of A/P, and the concentration of butyrate, isobutyrate, isovalerate, valerate, total VFA, and NH₃-N (p < 0.05), but a highly negative correlation with pH (p < 0.01). Differential metabolic pathways analysis suggested that metabolic pathways related to crude protein utilization, such as L-glutamate degradation VIII (to propanoate), L-tryptophan degradation IX, and urea cycle, increased with 5-ALA levels. In summary, including 5-ALA in the diet might improve energy and protein utilization by reducing the abundance of Actinobacteriota, the Bacteroidales BS11 gut group, the Lachnospiraceae NK3A20 group, and certain pathogenic bacteria and increasing the abundance of WPS-2.

Slaughter performance is a critical economic trait that varies across breeds, yet the rumen metabolic mechanisms driving these phenotypic differences remain unclear. The study involved 30 healthy 12-month-old beef cattle, with 10 animals from each of the three breeds: Chinese Simmental (ST), Taihang Yun (TY), and Charolais (CL). The cattle were randomly assigned into three groups using a completely randomized design, and the average initial body weight was 549.78 ± 59.45 kg. A 130-day feeding trial (10-day pre-feeding period, 120-day main trial period) was conducted. By comparing the slaughter performance, relative organ weight, and rumen fluid metabolomic profiles, the study investigated breed-specific differences in meat quality and potential underlying metabolic patterns. The results showed that CL exhibited a superior carcass yield, with a significantly higher dressing percentage (62.38%, p = 0.013) and net meat percentage (56.54%, p = 0.028) than ST and TY, and a significantly lower backfat thickness (p = 0.006); ST had the highest proportion of premium cuts, relative to carcass weight (72.97%, p = 0.014), with prominent economic value, while TY had significantly higher weights of visceral organs, such as liver, kidney, small intestine and omasum, than CL (p < 0.05). Metabolomic analysis revealed that CL and ST had elevated levels of purine metabolism, nucleotide synthesis and cofactor biosynthesis compared to TY. In conclusion, CL and ST possess advantages in carcass yield supported by upregulated anabolic metabolism in the rumen, whereas TY prioritizes visceral organ development. These findings provide valuable insights into the physiological and metabolic divergences regulating the slaughter performance and regional adaptability across cattle breeds.