The Shorthorn cattle, a prominent breed of beef cattle, originated in the historic counties of Northumberland, Durham, Yorkshire, and Lincolnshire in northeastern England. Empirical evidence suggests that through systematic selective breeding during the late Victorian agricultural revolution, Shorthorn cattle evolved into a globally recognized beef genotype by the early 20th century—a period characterized by proto-industrialization (Deerry, 2018). In comparison to indigenous Chinese cattle breeds, Shorthorn cattle demonstrate superior growth rates and meat quality. This makes them an essential genetic resource for enhancing beef production in China, particularly through hybrid breeding programs.

Traditionally, these cattle are raised on artificial grasslands. However, during the six-month forage withering period, their weight experiences a significant decline, which adversely affects subsequent fattening efficiency. Therefore, it is essential to optimize feeding strategies to mitigate these seasonal nutritional deficits in order to sustain Shorthorn cattle production. By aligning feed composition with the physiological needs of the cattle, intensive feeding regimes can reduce nutrient wastage, enhance metabolic efficiency, and minimize risks of environmental contamination (Moorby and Fraser, 2021).

The microbial ecosystem in the rumen plays a vital role in nutrient digestion and metabolism, with its composition influenced by factors such as host genetic background, developmental stage, dietary composition, and environmental conditions (Lin et al., 2019; Zhang et al., 2018). Changes in dietary nutrient composition can alter the abundance of specific microbial populations. For instance, studies have shown that the energy level of the ration significantly affects the rumen microbial composition in yaks (Ahmad et al., 2020). Additionally, feedlot fattening has been found to impact rumen fermentation, microbial diversity, and meat quality (Hu et al., 2021). Furthermore, Yang et al. (2024) observed that intensive feeding regimes led to an increase in metabolites associated with amino acid metabolism, such as L-glutamate (Yang et al., 2024). The proportion of dietary concentrate has also been reported to influence rumen microbial abundance, diversity, metabolite concentrations, and metabolic pathways (Pang et al., 2022). To gain deeper insights into these diet-driven microbial and metabolic dynamics, multi-omics approaches, integrating genomic, transcriptomic, and metabolomic data, have proven essential. These methods help link functional gene activity with microbial communities and metabolic processes within the rumen (Denman et al., 2018). By applying these approaches, researchers can better understand how dietary alterations affect microbial populations and metabolic functions, leading to a more comprehensive understanding of the rumen ecosystem in ruminants.

Despite significant advancements in ruminant nutrition, Shorthorn cattle, with their distinct genetic and metabolic characteristics, remain underexplored. Most existing studies on Shorthorn cattle have focused on genetic traits and growth performance, often overlooking the functional role of rumen microbiota and metabolites in nutrient utilization. To address this gap, our study integrates 16S/ITS sequencing and LC–MS metabolomics to investigate: (1) the impact of natural grazing versus intensive feeding on rumen microbial diversity and metabolite profiles in Shorthorn cattle, and (2) the relationships between microbial communities and key metabolites and metabolic pathways. This research represents the first multi-omics characterization of the Shorthorn cattle rumen ecosystem, providing valuable insights that can help refine feeding strategies and improve production efficiency.

The microbial community within the rumen is essential for sustaining the physiological health and normal digestive function of ruminants (Ley et al., 2008). In this study, our multi-omics analysis revealed distinct shifts in rumen microbiota and metabolomes in Shorthorn cattle under different feeding regimens, thereby providing novel insights into the interplay between dietary factors, microbial dynamics, and metabolic outcomes. The taxonomically rich rumen bacterial community drives anaerobic digestion to generate bioavailable substrates for host metabolism (Saleem et al., 2012; Simon et al., 2017). Alpha diversity serves as a key indicator of microbial community robustness, with higher values indicating greater structural stability and functional resilience against environmental perturbations (Jiang F. et al., 2021). Our results showed that Chao1 and Shannon indices were significantly higher in the naturally grazed group (DJCF) than in the intensively fed groups (DJCY, DJC), consistent with previous findings that high-fiber diets enhance community stability. Liu et al. observed that increasing dietary concentrates reduced rumen microbial diversity in yaks (Liu et al., 2019); similarly, He et al. reported higher microbial diversity in naturally grazed cattle (He et al., 2024a). This effect may stem from a decrease in rumen pH due to high-concentrate diets, which in turn inhibit the growth of fiber-degrading bacteria (Sonnenburg and Bäckhed, 2016). The PCoA results further confirmed the distinct bacterial and fungal community structures between DJCF and the intensive-feeding groups, with the first two principal components explaining 24.76 and 19.76% of bacterial variance, respectively, which indicates a strong dietary impact on microbial assembly.

At the phylum level, we found that Bacteroidota and Firmicutes were dominant; together, these phyla have been reported to comprise more than 80% of the bacteria in the gastrointestinal tract (Eckburg et al., 2005; Jiang C. X. et al., 2021; Liu et al., 2021). These observations are consistent with previous research (Chen et al., 2015; Ma et al., 2023; Zhou et al., 2017). Bacteroidota utilize carbohydrate-active enzymes (CAZymes) to ferment dietary polysaccharides into short-chain fatty acids (SCFAs) while mediating protein, bile acid, and oligosaccharide metabolism (He et al., 2024b; Hess et al., 2011; Thomas et al., 2011). Synergistically, Firmicutes employ specialized hydrolases to bioconvert complex substrates (e.g., cellulose, proteins) into bioavailable nutrients (He et al., 2024b; Huo et al., 2014). This cross-phylum partnership optimizes host digestive efficiency through complementary metabolic pathways (He et al., 2024a; Nagaraja and Titgemeyer, 2007). The DJCF group had the highest Bacteroidota abundance (57.62%) and the lowest Firmicutes abundance (34.07%), while the DJC group showed the opposite trend (Bacteroidota 48.84% and Firmicutes 43.08%). This shift coincided with changes in metabolic profiles: higher Bacteroidota in DJCF was associated with elevated fibre-degradation metabolites (e.g., cellobiose), whereas increased Firmicutes in the intensive-feeding groups correlated with higher purine metabolites (AMP, dAMP) and carbohydrate intermediates (maltose, glucose). These findings suggest that the Bacteroidota–Firmicutes balance acts as a key regulator of nutrient-utilization pathways in response to dietary change.

At the genus level, Prevotella and Rikenellaceae_RC9_gut_group were the dominant taxa, both belonging to Bacteroidetes, in agreement with the results of the present study (He et al., 2024a; Yang et al., 2024). Functionally, Prevotella is a key rumen bacterium mediating proteolysis and amylolysis via secreted enzymes (proteases, amylases, hemicellulases), thereby enabling the degradation of dietary proteins, starch, and hemicellulose; its abundance typically increases with higher dietary protein (Bekele et al., 2010; Cholewińska et al., 2020; He et al., 2024a). Rikenellaceae_RC9_gut_group, in turn, has been linked to lipid metabolism and the production of short-chain fatty acids, notably propionate, and may also contribute to protein degradation (Conte et al., 2022; Fan et al., 2017). Diet appears to modulate the relative dominance of these genera: Fernando et al. reported that concentrate-fed beef cows had a higher proportion of Bacteroidetes than those fed green hay, with Rikenellaceae_RC9_gut_group as the dominant genus (Fernando et al., 2010; He et al., 2024a), whereas in our study Prevotella predominated in the DJCY and DJC groups. This discrepancy likely reflects the higher crude-protein and starch contents of the DJCY/DJC total mixed-ration diets, which favor protein- and starch-degrading microbes such as Prevotella. Correlation analyses further supported these functional inferences: Prevotella, most abundant in DJCY (22.52%), showed significant positive correlations with cytidine 5′-monophosphate (CMP) and 2′-deoxyguanosine 5′-monophosphate (dGMP), and negative correlations with phytanic acid and caprolactam, suggesting a context-specific role in nucleotide metabolism under intensive feeding. By contrast, Rikenellaceae_RC9_gut_group, most abundant in DJCF (12.56%), associated with higher levels of propionate precursors, consistent with its contribution to lipid metabolism and short-chain fatty acid (SCFA) synthesis (Conte et al., 2022). Taken together, these patterns align with evidence that altering feed type and concentrate proportion (e.g., TMR with higher crude protein and starch) reshapes the Bacteroidetes/Firmicutes balance and modulates carbohydrate and purine-related pathways, thereby favoring Prevotella-driven metabolism under intensive diets.

Anaerobic fungi are pivotal decomposers in the rumen, and Neocallimastigomycota typically predominate; in our dataset they accounted for 75.68–86.74% of the fungal community, in line with reports that this phylum is the dominant fungal group in ruminants (Boots et al., 2013; Denman et al., 2018; Wallace et al., 2019). Diet remodels fungal diversity and composition: intensive feeding reshapes the rumen microbiota, including the mycobiome, whereas seasonal and forage quality shifts also modulate fungal taxa (Cengiz and John, 2002; Faichney et al., 1997; He et al., 2024b; Wang et al., 2023). In our study, the natural-grazing group (DJCF) displayed higher Neocallimastigomycota abundance than the intensive-feeding groups, consistent with literature showing context-dependent fungal responses to dietary inputs in high-altitude ruminants. Functionally, Neocallimastigomycota enhance nutrient bioavailability by secreting potent cellulases and cellulosomes that depolymerize recalcitrant plant cell walls (e.g., Orpinomyces systems) (Abhaya et al., 2017; Boots et al., 2013). Beyond classic fiber degradation, our integrative analyses revealed fungus–metabolite couplings that highlight ecological specialization: Orpinomyces (enriched in DJCF) correlated positively with 2-oxoadipic acid and luteolin—metabolites linked to plant-matrix breakdown—whereas Caecomyces (elevated under intensive feeding) correlated with phosphatidylcholine (PC) species, suggesting adjustment of lipid-related metabolism under high-concentrate diets. These findings extend current evidence that feed type and concentrate proportion restructure the rumen community and its metabolic outputs, and they pinpoint fungal lineages that track specific metabolite pathways, thereby underscoring fungi’s contribution to host energy harvest beyond lignocellulose turnover (Denman et al., 2018; He et al., 2024a; He et al., 2024b; Wallace et al., 2019).

Intensive or high-concentrate feeding reshapes rumen carbohydrate metabolism and fermentation; in yaks, multi-omics shows that intensive systems elevate dietary starch/protein and reconfigure the microbiome–metabolome interface (Yang et al., 2024). Consistent with this paradigm, our intensive-feeding groups (DJCY/DJC) accumulated carbohydrate intermediates (e.g., maltose, glucose), and pathway analysis indicated significant shifts in starch and sucrose metabolism together with purine metabolism, a pattern widely observed when feed type or forage-to-concentrate ratios change (Liu et al., 2019; Penner et al., 2009). Similar to the results of this experiment, we observed a significant increase of carbohydrate metabolites in the DJCY group, such as maltose, glucose, which are important intermediates of carbohydrate metabolism. The metabolic pathway is primarily sucrose and starch metabolism. In the rumen, microbial enzymes break down starch and cellulose into glucose, which then undergoes glycolysis to produce pyruvate. This pyruvate, derived from carbohydrate metabolites, acts as the key substrate for VFA production (Xue et al., 2018). Metabolic pathway analysis revealed that sucrose and starch metabolism and purine metabolism were significantly affected by feeding regimens. Four metabolites (Cellobiose, Trehalose, Maltose, Isomaltose) in the sucrose and starch pathway were more abundant in DJCF, aligning with the higher fiber-degrading microbiota in this group. Meanwhile, purine metabolites (AMP, dAMP, Adenosine) were enriched in DJCY and DJC, which corresponded to the elevated Prevotella abundance and were consistent with the role of these metabolites as biomarkers for microbial protein synthesis (Ametaj et al., 2010). The significant correlation between Prevotella and purine metabolites further confirms the direct involvement of specific taxa in regulating these pathways. Notably, our study identified several low-abundance microbial taxa with potential functional significance. LEfSe analysis revealed nine differential species in DJCF, including Fibrobacter succinogenes, which showed positive correlations with phytanic acid and eupatilin despite its low relative abundance. Similarly, the DJC group had specific low-abundance oscillospiraceae taxa associated with octanoylglucuronide, a metabolite involved in lipid conjugation. These findings highlight that even low-abundance microbes may play critical roles in metabolic regulation, but their actual metabolic functions remain unclear due to limitations in current multi-omics approaches.

Of particular interest is the discovery of changes in several metabolites associated with the purine metabolic pathway. Ruminal microbes degrade nitrogenous compounds from feed and channel them into nucleic-acid metabolism through de novo and salvage pathways; nucleosides are then catabolized to bases (e.g., xanthine, hypoxanthine, guanine, adenine) and further to degradation products (Fujihara and Shem, 2011; Stentoft et al., 2015). We found higher levels of purine-related metabolites in the concentrate-fed groups, consistent with previous reports of elevated xanthine and hypoxanthine under high-concentrate diets (Ametaj et al., 2010; Zhang et al., 2017). In addition, adenine and xanthine—and likely pseudouridine—identified among the feed-intake groups likely originate from bacterial nucleic-acid degradation (Noel et al., 2017; Sutton et al., 1975). In the above results, it was shown that concentrate feed contributes to rumen purine metabolism. Arachidonic acid functions as a sensitive and specific plasma biomarker for the average daily gain (ADG) in steers (Artegoitia et al., 2016). Furthermore, Naturally occurring in all mammalian cells, arachidonic acid (an ω-6 tetra-unsaturated fatty acid) maintains membrane fluidity while serving as the primary biosynthetic precursor for lipid-derived signaling molecules, including prostaglandins and leukotrienes (Martin et al., 2016). In this study, we found a higher concentration of prostaglandin in the DJCF, DJCY group compared with that in the DJC group. It may be related to the effect of castration of Shorthorn cattle on androgen. Phosphatidylcholine (PC), a glycerophospholipid, serves as a critical structural element in cellular lipid bilayers. Choline constitutes a primary component of PC, and adequate PC levels are essential for supporting lipid transport mechanisms (Shiau and Cho, 2002; Yeh et al., 2015). PC functions as a choline reservoir, with the stored choline being further processed into organic osmolytes through metabolic pathways involving glycine, serine, and threonine (Athamena et al., 2011; Jiang et al., 2018). Ren et al. showed that PC concentration increased linearly with increasing concentrate levels and that this metabolite, which is related to lipid metabolism, plays an important role in yak growth and rumen health. The results of the present experimental study showed that the PC concentration in the two fattening groups were significantly higher than that in the grazing group, indicating that the PC concentration increased with the nutrient level of the diets. To further explore the potential connection between rumen dominant microflora, and metabolites, Pearson correlation analysis was carried out. Dai et al. indicated that differential metabolites are significantly correlated, either positively or negatively, with dominant microorganisms (Dai et al., 2023). Emerging evidence from integrated multi-omics investigations has revealed utilization or production associations between bacteria and metabolomes in the rumen (Liu et al., 2019; Ma et al., 2021). Saccharomycetales_gen_Incertae_sedis in the DJCY group showed positive correlations with 24 metabolites (including CMP and turanose), implying a potential role in nucleotide and carbohydrate metabolism. However, the regulatory mechanisms driving these associations—whether through direct metabolic conversion or indirect ecological interactions—remain to be elucidated.

Overall, these patterns highlight the effects of different feeding strategies in Shorthorn cattle and provide insights into the mechanisms by which feeding practices shape rumen microbial processes. While our study integrates 16S rRNA gene and ITS amplicon sequencing with metabolomics, directly inferring in situ metabolic functions and the specific regulatory roles of low-abundance bacteria and fungi remains challenging. In future work, we will combine metagenomic and metatranscriptomic approaches to delineate links between microbial gene expression and metabolic pathways, and we will elucidate organism-level functions in the rumen microecosystem through isolation and in-vitro metabolic validation of key strains, thereby providing a theoretical basis for precision nutrition in Shorthorn cattle.

This study used multi-omics analysis to reveal the adaptive mechanisms of the rumen microbiome and metabolites of Shorthorn cattle in intensive feeding systems. High-concentrate diets enriched proteolytic bacteria Prevotella and suppressed fiber-degrading fungi Neocallimastigomycota, activating purine metabolism pathways, indicating enhanced microbial protein synthesis efficiency. Concurrently, Shorthorn cattle maintained Bacteroidota functionality under high-starch diets through host genetic adaptation. Thus, an intensive feeding system altered the rumen microbiome and rumen metabolism, resulting in improvements of Shorthorn cattle growth. These findings provide novel strategies for precision feeding based on metabolic biomarkers, though further validation is required to address limitations such as limited sample size and potential confounding effects of castration.