The gut microbiota and miRNAs are emerging as promising targets for managing and preventing inflammatory and metabolic disorders in mammals. In the present study, we identified functional fecal miRNAs associated with the feed efficiency phenotype residual feed intake and linkages between the miRNA profile and microbiome gut composition of Nelore cattle belonging to divergent feed efficiency groups.
The bovine gut microbiome consists of trillions of microorganisms, most of which are bacteria12. As expected, in ruminants, Firmicutes, Proteobacteria, and Bacteroidetes were the main bacterial phyla found in the microbiomes of the efficient and inefficient groups of Nelore bulls. Firmicutes is a phylum in which many members produce butyrate, an important substance that keeps the colon healthy and plays a significant role in animal health13. They also breakdown carbohydrates that cannot be digested by enzymes in the gut, such as dietary fiber and resistant starch14. Proteobacteria is a phylum that digests/degrades proteins through the process of decarboxylation of amino acids15, while Bacteroidetes is also one of the predominant phyla with fermentative characteristics and the ability to modulate the immune system16. Here, we did not observe significant differences in the richness of bacterial populations between efficient and inefficient animals, which could have been influenced by the small sample size used in this study. Nonetheless, Clemons et al.17 suggested that the lack of diversity differences regarding feed efficiency phenotypes may be due to dissimilarities at a finer resolution, such as for individual taxa and metabolites, rather than global changes in microbial communities and metabolites.
Functionality of fecal miRNAs
Fecal miRNAs have been characterized in bovine feces and identified as biomarkers for intestinal diseases18, however, little is known about fecal miRNAs and their relationship with feed efficiency traits in bovines.
The bta-miR-27b, bta-miR-30a, bta-miR-126, bta-miR-143, bta-miR-155, bta-miR-205 and bta-miR-196a were all up-regulated in the inefficient group. The target genes of bta-miR-126 and bta-miR-155 were predicted to be involved in signal transduction pathways associated with muscle development, such as the mTOR and Wnt signaling pathways. Mammalian target of rapamycin (mTOR) regulates cell proliferation, autophagy, and apoptosis by participating in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K)/Akt and AMPK pathways19. mTOR in conjunction with Akt, a protein kinase B, is required for skeletal muscle cell development20. Based on the higher expression of bta-miR-126 and bta-miR155 and given the most canonical post transcriptional downregulation mechanism of miRNA-mRNA interaction, we can speculate that the muscle development pathway is downregulated in inefficient animals, corroborating the idea that they exhibit less muscle in the adult phase than the efficient ones.
The target genes of bta-miR-126 were also related to the focal adhesion pathway. Focal adhesions (FAs) are points of contact between the cell and the extracellular matrix that regulate cell communication with the extracellular environment and cellular processes21. The digestion and absorption capacities of the small intestine are closely related to feed efficiency traits in pigs, and small intestine structures such as microvilli, focal adhesions, and intestinal mucosa are important factors affecting the absorption of nutrients in the intestine22. Pathways associated with small intestinal structure also include those involved in the regulation of the actin cytoskeleton, adherens and tight junctions22. Thus, the bta-miR-205 could also have a role in this structure by targeting genes associated with the adherens junction pathway. In cattle, the microarchitecture of the small intestine is related to improved feed efficiency. Greater cellularity indicates a more metabolically active small intestine in cattle with higher feed efficiency23.
Some upregulated miRNAs in inefficient animals were predicted to play important roles in metabolic homeostasis, including insulin and glucose metabolism. Among them, bta-miR-143 and bta-miR-27b were also upregulated in inefficient cattle in a previous study24. These authors speculated that the increased expression of btamiR-143 impaired insulin and glucose homeostasis by targeting the insulin signaling pathway and its regulation. This miRNA has also been reported with a role in intestinal epithelium regeneration by modulating the insulin growth factor signaling pathway25. In this study, bta-mir-143 was predicted to regulate genes related to EGFR tyrosine kinase inhibitor resistance, regulation of the actin cytoskeleton and the PI3K-Akt signaling pathway, while bta-miR-27b was predicted to regulate genes associated with type II diabetes mellitus, insulin resistance and insulin pathway. The regulation of feed intake and feed efficiency by insulin has been described in many species, including cattle26 and pigs27. Here, the predicted downregulation of the insulin pathway by bta-miR-27b in inefficient animals is consistent with findings in the literature that indicate increased insulin metabolism with reduced feed intake in efficient animals26.
The target genes of bta-miR-126 were associated with the FoxO signaling pathway. FoxO transcription factors regulate genes associated with glucose metabolism and resistance to oxidative stress28, and this pathway has already been associated with increased feed efficiency in Nelore cattle. Similarly, Casal et al.29 reported that efficient steers had better hepatic oxidative status associated with greater antioxidant ability and reduced oxidative stress, which would reduce maintenance requirements due to lower protein and lipid turnover, resulting in better energy use efficiency. Therefore, the downregulation of the FoxO signaling pathway in inefficient animals may result in higher oxidative stress, lowering feed efficiency in Nelore bulls.
Other enriched signaling pathways related to RFI through bta-mir-205 and bta-mir- 196a target genes were Rap 1 and Ras-related protein 1, respectively. Ras-proximate-1 or Ras-related protein 1 (Rap1) are small cytosolic proteins that act as cellular switches, being essential for effective signal transduction30 and related to leptin, which regulates body weight and feed intake in bovines. Both pathways were previously associated with increased feed efficiency in Nelore cattle31. In our study, based on the upregulation of bta-mir-205 and bta-mir-196 in the inefficient group, the Rap 1 and Ras signaling pathways were predicted to be downregulated, suggesting a mechanism for the previously observed differences in pathway modulation.
Relating modules to feed efficiency and finding potential biomarkers
Module–trait relationships are estimated by Spearman’s correlations between the MEs and the animals’ phenotypic information to select potential biologically interesting modules that could explain the phenotypic differences between groups, while hub miRNAs or ASVs are those with the highest correlation within the module. Therefore, hub miRNAs and hub ASVs identified by WGCNA can be considered principal components and, consequently, potential biomarkers for feed efficiency. In the efficient group, negative and positive correlations, respectively, were detected between the RFI and hub ASVs classified as Bacillales from MEblack and Succinispira mobilis from MEred, whereas in the inefficient group, negative correlations between the RFI and the hub bta-mir-16a from MEturquoise were detected.
Bacillales is an order of gram-positive bacteria from the phylum Firmicutes, and representative genera, including Bacillus are the core of the human gut microbiome and are found in bovine feces32. This genus was reported to have antimicrobial activity against microbes that promote nutrient absorption, and the order Bacillales was associated with inefficient beef cattle32. Succinispira mobilis is a succinate-decarboxylating anaerobic bacterium33, and previous reports mention acetate and succinate (a precursor of propionate) as the major products of ruminants fed high-starch diets34; therefore, S. mobilis might play a role in propionate synthesis, thereby improving feed efficiency in efficient Nelore bulls.
The bta-miR-16a and bta-miR-16b have been reported to regulate milk fat metabolism, with a negative effect on fatty acid metabolism and adipocyte differentiation35. The biological mechanisms driving the synthesis of fatty acids and triacylglycerols are complex and partially regulated by miRNAs. Several miRNAs, including miR-16b, were predicted to target genes related to lipid metabolism and/or adipogenesis, and as the adipose tissue modulates a variety of processes related to feed intake, energy homeostasis, and physiology, are also associated with feed efficiency traits36. Previous studies also indicate a potential role for miR16 in inflammatory processes, with this miRNA increasing T-cell subtypes, and influencing the degradation of mRNAs from immune response pathways37. These results indicate that bta-miR-16a may contribute to reduced feed efficiency due to its functional effects on fatty acid metabolism and the immune response.
miRNA-microbiome interactions
The relationship between host miRNAs and the gut microbiota has been investigated, being Liu et al.7 the first to propose a linkage between miRNA expression and the gut microbiota composition (and its metabolites). Since then, many manuscripts have been published 38–41 and, to support this, in this study, we identified high and significant correlations between miRNA expression and the gut microbiome and its relationship with feed efficiency in Nelore cattle. According to the canonical view, eukaryotic miRNAs negatively regulate mRNA translation via complementary binding to 3’ untranslated regions (UTRs), which results in either translation repression or degradation of the mRNA transcript42. However, the role of miRNAs in bacterial gene regulation is yet to be fully understood. Host miRNAs can enter bacteria in different ways, including through extracellular vesicles, and can specifically regulate bacterial gene transcripts that control bacterial growth7. Conversely, changes in the microbiome may also induce differences in miRNA expression43, demonstrating the power of miRNA-microbiome interactions.
In coexpression analysis, module eigengenes are considered important biological clusters, and microorganisms in the same modules have strong relationships, which provides an opportunity to investigate and explore highly related taxa within a microbial community44. The roles of miRNAs in regulating host–microbe interactions were further evaluated, exploring the relationships among the expression of miRNAs and bacterial composition. No direct relation between the microbiome and the described miRNAs has been reported in literature.
miRNA-microbiome interactions in the efficient group
In the efficient group, DE bta-mir-205 from MEbrown was negatively correlated with Prevotella, Clostridiales, Lachnospiraceae, Firmicutes, and Gammaproteobacteria from MEred. With a role in digesting complex polysaccharides, such as cellulose and hemicellulose, the genus Prevotella has been associated with lower feed efficiency in cattle45 and pigs 46. However, Prevotella was recently identified as a potential biomarker for efficient beef cattle47. The Prevotella genus, with 29 known species, contains cellulolytic bacteria that degrade cellulose into acetic, isobutyric, isovaleric, and lactic acid, providing energy for the host48. In addition to increasing glycogen storage and glucose tolerance, Prevotella-rich microbiota can improve growth performance, which is important for regulating RFI in beef cattle 49. In our study, we are still determining which species of Prevotela was identified as, in general, 16S rRNA gene sequences allow differentiation between organisms at the genus level. Gammaproteobacteria is a class of Proteobacteria identified in a study of feed efficiency phenotypes in beef cattle and the relative abundance of this phylum has also been associated with high-efficiency steers17. The DE bta-mir-205 was positively correlated with Hespellia porcina, Alistipes, Peptostreptococcaceae, Ruminococcaceae and Clostridium saccharogumia from ASV MEblue. Alistipes is a genus of bacteria in the phylum Bacteroidetes that colonizes the human gastrointestinal tract and has protective effects against intestinal inflammation50, while the species Clostridium saccharogumia is associated with increased body weight and abdominal fat in chickens 51.
In a study with efficient steers, Lourenco et al.52 demonstrated increased Peptostreptococcaceae and Ruminococcaceae populations. The greater abundance of some members of the Peptostreptococcaceae family may contribute to increased ammonia availability in the hindgut, allowing for the development of structural carbohydrate-fermenting bacteria in more efficient steers52. Ruminococcaceae is a family composed of both fibrolytic organisms and involved in starch hydrolysis, which produces acetate, formate, and succinate. contributing to increased feed efficiency. In our study, Ruminococcaceae from MEyellow was a unique taxon negatively correlated with DE bta-mir-155 from MEblue. On the other hand, in the efficient Nelore bulls, DE bta-mir-155 was positively correlated with Coriobacteriaceae from MEred. This family of bacteria and different phylotypes are considered regulatory targets for improving host feed efficiency, as they are more abundant in efficient steers53. DE bta-miR-126 from MEturq was positively correlated with Lachnospiraceae, Bacteroidale and Clostridiales from MEyellow. Myer et al.54 also reported that Lachnospiraceae and Clostridiales were more abundant in efficient steers. Acetogens can be found in the Lachnospiraceae and Ruminococcaceae families and serve as hydrogen sinks, which may increase with reduced methane production55. The relationship between methane production and feed efficiency is known, where the energy not lost as methane can be converted into weight gain, increasing animal efficiency32. Furthermore, the ASV MEred was negatively correlated with RFI in the module-trait association analysis. Overall, the positive effect of these microorganisms on feed efficiency biological processes indicate that these miRNAs and these taxa might contribute to increased feed efficiency in Nelore cattle.
miRNA-microbiome interactions in the inefficient group
In the inefficient group, DE bta-miR-196a from MEyellow was negatively correlated with Lachnospiraceae and Ruminococcaceae families from MEyellow, and the DE bta-miR 126 from MEblue was negatively correlated with Anaerorhabdus furcosa and Bifidobacterium, in addition to the Lachnospiraceae and Ruminococcaceae families. The miR-126 was recently implicated as potential biomarker in an inflammatory bowel disease (IBD) study, reported to inhibit leukocyte adhesion pathways56. A. furcosa has been associated with human infection and the production of short-chain fatty acids (SCFAs). SCFA production improves intestinal homeostasis and weaning stress in piglets and is associated with the modulation of intestinal microbiota composition and immune system genes 57. Bifidobacterium species are known to produce carbohydrate-degrading enzymes, which facilitate carbohydrate metabolism and efficiently extract energy, contributing to the host's feed efficiency58. Furthermore, Bifidobacterium is also a significant producer of SCFAs and decreased in abundance in a study of IBD patients 56. SCFAs may affect the differentiation of epithelial cells, which are known to play an essential role in intestinal homeostasis. In IBD patients, the host inflammatory response produces oxidative stress for the host and the intestinal microbiota, leading to intestinal dysbiosis with a reduced abundance of Firmicutes and Bacteroidetes species56. Based on the idea that inefficient animals may present intestinal dysbiosis due to metabolic processes of oxidative stress, we can speculate that in our study, decreased Bifidobacterium and A. furcosa populations may reflect the effect of bta-miR-126 in the inefficient animals. Consistent with our results, E. Hernandez-Sanabria et al.34 also found that Bifidobacterium was associated with inefficient steers, while A. furcosa spp. have never been linked to feed efficiency.
In addition to the negative correlations in the inefficient group, we found most of the positive correlations of DE bta-miR-30a from MEturquoise and bta-mir-196a with ASVs classified as Lachnospiraceae and Ruminococcaceae families, and with Bacteroidales, Bacillaceae and Clostridium. Furthermore, the miRNA MEturquoise was negatively correlated with RFI in the module-trait association analysis. Bacteroidales is an order of bacteria that includes the genus Prevotella and is commonly associated with feed efficiency in bovines47. This genus is one of the most abundant taxa in the rumen, with species that grow on starch, protein, peptides, hemicellulose, and pectin and, similar to what we found in our study, can be both positively and negatively correlated with FE in beef and dairy cattle59. The order Bacillales and the family Bacillaceae have been associated with inefficient cattle32, while the presence of the Clostridiaceae family in the digestive tract of ruminants is well documented. Clostridiaceae are essential commensals in the digestion of carbohydrates and proteins, and numerous species are involved in bile acid metabolism, being related to a higher feed efficiency60. The genus Clostridium is more frequently associated with feed efficiency in poultry61. Considering that the Lachnospiraceae and Ruminococcaceae families exhibited both positive and negative correlations in the feed efficiency groups, we suggest that these ASVs may belong to different genera, species or lineages and be physiologically different within the groups, which could not be observed here due to the limitations of 16S taxonomic signals.
Final considerations
The role of miRNAs and their interactions with the host and its microbiota have been gaining prominence, and several studies have demonstrated that miRNAs can modulate the intestinal microbiota, while the intestinal microbiota, in turn, may regulate miRNA expression. Fecal miRNAs can regulate bacterial composition by targeting bacterial genes, and conversely, the gut microbiota can regulate host gene expression and miRNAs through gut microbiota metabolites 38.
In humans, miRNAs have been associated with several biological processes, such as the immune system, cancer, and obesity, and due to their increasing relevance, in the last decade, they have been associated with production traits in livestock species. Some studies in beef cattle have implicated miRNAs as potential regulators of important biological pathways related to feed efficiency, such as muscle development and adipogenesis. In this study, some of the upregulated miRNAs correlated with bacteria that contribute to lower feed efficiency in the inefficient group were also correlated with bacterial microbiomes that increased feed efficiency in the efficient group, suggesting that these miRNAs and bacteria are somehow related to biological processes that influence feed efficiency. Furthermore, differences in richness and diversity between feed efficiency groups would be expected from the correlations found with miRNAs. However, the expected effects of miRNA would be on gene expression and thus on the functionality of the microbiome. This hypothesis could not be confirmed as the method used to access microbiomes in our study does not allow for identification of functional differences. Also, if slight differences in individual microorganisms’ abundance would result from this modulation, they would probably not have overpassed the multiple tests correction due to the limited sample size of the study, since the number of microorganisms was far higher than the number of miRNAs per sample.
Our results suggest a complex link between host miRNAs and the bovine microbiota and the taxa Prevotella, Ruminococcaceae, Lachnospiraceae, Anaerorhabdus furcosa, Bifidobacterium, Bacillales, Succinispira mobilis, Peptostreptococcaceae and Coriobacteriaceae appear to influence feed efficiency. The miRNAs and taxa identified from network analyses may serve as potential candidates for exploring host–microbe interactions. Although our exploratory study has limitations, our findings could serve as a basis for future studies on the development of strategies to manipulate the microbiome and improve feed efficiency traits of bovines.