Unveiling the rumen-microbiome-brain circuit: a unique dimension of gut-brain axis in ruminants

Apart from direct neurotransmitter synthesis, gut bacteria can also synthesize neuroactive precursors that serve as substrates for neurotransmitter production and neuromodulators that indirectly regulate nervous system. Over 90% of host serotonin is produced by enterochromaffin cells from tryptophan, a precursor that can be synthesized by bacteria such as Bacillus subtilis through tryptophan synthase [67]. Indole, both a precursor of serotonin and a neuromodulator that regulates brain cognition, can be produced by diverse gut bacterial species, such as E. coli, Clostridium sporogenes, and Bacteroides spp. which harbor tryptophanase (TnaA) [68]. Clostridium sporogenes can also decarboxylate tryptophan to produce tryptamine, a neurochemical molecule affecting the neurological activity of the host [69]. In addition, SCFAs (e.g., acetate, propionate, and butyrate), the major end products of carbohydrate fermentation by gut bacteria, as well as secondary bile acids (e.g., lithocholic acid and deoxycholic acid), synthesized from primary bile acids by gut bacteria such as Clostridium scindens and Clostridium sordellii, are important neuromodulators that regulate host neurotransmitter synthesis and neuronal development [41, 70, 71].

Moreover, advances in metagenomic sequencing and bioinformatics have enabled the identification or prediction of other gene clusters corresponding to the production or degradation of neuroactive compounds in human gut microbiota [6]. Further studies are needed to confirm their functional roles.

Gut microbe-derived neurotransmitters can directly activate receptors on enteric neurons and relay signals to vagal afferents, which are in close proximity to the myenteric plexus of ENS, further affecting the CNS [41, 60]. Neurotransmitters in the gut lumen can also be uptaken by gut epithelial cells by corresponding transporters, and further reach vagal afferent and activate their neurotransmitter receptors expressed on the end terminals of vagal afferent [72]. Gut microbe-derived EVs can cargo microbial synthesized neurotransmitters to the CNS via activation of vagal afferent fibers in the myenteric plexus of ENS [73] or can enter the systemic circulation and cross the blood–brain barrier delivering the protected cargo neurotransmitters directly to the CNS [66]. Gut microbe-derived neurotransmitter precursors such as tryptophan and tyrosine can be converted into serotonin and dopamine by enteric neurons via tryptophan hydroxylase and DOPA decarboxylase [74], and subsequently regulate the CNS through the vagal pathway. Additionally, neuromodulators SCFAs, indole, and secondary bile acids that are uniquely produced by gut microbes can activate specific receptors on enteroendocrine cells, such as free fatty acid receptors (FFARs), Aryl hydrocarbon receptor (AhR), and Farnesoid X receptor (FXR) bile acid receptors, which regulate the expression of gene involved in neurotransmitter, thereby modulating the neurotransmitter synthesis and influencing the activation of ENS and vagal afferents [28, 72]. Moreover, gut epithelium contains specialized neuropod cells [48, 52, 53] that can sense microbial-derived neurotransmitter signals within the lumen and form direct synaptic connections with vagal afferent neurons through axon-like basal processes in the gut mucosa, thereby relaying gut microbial signals to the brain [75]. In addition to transmitting sensory signals, the vagus nerve serves as a physical link in the microbiota-gut-brain axis, presenting a promising therapeutic target for drug delivery via the gut microbiota-vagal pathway [72].

Certain gut microbe-derived neuroactive compounds can be transported to the bloodstream through gut epithelium, across BBB, and influence CNS. Most gut-derived neurotransmitters, such as glutamate, serotonin, dopamine, and acetylcholine, cannot cross the BBB due to their size, polarity, and lack of specific transport mechanisms [76]. However, GABA in the gut lumen can cross the BBB through the GABA transporter 2 (GAT2) system expressed on brain capillary endothelial cells [77, 78] and bind to the specific GABA receptors, such as GABA-A and GABA-B receptors in the pre- and post-GABAergic neuronal terminals, modulating CNS neurotransmitter homeostasis [79, 80]. Microbe-derived tryptophan can cross the BBB via large neutral amino acid transporter 1 and is subsequently converted to serotonin in the brain [67, 72, 81]. SCFAs, indole, and secondary bile acids can also pass through the BBB and regulate brain functions. Acetate improves maturation of astrocytes by serving as an energy source [82] and also enhances the synthesis of neuroactive peptide pro-opiomelanocortin while suppressing neuropeptide Y, further regulating appetite [83]. Propionate and butyrate exhibit neuroprotective properties by attenuating neuropeptide Y depletion [84], with butyrate also modulating brain serotonin production [85]. Additionally, SCFAs enhance BBB integrity by increasing the expression of tight junction proteins, reducing paracellular permeability, thereby regulating the availability of neurotransmitters and their precursor in the brain and further regulate neurodegeneration [86 –88] (Fig. 1). In addition, indole derivatives cross the BBB via passive diffusion or by efflux transporters such as P-glycoprotein [89] and can activate AhR signaling in the brain, influencing cognition and neuroprotection [90]. Secondary bile acids have been shown to exert neuroprotective effects in neurodegenerative diseases [91, 92]. They can cross the BBB using the apical sodium-dependent bile acid transporter (ASBT) in enterocytes, modulating the activity of neuronal transporters, such as the dopamine and GABA transporters (DAT, GAT1), thereby enhancing the transport efficiency of these neurotransmitters across neuronal membranes in the brain [93 –95].

Taken together, gut microbiota plays important roles in regulating the nervous system, as well as influencing behavior and emotional states. This has led to the concept of psychobiotics, microbes with the capacity to confer mental health benefits [96]. For example, it has been demonstrated that administration of probiotic strains Bacillus clausii and Lactobacillus fermentum NMCC-14 (10¹⁰ CFU/mL/d) to mice subjected to acute and subacute restraint stress led to a significant increase in norepinephrine, serotonin, and dopamine levels in the hippocampus and prefrontal cortex [97]. These findings underscore the therapeutic promise of developing gut microbe-derived psychobiotics as interventions to regulate central nervous system function through the gut-brain axis.

In contrast to monogastric animals, ruminants heavily rely on their stomach, particularly the biggest compartment, the rumen and the large microbiome that inhabits it for nutrient digestion. The intricate innervation between the CNS and ENS is essential for the unique motility patterns and circadian rhythms in the rumen [98, 99], especially for coordinating rumination, the unique physiological process of regurgitating, remasticating, and reswallowing feed to optimize fermentation in ruminants. The myenteric plexus, located between the inner circular and outer longitudinal muscle layers of the rumen, comprises ganglia, neuronal soma, and connecting nerve fiber strands (NFS) [98, 100]. The submucosal plexus includes an inner layer, containing ganglia connected by major NFS, and an external layer, innervating the submucosal and mucosal layers in the ruminal wall [98]. In addition to the ruminal wall, the ruminal pillar, a muscular division in the rumen that separates the stomach into different sacs, contains a denser network of primary NFS, arranged in parallel at regular intervals. Anatomical and quantitative distribution of vagus nerve fibers across the rumen was reported in sheep several decades ago [101]. Together, the rumen possesses its own enteric nervous system, which is extensively integrated with vagal innervation to coordinate its complex motor functions, such as rumination.

The similarities in the distribution of ENS and vagal afferents in the rumen, compared with those in the intestine, suggest that ruminal afferents are capable of sensing luminal signals. However, with more layers of ruminal epithelial cells, neurotransmitters and other neuroactive compounds produced in the ruminal lumen must diffuse across several epithelial layers before reaching the underlying afferents, which may reduce the efficiency of direct signaling to enteric neurons and vagal afferents. Furthermore, the relatively low abundance of enteroendocrine cells in the rumen may limit the production of host-derived neuroactive hormones and constrain enteroendocrine-CNS signaling pathways. Nevertheless, the unique physiology of rumination, the higher expression of transporters and receptors for certain neurotransmitters and microbe-derived neuroactive compounds in rumen tissue, along with its dense and metabolically active microbiota collectively suggest the existence of a distinct rumen-microbiome-brain axis.

The rumen hosts a vast and diverse microbial community, dominated by bacteria but with comparatively higher abundances of fungi and protozoa than the intestinal microbiota [106]. While these microbes are essential for fermentation, their potential neuroactive roles remain largely unclear.

In addition to bacteria, rumen also hosts up to 50% biomass of protozoa, predominantly represented by genera like Entodinium, Isotricha, Diplodinium, Epidinium, and Dasytricha [121 –123]. These ciliated protozoa directly produce neuromodulator SCFAs through fermentation of fiber and starch [122, 124] and also indirectly regulate the ruminal SCFAs concentration through engulfing the rumen fiber-digesting bacteria and interacting with methanogens that shift the rumen fermentation capability [125]. Though it is largely unknown whether these commensal rumen protozoa can synthesize other neuroactive compounds and further regulate the nervous system of ruminants, several protozoan species detected in the rumen have demonstrated neuroactive potential. For example, Entamoeba invadens and Entamoeba histolytica, detected in the rumen epithelium of calves [126], have been reported to produce glutamate and GABA in vitro [127]. Toxoplasma gondii and Dictyostelium discoideum, which account for 5%–7% of classified protozoa species in the rumen fluid of yak and cattle [128], express glutamate decarboxylase isoforms (GadA, GadB) responsible for GABA production [129] and a tyrosine hydroxylase-like enzyme involved in dopamine biosynthesis [130], respectively. However, these protozoa are not classical resident rumen protozoa and may act as transient passengers in the rumen [131]. Further studies are required to determine whether rumen commensal protozoa share similar capabilities and contribute to host neurophysiology.

Furthermore, although the majority of SCFAs are absorbed across the rumen epithelium [132], nutrients that are not fully degraded in the rumen or absorbed therein pass into the intestine, where the rumen microbiota can indirectly influence the ENS and CNS through their contribution to the gut-brain axis. In particular, the small intestine remains the primary site of nutrient absorption, giving certain rumen-derived microbial neuroactive compounds a distinct advantage over those produced in the hindgut, which cannot return to the foregut for absorption and are largely excreted in feces. Although neuroactive compounds generated by the hindgut microbiota can activate enteric and dorsal root nerve signaling, their systemic impact may be limited if absorption in the hindgut is insufficient [133, 134]. For example, rumen-derived microbial GABA and tryptophan, which can cross the BBB, are efficiently absorbed in the small intestine, whereas those synthesized in the hindgut are transported inefficiently across the hindgut epithelium and therefore have limited access to the circulation and CNS [135, 136].

Advances in techniques continue to push this field forward. Untargeted metabolomic analysis discovered additional neuroactive compounds in rumen fluid, such as N-arachidonyl dopamine, an endogenous agonist of Vanilloid Receptor 1 involved in pain sensation and a neuroprotectant [137], and microbe-derived indole derivatives such as 3-indoxyl sulphate, methyl indole-3-acetate, involved in the serotonergic synapse [138]. Further research integrating genomics and metabolomics is necessary to uncover the neuroactive compounds uniquely synthesized by rumen microbes, corresponding microbial genes involved in their biosynthesis, and the regulatory effects on the receptors of the host.

Transportation is one of the major stressors for farm animals, affecting their health, performance, and product quality. Acute responses to transportation stress lead to elevated stress hormones, increased proinflammatory cytokines, and altered rumen microbiota immediately after transport [22, 146, 147]. These stress responses can last several weeks and cause chronic issues [22, 147]. Stress-induced alterations of rumen microbial population, such as increased lactic acid-producing bacteria Lactobacillus and reduced fiber-digesting bacteria Prevotella, result in reduced rumen pH and elevated levels of SCFAs, lipopolysaccharide (LPS), and glutamate [22, 146, 147]. These alterations may influence rumen motility, appetite, and emotion of ruminants through activating ENS, vagal, or humoral pathways. The ruminal bacterium Saccharopolyspora rectivirgula has been positively correlated with elevated glutamate concentrations in the rumen of beef cattle 16 days post transportation [147]. This species has also been linked to increased oxidative stress post-transport. However, whether elevated ruminal glutamate during transportation intensifies stress responses via vagal pathways and subsequently contributes to oxidative stress remains to be elucidated.

Weaning stress, caused by a shift in diet and separation from mothers, leads to increased vocalization as animals call out for their dams or social companions [152], reduced time grazing, ruminating, and daily feed intake of the calves [150]. These stressful events lead to reduced rumen pH, altered rumen microbiome (e.g., increased Butyrivibrio and reduced starch-degrading bacteria such as Ruminobacter), increased SCFA levels, and slowing the growth of the calves [139, 148, 149, 153]. A recent study observed increased glutamate concentration in the plasma of weaned calves compared to non-weaned calves [150]. However, the extent to which changes in the rumen microbiota influence the profiles of ruminal neuroactive compounds has yet to be elucidated.

Heat stress has a considerable impact on the livestock industry, particularly for dairy cattle with high milk production and elevated internal heat loads, an issue which is worsened due to global warming [154]. Heat-stressed ruminants experience discomfort, with increased respiration rates, decreased feed intake, and altered rumination behavior, further adversely affecting dairy cow reproduction and milk yield [155, 156]. Rumen microbial communities are also usually disrupted during heat stress [151, 157], with an increase of lactate-producing bacteria, such as Streptococcus spp., and amylolytic bacteria, such as Ruminobacter spp., that results in higher ruminal lactate concentrations, reduced acetate level, and lower ruminal pH [151]. Neurotransmitter glutamate supplementation can relieve heat stress conditions by improving daily feed intake and weight gain, and reducing stress hormone corticosterone level in sheep, due to its potential role in enhancing rumen fermentation [158]. However, the potential role of rumen glutamate as an excitatory neurotransmitter to activate ENS and enhance rumen contraction during heat stress, and further improve appetite, is largely unexplored. Heat stress alters serotonin/5-HT metabolism by reducing the circulating serotonin levels in dairy calves [159]. Interestingly, the rumen fluid metabolome showed the serotonergic synapse pathway as the most enriched metabolic pathway in growing Holstein heifers experiencing heat stress compared to a non-heat-stressed group [138]. Moreover, heat-stressed heifers exhibited increased levels of indole derivatives, including 3-indoxyl sulfate and methyl indole-3-acetate [138], suggesting that heat stress reshapes rumen microbial metabolism of tryptophan and serotonin, which may in turn influence the CNS through serotonin vagal pathways, as well as the availability of tryptophan and indole metabolites for brain serotonin synthesis.

Emerging evidence suggests that stress-induced shifts in the rumen microbial population likely contribute to changes in the profiles of neuroactive compounds in both rumen fluid and blood. These neuroactive compounds may provide feedback to the brain via the nervous system, influencing behavior, mood, and other physiological states. A recent study showed that oral supplementation of probiotics, particularly the lactic acid-producing bacteria Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus plantarum, resulted in overall slower chute exit speed and less frequent vocalizations, suggesting that dietary probiotics can improve cattle performance and benefit cattle handling [160]. Future research should aim to uncover whether these supplemented probiotics colonize the rumen and produce neuroactive compounds that exert behavioral and stress-reducing effects.