Pre-weaning is the most important period for the growth and development of calves. Intestinal morphology, microbial community and immunity are initially constructed at this stage, and even have a lifelong impact on calves (1, 2). The living environment of the calf changed from a sterile uterus to the outside world, and the nutritional conditions changed from maternal provision to the calf’s own feeding of feed and milk. Due to the incomplete development of the immune ability and digestive system of calves, newborn calves are very sensitive to external stimuli, and any external interference will seriously affect the growth of calves (3, 4). If the appropriate feeding mode is not adopted at this time, it will lead to low daily weight gain, diarrhea (5). It is reported that 30% of the deaths of pre-weaning cows are caused by diarrhea, which highlights the importance of intestinal health (6). The development of the digestive tract of calves is a unique process. With the development of the gastrointestinal tract and the settlement of the microbial community, calves gradually change from pseudo-ruminant animals to functional ruminants in physiology. The morphology of intestinal epithelial villi, the development of intestinal smooth muscle and rumen volume are basically completed at this stage which was ac-companied by the development of salivary organs and the development of rumination behavior. These factors directly affect the feed intake, nutrient absorption rate and digestive ability of calves after weaning (7).

Studies have shown that early feeding patterns have a significant impact on gastrointestinal development and microbial communities. Differences in dietary composition and feed physical morphology will stimulate intestinal structure development and microbial community composition (8–10). For example, alfalfa hay can promote the development of intestinal epithelial villi; the feeding of high carbohydrate and low fiber feeding will be detrimental to the rumen development of calves; additives can directly or indirectly increase the proportion of probiotics (11–13). Research shows that intestinal microbial community can be directed to change, adjusting feeding patterns can help animals form beneficial microbial communities before weaning (14). This can not only promote the development of calf digestive ability, but also is indispensable for the development of intestinal immune function. Deng’s research on giant pandas has proved that Streptococcus alactolyticus can promote the dietary adaptation of giant pandas by participating in protein metabolism (15). Recent studies have also found that microbial communities also communicate biological information with the nervous system, it points to a new field: neuro microbiology, this further illustrates the huge impact of gut microbes on the host (16).

There is no fixed feeding mode for newborn calves. Appropriate feeding mode should be comprehensively formulated according to their nutritional needs, breed characteristics, feeding plan and other factors (17). For calves at this stage, growth is important, but more importantly, the healthy development of the digestive system based on the gastrointestinal tract. This will have a long-term impact on the subsequent growth and production of calves (18, 19). Therefore, it is very important to develop a suitable feeding mode. In this study, three different feeding modes were used to explore the effects of different feeding modes on the growth and development of ileum and blood metabolism of calves from the perspective of metagenomics and metabolomics. The relationship between intestinal development, digestive function and microbial community composition was explored to provide a theoretical basis for further improving the feeding pattern of calves. At the same time, it inspired us to explore the relationship between intestinal microorganisms and the nervous system from a new perspective of neurobiology.

The ileum is located in the posterior part of the small intestine and is the main place for digestion and absorption of nutrients. Therefore, the development of ileal function is essential for the health and growth of calves. We observed from the sections after HE staining that the intestinal epithelial villi in the SF and SH groups were better developed, the villus height was higher, and the goblet cells in the villi were more closely arranged. The goblet cells can secrete mucus and form a physical barrier on the surface of the intestinal mucosa, which is the main component of the intestinal barrier (34). These characteristics mean that the ileum of SF group and SH group has a larger absorption and digestion area, and the intestinal morphology is more conducive to the absorption of nutrients, and has a stronger barrier function, which is the characteristic of healthy development of calf intestine, which is conducive to the development of digestive ability and gastrointestinal health of calves (35, 36). The research of Ma showed that the intestinal tract of calves must undergo development, physical and physiological changes, before they can better adapt to the transition from milk to solid feed (37). Studies have shown that the healthy development of intestinal epithelial environment is conducive to the development of intestinal probiotics and the stability of microbial communities (38). We observed a more obvious inflammatory response in the TMR group, and the intestinal wall in the SH group was relatively complete. After subsequent analysis, we believe that it may be due to the digestive ability of calves and maturity, which cannot adapt to the inflammatory response caused by TMR feed. The parasite phenomenon may be caused by the invasion of pathogens. Whether it is related to the total mixed feed needs further study.

Metagenomic analysis compared the microbial community differences among the three groups. The results showed that SH group was enriched in Actinobacteria and more than the other two groups. In previous studies, except for a small number of pathogenic bacteria, Actinobacteria is usually a probiotic that is conducive to the digestion and absorption of organic matter. In some farms, probiotics are used to selectively stimulate the growth of Actinobacteria (39). Moreover, Actinobacteria can also produce secondary metabolites such as natural drugs, enzymes, and active factors, and is considered to be a potential probiotic (40). Bifidobacterium under the classification of Actinobacteria plays an important role in regulating the immune system and preventing intestinal diseases. It can reduce the inflammatory response by producing short-chain fatty acids and regulating inflammatory factors. The proliferation of Actinobacteria may be the key to regulating immunity and reducing inflammatory response in SH group. Some studies have shown that Actinobacteria also plays a role in reducing pathogens such as Escherichia-Shigella, indicating that it can also have a positive effect on preventing diarrhea (41). Olsenella has obvious advantages in SH enrichment. Olsenella belongs to probiotics under the classification of lactic acid bacteria. Studies have shown that Olsenella is positively correlated with butyrate. Butyrate is one of the main volatile fatty acids produced by rumen fermentation, and ileum is also the main part of absorbing butyrate. Butyrate can provide energy for Olsenella and can strengthen the intestinal barrier function, which is conducive to intestinal health (42). The study of Zhang also showed that individuals with higher relative abundance of Olsenella have a stronger ability to produce short-chain fatty acids, which can effectively affect the structure of the intestinal epithelial mucus layer, stimulate the development of intestinal epithelial tissue, nourish the development of intestinal epithelial cells such as goblet cells, and reduce the risk of inflammatory bowel disease in individuals (43).

The dominant bacteria in the SF group were Chlamydiae and Proteobacteria. Chlamydiae is a pathogen that may cause enteritis (44, 45), but no diarrhea symptoms were found in this individual, and there may be a case of invisible infection of Chlamydiae (46). At the same time, other pathogens with high correlation with Chlamydiae, such as Campylobacter, were not found. Chlamydiae, as a potential pathogen, may be related to the local inflammation observed in the SF group, which may lead to impaired intestinal absorption and induce other diseases (47). Timely treatment is needed. The study of Xia in weaned piglets showed that the addition of plant essential oils containing substances such as tributyrin to the feed can resist Chlamydia, regulate microbial morphology, and improve intestinal villus morphology (48).

Proteobacteria in both TMR group and SF group have high abundance. Previous studies have shown that many intestinal pathogenic bacteria belong to Proteobacteria, and the relative abundance of Proteobacteria is too high, which may cause inflammation and cause calf diarrhea (49, 50). Shin used the enrichment of Proteobacteria as one of the important markers of intestinal microflora imbalance in healthy individuals. In general, Proteobacteria will not be significantly enriched in healthy individuals (51). The study of Han on weaned piglets showed that Proteobacteria could activate inflammation-related cytokines and cause intestinal inflammation damage. At the same time, the daily weight gain of piglets with enrichment of Proteobacteria was significantly reduced, which indicated that the intestinal nutrition absorption function was also affected (52). The study of Varada on newborn buffaloes found that feeding probiotics can regulate the composition of intestinal microbial communities by reducing the abundance of Proteobacteria (53). Numerous studies have shown that the enrichment of Proteobacteria can lead to inflammatory response and cause intestinal epithelial cell dysfunction (54, 55). This may be one of the reasons why the TMR group had a large number of inflammatory cell infiltration, intestinal epithelial cell shedding, and even diarrhea symptoms. Sarcina enriched in TMR is a pathogen that has been reported many times. Katharine found that Sarcina was associated with gastric ulcer and fungal infection in calves and goats, which may be the cause of gastritis and enteritis (56). The study of Simpson also found that Sarcina may cause necrosis of the intestinal epithelial surface and may be related to parasitic infection (57). Many studies have pointed out that Sarcina is associated with diarrhea, inflammation and other symptoms. Sarcina is significantly enriched in the intestinal tract of horses parasitized by ascaris, and Sarcina is often found in goats, rabbits and other animals with diarrhea symptoms or fungal infection (58, 59). The study of Zhuo on piglets directly pointed out that the enrichment of Sarcina is not conducive to the healthy development of the intestinal tract, affects the production performance and feed absorption rate, and increases the probability of intestinal pathogen infection. They also pointed out that Sarcina has a great relationship with the reduction of microorganisms that produce short-chain fatty acids and feeding patterns (60). This is similar to our findings. Therefore, we believe that the inflammatory response, parasitic infection and diarrhea symptoms in the TMR group are closely related to Sarcina.

After comparing the enrichment of KEGG signaling pathway, we found that in Cell cycle-Caulobacter (pathway ID: ko04112) and Necroptosis (pathway ID: ko04217) signaling pathways, SH group was less, SF group and TMR group were more. Cell cycle-Caulobacter is a key pathway that can regulate DNA replication, cell cycle and cell topology. Cell cycle response regulator is the core of his network. Cell cycle-Caulobacter regulates the asymmetric division of bacterial cells, resulting in stalked cells and swarmer cells with different fates (61). The stalked type of progeny cell type will enter the S phase, while the swarmer type will remain in the G1 phase (62). We compared the metabolites annotated in the three groups, and found that the unique metabolites in the SF group and the TMR group eventually pointed to polar morphogenesis and pili biogenesis, while the unique metabolites in the SH group pointed to flagellar ejection holdfast biogenesis, stalk formation, that is, the cells in the SF group, TMR group and SH group went to different divisions, and different daughter cells had different cell membrane structures and cell morphology. This leads to differences in subsequent functions (63, 64). Studies have shown that phages with pili and flagella structures are more susceptible to the host through CtrA regulation (65). This molecular network regulation has a direct relationship with bacterial infection. McAdams ‘s study pointed out that the function within the Cell cycle-Caulobacter network is conserved, but after asymmetric division, due to differences in cell function and protein, downstream specific coupling species will be very different (66). Therefore, we boldly speculate that the Cell cycle-Caulobacter pathway in the SH group is not as active as the other two groups, and the final cell division results are different, which may be one of the important reasons for the differences in bacterial infection and inflammatory response between the SH group and the TMR group and the SF group. The difference in Necroptosis is clearer. SH has no unique metabolites, while the unique metabolites of SH group and TMR group are the same, which are H2AX, VDAC, Drp1, ESCRT-III. These metabolites directly or indirectly point to inflammatory response, mitochondrial damage, etc., and their activity may lead to cell damage, and ultimately necroptosis (67). Yi’s study found that plasma pathogens such as Staphylococcus aureus can lead to cell necroptosis by affecting mitochondrial membrane potential and membrane permeability. This necroptosis is regulated by calmodulin-dependent protein kinase II (CaMKII) and mixed lineage kinase domain-like (MLKL) (68). The unique metabolites we found in TMR group and SF group were involved in the above reaction process. Huang’s study also pointed out that activation of MLKL and CaMKII can lead to mitochondrial dysfunction and pro-inflammatory response (69). We also found that the unique metabolites of TMR group and SF group were concentrated in cytochrome c oxidase \ reductase after comparing the Oxidative phosphorylation (pathway ID: ko00190) of the three groups. We believe that this may be due to bacterial infection that activates pathways such as MLKL, which leads to necroptosis caused by mitochondrial dysfunction and promotes inflammatory response, and ultimately leads to damage to intestinal epithelial structure (70). In addition, intestinal flora may also interact with metabolic intermediates in Oxidative phosphorylation. Existing studies have found that NAD (nicotinamide adenine dinucleotide) can promote the secretion of intestinal mucin 2 (MUC2), enhance the ability of antibacterial infection, and alleviate intestinal inflammation by inhibiting NF-kB. Oxidative phosphorylation abnormalities and mitochondrial damage are likely to affect the levels of metabolites such as NAD, increasing the risk of intestinal inflammation and bacterial infection (71).

In the two signaling pathways of Purine metabolism (pathway ID: ko 00230) and Pyrimidine metabolism (pathway ID: ko00240), the three groups have more enrichment, but their metabolic process is different. As shown in Figure 10, some of the reactions in the TMR and SF groups were more active than SH in these two pathways, including several key enzymes that catalyze one-way reactions, such as apyrase (EC: 3.6.1.5) and AMP deaminase (EC: 3.5.4.6), which are unique to the SF and TMR groups. Studies have shown that when the body is in an inflammatory or stress state, the demand for nucleotides will increase to promote intestinal cell proliferation and tissue repair (72, 73). The activity of apyrase (EC: 3.6.1.5) will lead to excessive metabolic intermediates such as GMP and AMP. Their final products all point to uric acid, but the enzyme for the next metabolism of uric acid is not active. Although nucleotide metabolism in the SF and TMR groups may be an autonomous repair of inflammation, it also increases the risk of accumulation of substances such as uric acid. In general, purine catabolism produces metabolites with potential toxicity (74, 75). Excessive accumulation of uric acid may lead to the formation of sodium salt deposition in shutdown, skin and other parts, causing pain. It may also cause kidney damage and lead to the occurrence of uric acidemia, which is common in long-term high protein intake of livestock and poultry (76, 77); in serious cases, it may also lead to atherosclerosis (78). Apyrase is involved in the dephosphorylation of GTP, ATP, UTP, CTP and other substances, and is a one-way reaction. ATP, GTP and ITP were dephosphorylated to produce Uric acid. UTP and CTP were also dephosphorylated to produce Uracil, which was converted into β-Alanine to participate in the next reaction. After dephosphorylation, dTTP is dephosphorylated to generate dTMP, which will further generate Thymine, and then generate (R)-3-Amino-2-methylpropanoate to participate in Valine, leucine and isoleucine degradation pathway. Therefore, we inferred that the Nucleotide metabolism of the three groups was more enriched because the intestine of the calves was in the developmental stage, and the metabolism was vigorous because it responded to stimuli such as feed differences and microbial flora changes. However, the SF group and the SH group showed uniqueness in some metabolic processes due to the need for anti-inflammatory and epithelial cell repair. The activity of enzymes such as apyrase (EC: 3.6.1.5), and the enzymes not annotated to uric acid catabolism may lead to the risk of uric acid accumulation in the SF group and the TMR group.

By comparing the analysis results of the three groups of CAZy databases. The differences are mainly reflected in the GH1 family and the GH19 family. Under level 3 classification, the enrichment of β-glucosidase (EC 3.2.1.21) belonging to GH1 family in SH group was more than that in the other two groups. β-glucosidase mainly plays a role in glycolipid metabolism and is a key enzyme that catalyzes cellulose to produce glucose. We believe that the higher β-glucosidase activity in the SH group is closely related to its microbial community. Studies have shown that adding β-glucosidase to feed or activating β-glucosidase through diet can improve the digestive capacity of the gastrointestinal tract and benefit animal growth (79). β-glucosidase can also increase the activity of intestinal amylase (80). This is also consistent with our found, the amylase content of SH group was the highest. Zhang ‘s study found that castration changed the cecal digestion environment of dairy cows, significantly reduced the activity of β-glucosidase, reduced the relative abundance of Ruminococcaceae and other bacteria related to β-glucosidase, and reduced the daily gain of dairy cows (81). Therefore, the enrichment of β-glucosidase has a positive effect on calves. The chitinase (EC 3.2.1.14) in GH19 family showed more obvious differences. The enrichment of chitinase in SH group was much less than that in SF group and TMR group. Chitinase has strong specificity and can cleave chitin glycosidic bonds. Chitin is a component of invertebrates, arthropods, fungi, etc., but mammals can hardly synthesize chitin themselves. Therefore, attention should be paid when excessive chitinase is enriched in mammals, and it may be food containing chitin or microbial pathogens invade the body (82). Recent studies have shown that chitin protein is related to gastrointestinal diseases. Chitinase can play an important role in gastrointestinal digestion and diseases due to its specificity, and it is likely to become a new idea for the treatment of gastrointestinal diseases (83, 84). The study of Beier found that microorganisms can also produce chitinase, including Firmicutes, Actinobacteria, etc. Chitinase can specifically hydrolyze chitin and make it lose pathogenicity, which is particularly critical for mammals (85). We observed local inflammatory reactions in both the SF group and the TMR group. Parasites also appeared in the TMR group. It is most likely that foreign pathogens produce chitin, which stimulates some of the bacteria in the intestine that can release chitinase to respond, causing differences in intestinal flora.

In addition to this, there is a more critical phenomenon, we found that in the TMR group, a unique enrichment of many neurotransmitters and their precursors: Dopamine, Noradrenaline, Levodopa, etc., which may point to a new research field: neuro microbiology. In recent years, more and more studies have shown that microorganisms not only have the functions of assisting digestion and maintaining barriers, but also participate in the regulation of the enteric nervous system and the central nervous system by producing neurotransmitters or neurotransmitter precursors (86). It is reported that intestinal microorganisms can synthesize neurotransmitters such as dopamine, norepinephrine, 5-hydroxytryptamine and compounds involved in the metabolism of neurotransmitters in the body, which can act on the local intestinal nervous system and regulate the intestinal environment (87, 88). Taking the synthesis mechanism of dopamine (phenylalanine-tyrosine-L-dopa-dopamine) as an example, phenylalanine hydroxylase converts L-phenylalanine into L-tyrosine, which can cross the blood–brain barrier and enter the brain. In the brain, it is converted to (s)-3,4-dihydroxyphenylalanine (L-DOPA) by tyrosine hydroxylase, and then L-DOPA is converted to dopamine by dopa decarboxylase (89, 90). Although the pathway by which these neurotransmitters are synthesized by the gut microbiome is unclear, it is likely that the gut microbiome also has the ability to convert local dopamine to norepinephrine, such as dopamine-beta oxidase (91). Intestinal microorganisms can also synthesize neurotransmitter precursors such as L-tyrosine. Due to the lack of relevant transporters, some neurotransmitters circulating in the blood, such as dopamine and norepinephrine, cannot cross the blood–brain barrier. However, the precursors of these neurotransmitters can cross the blood–brain barrier, synthesize related substances in the brain, and then regulate, which may be the way that microorganisms connect the central nervous system.

Intestinal flora communicates with the central nervous system, sympathetic nerve and vagus nerve through neurotransmitters, regulates the level of neurotransmitters such as auxin, and affects the growth and development, gastrointestinal digestion and immune function of animals (92). For example, slight changes in the vagus nerve will lead to dramatic changes in the release of downstream neurotransmitters, which will affect digestive level, intestinal permeability, intestinal motility and immune response (93, 94). The central nervous system receives signals from these neurotransmitters and regulates the digestive environment and microbial community composition in the gastrointestinal tract by producing neurochemicals such as 5-hydroxytryptamine, cholecystokinin, and dopamine. Microorganisms also stimulate intestinal endocrine cells to produce hormones and complete feedback to the central nervous system through neural afferent fibers. For example, in the presence of dopamine and norepinephrine, Escherichia coli will proliferate faster and may also enhance its motility and toxicity (95). Liu ‘s study found that abnormal casein synthesis aggravated enteritis, disrupted microbial flora and its metabolic function, and led to abnormal immune response (96). The intermediate compound of neurotransmitters: short-chain fatty acids, also participate in the regulation of microorganisms and neurotransmitters. Studies have shown that short-chain fatty acids can regulate the metabolism of many neurotransmitters by acting on the response element binding proteins in the cAMP signaling pathway (97, 98). Taking dopamine as an example, short-chain fatty acids can regulate the production of dopamine and the conversion of dopamine and norepinephrine by regulating the expression of tyrosine carboxylase and dopamine-β-carboxylase (99). These metabolic pathways are reflected in the differential metabolic pathways in the TMR group. Especially for ruminant mammals such as calves, the short-chain fatty acids in the body will be more abundant, and the regulation mechanism of short-chain fatty acids will be more complicated. In some animal model experiments, it has also been found that metabolic abnormalities caused by intestinal microbial community disorders and the release of monoamine substances may lead to depression (100). This proves the deep relationship between intestinal microbial changes and host mental disorders. In animal husbandry, whether it will affect animal feed intake and estrus cycle needs further study.

In recent years, many studies have shown that intestinal microorganisms can communicate and regulate with the gastrointestinal tract and brain through neurotransmitters, and interact to maintain the dynamic balance of the intestinal environment. One of the difficulties in the field of neurobiology is to identify whether the source of various neuroactive compounds is host or microorganism. Secondly, the influence between intestinal microorganisms and neurotransmitters, and the complex communication between intestinal microorganisms and the brain are difficult. Combining the analysis results of metabolomics and metagenomics will help us understand the operation relationship between the microbial-intestinal-brain axis from the perspective of neuro microbiology. In the future, specific microbial targeted interventions can be performed on the host from the perspective of neuro microbiology, which can be used to adjust the intestinal flora and improve digestion. It is even possible to regulate specific microbial communities by understanding the relationship between gut microbes and brain health, and to treat neurological diseases through microbial regulation of the central nervous system.

In summary, our study found that the feeding mode of ‘starter + hay + milk ‘was more conducive to the development of calves. The calves in the SH group had better intestinal development, intestinal microbial community was more conducive to digestion and absorption, and immunity was stronger. The feeding mode of the SH group was conducive to the healthy development of the calves. Therefore, the calves in the SH group had higher daily weight gain, stronger digestive ability, and no pathological inflammatory reaction was found. For calves, daily weight gain is important, and more importantly, there can be a stronger digestive system and immune system to ensure that calves can have better growth and development after weaning, which is the advantage of SH group compared with SF group. The enrichment of neurotransmitters found in the TMR group also inspired us to study intestinal microorganisms from a new perspective. The research ideas of neuro microbiology will be one of our future focuses. Therefore, the results of this study provide a basis for improving the feeding mode of calves, and inspire us to explore the relationship between microorganisms and hosts from the perspective of neural metabolites.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was approved by the Institutional Animal Care and Use Committee of the College of Animal Science and Technology. The study was conducted in accordance with the local legislation and institutional requirements.

JW: Data curation, Writing – review & editing, Writing – original draft. YC: Data curation, Writing – original draft, Writing – review & editing. ML: Data curation, Investigation, Project administration, Writing – review & editing. SX: Investigation, Software, Writing – review & editing. KZ: Investigation, Software, Writing – review & editing. HF: Investigation, Software, Writing – review & editing. JN: Investigation, Software, Writing – review & editing. WS: Conceptualization, Investigation, Software, Writing – review & editing. XJ: Conceptualization, Investigation, Software, Writing – review & editing. SL: Methodology, Project administration, Supervision, Writing – review & editing.

This study was supported by the key research and development project of Sichuan beef cattle genome selection technology system (2022YFYZ0006).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.