Due to the incomplete rumen system of calves prior to weaning, undigested food components reach the intestine where microbial metabolism produces many compounds to regulate calf growth and development, including FAs and AAs [35, 36]. Increased intestinal permeability and disturbance of the intestinal microbiota are key factors leading to disease and growth retardation in calves. Various studies have described the development of the gastrointestinal microbiome in calves [37–40], but limited information is available regarding the specific changes that occur in the faecal flora and metabolome of calves. In the current study, we found significant differences in the faecal microbiota and FA and AA metabolism between calves classified as healthy and sub-healthy, as well as those that died within two to three weeks of birth. This early establishment of cross-communication between microbes and metabolites may have profound implications for the health of cattle later in life.
As the newborn grows and is introduced to solid foods, microbiota diversity increases, and the intestinal microbial community converges toward an adult-like state . These changes in the composition and maturity of gut microbes are reportedly related to growth status . Our data confirmed that the microbiota composition changed gradually from weeks 1 to 8 and alpha diversity increased, reflecting maturation of the gut microbiota in healthy calves (Fig. 2a-c and Additional file 2: Figure S2a, b). In group H, the relative abundances of Rikenellaceae and Prevotellaceae in the microbiota gradually increased over time (Fig. 2e, g). Both decreases of these bacterial families have been previously linked to gut-related diseases, such as inflammation and oxidative stress [43, 44]. In contrast, the abundance of S. marcescens, a pathogenic organism that can cause a variety of inflammatory responses and has immunosuppressive effects [45–47], gradually decreased with weekly age and differed significantly between weeks 1 and 8 (Fig. 2f, g). Moreover, the abundances of A. shahii and P. bacterium DJF B175 gradually increased with the growth and development of calves (Fig. 2f, h, i). These strains and the genus to which they belong reportedly utilise some polysaccharides to regulate the intestinal barrier, improve its permeability, and reduce harmful metabolites, all of which are beneficial to host health [48–51]. However, additional studies are needed to determine whether these bacteria can promote healthy calf growth and their mechanisms of action.
Intestinal dysbiosis refers to an imbalance between beneficial and pathogenic bacteria, resulting in inhibition of beneficial flora and overproliferation of pathogenic bacteria [52–54]. Intestinal inflammation and other typical metabolic diseases have been reportedly associated with intestinal flora imbalance [55, 56]. Comparing the microbiota of calves in groups H and SH at week 8 revealed that the abundance of Firmicutes was higher in group SH, while that of Bacteroidota was lower (Fig. 3f). The Firmicutes to Bacteroidota ratio in the gut microbiome has been linked to a variety of diseases, such as inflammatory bowel disease and metabolic syndrome . The higher Firmicutes to Bacteroidetes ratio in the microbiota of group SH reflected the "unhealthy" state of the intestinal flora. Further, the abundances of E. shigella and Fusobacterium, typical pathogens closely associated with intestinal inflammation and even colorectal cancer, were higher at week 8 in the microbiota of group SH (Fig. 3g, h) [58, 59]. Notably, the abundance of P. bacterium DJF B175 was gradually upregulated over time in the microbiota of group H, but was lower in the microbiota of group SH at week 8 compared with group H (Additional file 3: Figure 3f). The same downregulation occurred with P. goldsteinii, a strain that is associated with enhanced intestinal integrity and promotes resistance against intestinal and respiratory inflammation [60, 61].
In a follow-up analysis, we conducted a comparison of the three groups at the first week after birth. Enterobacteriaceae, E. coli, and E. cecorum were more enriched in the microbiota of group D than in that of groups H and SH (Fig. 4c-e). Several of these bacterial genera and strains have been shown to disseminate genes encoding antimicrobial resistance and are associated with intestinal inflammation. Their high proliferation seems to reduce intestinal resistance to other intestinal pathogens, thereby aggravating several diseases related to inflammation, such as necrotising enteritis, sepsis, and bone infection [62–64]. As mentioned above, the abundance of P. bacterium DJF B175 in the microbiota decreased with decreasing calf health in a comparison of the three groups (Fig. 4e). In addition, UCG-005, Rikenellaceae RC9 gut group, and Akkermansia were enriched in the microbiota of group H in contrast to that of groups SH and D (Fig. 4f). Some of these strains have been verified to maintain the balance of intestinal flora, promote average daily weight gain, and exert positive effects on growth and development [65–67]. Overall, the balance of the gut flora is important for nutrient intake and normal physiological activities. Suppression or infringement of beneficial microbiota by harmful bacteria results in a serious imbalance that may lead to retardation of calf growth and development or even death. The study findings suggest that early death of calves may be related to intestinal microbiome dysregulation, particularly, severe upregulation of strains negatively associated with growth, such as E. coli, and relatively low abundance of beneficial strains. In contrast, enriched P. bacterium DJF B175, Akkermansia, and Bacteroides populations in the intestinal microbiome may promote the healthy growth of calves.
In addition to changes in microbiota composition, the faecal FA composition differed significantly between groups D and H at one week of age, with higher faecal FA levels in group D, especially MCFAs (Fig. 5). Microbes can metabolise FAs, regulate the absorption of FAs by intestinal cells, and affect host energy metabolism, all of which are closely related to the pathogenesis of some diseases [68, 69]. MCFAs are important substrates in mammalian energy metabolism and synthesis, and can also improve immune and inflammatory responses in intestinal cell lines [70, 71]. For example, lauric acid inhibits a variety of pathogens, including E. coli, can significantly reduce inflammatory responses, and improves serum levels of inflammatory cytokines IL-6, TNF-α, IL-4, and IL-10 [72, 73]. When the intestinal flora is in a healthy state, the body maintains an environment suitable for nutrient metabolite absorption. However, disordered intestinal flora impairs the function of the intestinal barrier, which weakens the absorption of FAs by intestinal epithelial cells and leads to loss of large amounts of FAs . Therefore, high FA concentrations in the intestine lead to an imbalance in host metabolic homeostasis and weakened disease resistance. Indeed, caprylic, nonanoic, and decanoic acid levels were shown to be higher in the watery faeces of patients with diarrhoea . A similar study reported higher levels of branched-chain fatty acids (BCFAs) in the faeces of diarrhoeal calves compared to those of healthy calves . Moreover, levels of faecal LCFAs in patients with colorectal cancer were higher than those in healthy subjects . These studies support that intestinal microbiome disorder may lead to intestinal FA imbalance, which may disrupt the metabolic homeostasis of calves and damage their health.
Changes in the AA metabolism calves in the current study further confirmed the faecal microbiota results. Levels of some faecal AAs were higher in one-week-old calves in group D than in groups H and SH (Fig. 6d). Intestinal bacteria are known to alter the distribution of free AAs (FAAs) in the GIT and affect the bioavailability of host AAs . For example, Clostridium sporosporum can reportedly degrade tryptophan and secrete indole propionic acid through the metabolic pathway of tryptophan . Therefore, higher AA concentrations in the intestine are thought to be the result of incomplete fermentation. Studies have reported that faecal FAA concentrations were significantly increased in patients with inflammatory bowel disease [80, 81]. Further, the remission of diarrhoea in calves receiving FMT was accompanied by varying reductions in faecal AA concentrations . Comparing the faecal AA results with the bacterial results, the abundance of Clostridium sp., the main AA-fermenting bacteria, was significantly decreased in the microbiota of group D, whereas the abundance of E. coli, the main bacteria responsible for methionine synthesis and transformation, was significantly increased (Fig. 4e) [82–84]. These findings may explain the abnormal increase in intestinal AA concentrations at one week of age and the intestinal bacteria imbalance in group D. Moreover, faecal AA concentrations were lower at week 8 than week 1 in groups H and SH (Fig. 6b-c) likely because the intestinal flora structure of calves tended to be stable and AA use increased at eight weeks of age. Combining the faecal FA and AA results, we speculate that differences and disorder of intestinal microbiota distribution in calves might lead to abnormal intestinal function, absorption of MCFAs and LCFAs, and influence AA catabolism, resulting in an increased risk of early death. However, due to the lack of detection of serum metabolites, we could not judge the absorption of FAs and AAs in calves.