A better understanding of the etiology of digestive disorders is crucial for safeguarding horse welfare and digestive health (1). Several factors related both to intrinsic to the individual and to management conditions can predispose the horse to the onset of digestive disorders (2). One of the main risk factors of digestive disorders is being fed a diet high in starch and low in fiber (3). In fact, the horse’s capacity to digest starch is highly limited due to their low production of pancreatic α-amylase (4, 5). Accordingly, several authors agree that starch consumption should be limited to not more than 1 gram of starch/kg bodyweight (BW)/meal (6–9). Despite this recommendation, horses are commonly fed diets characterized by much higher amounts of starch (10–12). Such a feeding practice results in an impairment of the gut health, causing hindgut acidosis, dysbiosis and colic (13). Systemic consequences are also possible, such as laminitis, weight loss and poor performance (1, 14, 15). The presence of indigested starch in the hindgut can trigger microbial population changes during the fermentation processes (5, 16), yet little research effort has been directed at characterizing the horse microbiota in the different compartments of the intestinal tract according to diet with respect to other animal species (17–19). De Fombelle et al. (20) applied a culture-based methodology to samples obtained from the jejunum, ileum, caecum, right ventral colon and left dorsal colon; this approach thus limited the output to cultivable species only (21). Dougal et al. (22) investigated microbial population communities using a PCR-based method on samples from the caecum, right dorsal colon and rectum of eight equines euthanized for reasons not related to metabolic or intestinal disturbances. However, neither culture-dependent nor PCR-based methodologies permit the full richness and diversity of the bacterial populations resident in the gut to be characterized (23). To the best of our knowledge, Dougal et al. (24) used an amplicon sequencing approach to identify the core bacterial community across seven different regions of the large intestine of ten equines. Moreover, the study carried out by Costa et al. (18) applied a sequencing approach to evaluate the microbiota of the horse in the different compartments of the intestinal tract. However, this latter study only involved eleven horses belonging to different breeds, characterized by a wide age range (2 to 30 years) and which were euthanized for pathologies (unrelated to gastrointestinal diseases). The goal of the present study was to deepen our understanding of the microbial differences in different intestinal tract compartments in healthy horses, homogenous for breed and age, according to the diet being fed, namely a high-fiber diet (HF) vs. a high-starch diet (HS). We hypothesized that that differences in the gut microbiota in the distinct intestinal tract compartments would be observed between the two dietary groups. This study aims to further our understanding of the microbial changes associated with a HS diet in the intestinal environment.

The present study forms part of a larger research project investigating the effects of a high-fiber diet (HF) vs. a high-starch diet (HS). The same horses were also investigated for other parameters of gut health as gut histomorphometry (25) and fermentation end-products (16); behavior (26) and growing performances (15).

The study followed the guidelines of the current European Directive (2010/63/EU) on the care and protection of animals, and it was approved by the Ethics Committee of the Department of Veterinary Sciences of the University of Turin (Italy) (Prot. N. 2,202/2019).

At the end of the fattening period, we revealed no significant difference in the mean ± SD slaughter BW of horses fed the HF vs. the HS diet: 344.40 ± 2.91 kg vs. 347.67 ± 6.71 kg, respectively (15). No clinical signs of colic or gastric ulcerations were recorded during the study period but the horses belonging to the HS group produced unformed feces. Moreover, histopathological evaluation of the different intestinal tract compartments showed higher level of inflammation in the jejunum and pelvic flexure of horse fed the HS diet compared with those fed the HF diet (25).

The present study looked for variations in the gut microbiota along the length of the digestive tract of healthy horses according to two diets. We compared the effects of a high starch diet (HS), which exceeds the safe recommended level of 1 gram of starch/kg BW/meal (8), and a high fiber diet (HF), which satisfies the nutritional requirements of the horse.

The available scientific literature confirms the negative effects of HS diets on the gut health and welfare of horses (35), but a systematic assessment of the microbiota across the different compartments of the digestive tract is lacking (19). Microbiota composition is influenced by age, breed and the feed management system applied (23, 36). To avoid effects associated with breed or age in the present study, we used horses of the same age (14.3 ± 0.7 months) and breed (Bardigiano breed). They were not homogenous for sex; however, this is unlikely to have influenced the results since no sex-dependent differences in the gut microbiota were found in different gastrointestinal tract compartments of the horse analyzed using the NGS approach (18, 37). Moreover, all the horses of the present study were housed under the same environmental conditions, and the only difference was the diet.

Microbial populations were analyzed from alpha and beta diversity, bacterial populations present, and their potential function within the digestive tract of the horses.

A first important finding was that the horses fed the HF diet showed higher alpha diversity indices in the caecum, pelvic flexure and right dorsal colon compared with those fed the HS diet. This is relevant because high levels of bacterial diversity have been associated with greater stability of the intestinal microbiota and greater colonization resistance against potentially pathogenic bacteria (38, 39). However, the Shannon index was increased in the ileum of the horses fed the HS diet, whereas the alpha diversity indices of the other intestinal tract compartments were not different between the two diets. This could be explained by the individual variability or by the fact that the duration of the feeding trial (72 days) was too short to induce any significant changes in the other intestinal tract compartments. Moreover, the measures of beta diversity revealed significant compositional differences between the different intestinal tract compartments studied according to diet (HF vs. HS). This was not surprising since the diet is well-known to be the main factor affecting microbiota composition in the horse (1).

As already reported in the scientific literature, our findings support the division of the equine digestive tract into two distinct regions according to microbiota composition: the upper (duodenum, jejunum and ileum) and the lower (caecum, sternal flexure, pelvic flexure, right dorsal colon and rectum) digestive tract (see Figure 1) (18, 37). Interestingly, cluster analysis (Figure 4) revealed further differentiation within the hindgut: one cluster comprised the caecum and sternal flexure, and another was made up of the pelvic flexure, right dorsal colon and rectum; once again, confirming the findings of Costa and colleagues (18) who showed that that neighboring intestinal tract compartments are more similar to each other.

We show Firmicutes to be the primary bacterial phylum in all intestinal tract compartments, in accordance with the findings by other authors (18, 24, 40). Bacteroidetes was the second most abundant phylum (41, 42), and the third was Proteobacteria in the small intestine and Verrucomicrobia in the hindgut (19).

When we looked at the microbiota composition at genus/family level, we found interesting differences according to the diet adopted (HF vs. HS). The small intestine (jejunum and ileum) of horses fed the HS diet displayed a higher relative abundance of Proteobacteria compared with horses fed the HF diet as a result of a higher relative abundance of Ralstonia and Enterobacteriaceae. The increased presence of bacteria belonging to Proteobacteria is reported to be associated with inflammatory intestinal diseases in the horse (41). In particular, Enterobacteriaceae comprises some pathogenic bacteria, such as Escherichia Coli and Salmonella spp., which can lead to dysfunctions of the intestinal barrier, generating a condition of leaky gut and enteritis (1). Previous research published by our research team on the same horses fed the HS diet showed they were characterized by greater levels of inflammation of the intestinal wall in the jejunum (25) and by higher levels of intestinal permeability (15). Thus, we can speculate that these conditions were triggered by shifts in the microbiota composition associated with the consumption of a HS diet characterized by an overabundance of bacteria belonging to the Proteobacteria.

Progressing toward the more distal compartments of the digestive tract, the caecum and sternal flexure appeared similar in terms of their microbiota compositions, as shown in the heatmap (Figure 4). However, significant differences at the genus level revealed some positive effects of the HF diet related to an enhancement in metabolic pathways involved in fermentation processes with the production of short chain fatty acids (SCFAs). Specifically, the horses fed the HF diet showed higher frequencies of Fibrobacter in their caecal microbiota communities with respect to those fed the HS diet. Moreover, the abundances of Fibrobacter, Prevotella and Paraprevotellaceae within the sternal flexure were also greater. Dougal et al. (39) identified Fibrobacteraceae and Prevotellaceae as being two of the most abundant members of the core bacterial communities in the horse intestinal tract. Therefore, in our study the HS diet seems to be associated with a reduction in the overall size of the core microbiota within the caecum and sternal flexure. Such a reduction has been reported to increase the risk for digestive diseases and metabolic dysfunctions in the horse (43). Moreover, we found higher abundances of Streptococcus in the caecum and Fusobacterium in the sternal flexure of horses fed the HS diet, both of which are potential pathogenic bacteria associated with the presence of colitis or diarrhea (44). Indeed, the feces of horses fed the HS diet were less formed compared with those fed the HF diet (16).

Considering the distal intestinal tract compartments (pelvic flexure, right dorsal colon and rectum), Succinivibrionaceae were more abundant in the pelvic flexure and rectum of horses fed the HS diet. This is not surprising since these bacteria are amylolytic, and their greater prevalence is likely to be associated with the presence of undigested starch in the hindgut (26, 45). The samples collected from the pelvic flexure and right dorsal colon of both groups of horses showed compositional differences related to genera belonging to Firmicutes and Bacteroidetes. The majority of these taxa (Clostridium, Clostridiales, Ruminococcus, Prevotella and Bacteroides) are typical members of the physiological microbiota of the horse. However, it is interesting to note the positive effect that the HF diet had on the right dorsal colon. Samples collected from the right dorsal colon of horses fed the HF diet displayed a greater relative abundance of Akkermansia, a mucin-degrading genus that helps to maintain the integrity of the mucin layer of the intestinal barrier (19, 46). Finally, the fecal samples collected from the rectum of the horses fed the HF diet displayed an increase in the relative frequency of bacteria belonging to the Lachnospiraceae and Ruminococcaceae families, which form part of the intestinal core microbiota of the horse (39). Moreover, Spirochaetes (in particular Treponema), although at a lower abundance frequency than Lachnospiraceae and Ruminococcacea, were higher in the fecal samples of the horses fed the HS diet. This was an unexpected finding since these bacteria are involved in fiber degradation.

To the best of our knowledge, the present study is the first to evaluate the microbiota composition of all digestive tract compartments in horses homogenous for breed, age and previous management practices. A limitation of the present study was its small sample size. A second was the fact that the animals were not used as their own controls through comparison of their fecal microbiota. Studying the fecal microbiota before and after the feeding trial would have enabled us to discern whether limited changes of the microbiota differences in certain areas of the digestive tract between HF and HS groups were indeed attributable to the microbial composition of the ecological niches or a reflection of variability between horses. Nonetheless, the study provides evidence sustaining the view that each compartment of the intestinal tract behaves as an ecological niche, as first suggested by Costa and colleagues (18). Moreover, we demonstrated that insights into changes occurring at the lower taxonomic level along the length of the digestive tract (such as those caused by diet) cannot be gained by analyzing fecal samples collected from the rectum (which revealed a reduction of the overall size of the core bacterial community and an increase in amylolytic bacteria in the horses from the HS group). This finding has important practical implications for the design of studies aimed at investigating changes in the horse microbiota associated with certain variables, such as diet.

This is the first study to evaluate the effects of a high-fiber (HF) vs. a high-starch (HS) diet on the microbiota within the different intestinal tract compartments of healthy horses, homogenous for age and breed. The results confirm that each intestinal tract compartment of the horse’s digestive system represents a distinct ecological niche, and that diet significantly influences the microbiota composition. In particular, the overall size of the core community and the indices of alpha diversity were lower in horses fed the HS diet, which also showed higher levels of amylolytic bacteria. From the clinical perspective, our findings underscore the need to be cautious when drawing conclusions about the intestinal health of horses based on the analysis of the fecal microbiota. Fibrous diets should be promoted to prevent dysbiosis of the microbiota associated with high-starch diets.

The data presented in the study are deposited in the NCBI repository, accession number PRJNA1105226.

The animal studies were approved by Ethics Committee of the Department of Veterinary Sciences of the University of Turin (Italy) (Prot. N. 2202/2019). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

FR: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft. SC: Data curation, Methodology, Resources, Writing – review & editing. DB: Project administration, Resources, Supervision, Writing – review & editing. PS: Methodology, Resources, Supervision, Writing – review & editing. IF: Formal analysis, Methodology, Software, Writing – review & editing. LC: Data curation, Formal analysis, Methodology, Software, Writing – review & editing. MC: Data curation, Formal analysis, Methodology, Software, Writing – review & editing. RM: Methodology, Writing – review & editing. DC: Data curation, Formal analysis, Writing – review & editing. EV: Conceptualization, Project administration, Resources, Supervision, Writing – review & editing.