Characteristics of bacterial community and volatile fatty acids in the gastrointestinal tract of Tarim wapiti

Abstract

Tarim wapiti (Cervus elaphus yarkandensis) may have a complex gut microbiota that contributes to its unique qualities due to its biological characteristics of tolerance to coarse feed and salt-alkali. The contents of the forestomach (rumen, reticulum, and omasum), abomasum, and small intestine (duodenum, jejunum and ileum) were taken in this investigation from six Tarim wapiti. The objective was to assess the content of volatile fatty acids (VFAs) and microbiological diversity through 16S rDNA. The results showed that acetate, propionate, butyrate, and valerate concentrations were much higher in the forestomach compared to the small intestine. The communities of bacteria were abundant and diversified in the forestomach and abomasum compared to the small intestine. Bacteroidetes were significantly more abundant in the forestomach and abomasum than in the small intestine, but Firmicutes were the reverse. The predominant bacterial genera identified in the forestomach and abomasum were Rikenellaceae RC9 gut group, Prevotella and F082; Clostridium sensu stricto 1, Acetitomaculum, Paeniclostridium, and Romboutsia were the main bacterial genera in the small intestine. The relative abundance of genes for microbial energy metabolism functions was substantially reduced across the rumen to the reticulum, across the abomasum to the duodenum for carbohydrate metabolism and lipid metabolism functions, and across the jejunum to the ileum for lipid metabolism and energy metabolism functions. In addition, VFAs were found to be positively correlated with dominant bacteria in forestomach and abomasum, and negatively correlated with dominant bacteria in small intestine. According to the study, Tarim wapiti have developed a unique and distinct dominant bacterial community in its forestomach, abomasum, and intestines.

HIGHLIGHTS

• This is the first report that provides a detailed analysis of the microbial communities present in different segments of the forestomach, abomasum, and small intestine of the Tarim wapiti.

• The study established baseline data on the Tarim wapiti, which is the only red deer species that can survive in desert environments.

Keywords:

• Tarim wapiti
• volatile fatty acids
• microbiota
• 16S rDNA
• gastrointestinal tract

Introduction

The gastrointestinal tract (GIT) of ruminants is divided into three main parts: the stomach, which includes the rumen, reticulum, omasum and abomasum; the small intestine, which comprises the duodenum, jejunum and ileum; and the large intestine, which consists of the caecum, colon and rectum (Bergmann 2017). The stomach and small intestine are the primary parts for nutritional digestion and absorption in the GIT of ruminants and are the main focus of study on ruminant microbiota. Different bacteria, pH, temperatures and oxygen concentrations exist in different segments of the GIT (Su et al. 2020). Variances in microbial diversity in the GIT of ruminants can result in variances in their roles in the body, such as the digestion and metabolism of nutrients in the body (Barko et al. 2018). For instance, bacteria in the rumen of ruminants digest cellulose and hemicellulose in plant cell walls, and ferment to produce volatile fatty acids (VFAs), which provide energy to the host (Doreau and Ferlay 1994; Jane and Van 1980). Acid-tolerant Firmicutes are the dominant microbial populations in the small intestine, and they play crucial roles in the breakdown of proteins and carbohydrates (Zhang et al. 2018). Furthermore, the intestinal microbiota assumes a crucial role in upholding the equilibrium of the gut flora, safeguarding the health of the intestinal tract, and augmenting the body’s immune response (Stanley et al. 2014).

Deer family animals are ruminants like cattle and sheep, but deer still have a unique digestive physiology. Studies on the gastrointestinal microbiota of cervids have mainly focused on the rumen owing to their strong wild instincts and lack of domestication. The rumen of Tarim wapiti is dominated by Prevotella (Qian et al. 2017). According to Wilson (2019), Ruminococcus is actually dominant in wild deer, while Ishaq and Wright (2014) found that the moose rumen has a high abundance of Prevotella. In addition, Zhen et al. (2022) and Jiang et al. (2022) reported that the dominant bacteria in deer faeces were Rikenellaceae RC9, while Ruminococcaceae was also found to be dominant by Zhen et al. (2022) and Li et al. (2017). However, it is important to note that faecal samples primarily represent the large intestine microbiome, as reported by Ahn et al. (2023). While the rumen plays a vital role in ruminant digestion, the reticulum, omasum, abomasum and the small intestine also contribute significantly (Ahn et al. 2023). Kim et al. (2019) conducted an analysis of the bacterial composition of eight segments of the elk digestive tract, including the rumen, reticulum, omasum, duodenum, jejunum, ileum, caecum and rectum. The study found that Prevotella was more abundant in the stomach than the small intestine, while Clostridium and Romboutsia were present in significant amounts throughout the digestive tract. Xie et al. (2021) analysed the microbiota of 10 segments throughout the GIT of Capreolus pygargus and Muntiacus reevesi. The study found that Prevotella and Fibrobacter were the dominant genera in the stomach, while Escherichia coli was dominant in the small intestine. Li et al. (2014) analysed the dominant phyla in the cervid digestive tract and found that Firmicutes and Bacteroidetes were prevalent. The study also found that the families Prevotellaceae and Anaerovoracaceae were relatively abundant in the rumen, while the families Ruminococcaceae and Bacteroidaceae exhibited higher abundances in the ileum, caecum, and rectum. Li et al. (2018) analysed the bacterial colonisation process of the jejunum and ileum of sika deer and found that the relative abundance of Firmicutes exhibited a trend of increasing and then decreasing over time. Firmicutes remained the dominant phylum at days 42 and 70, with Lactobacillus, Romboutsia, Intestinibacter and Clostridium sensu stricto 1 being the dominant genera. Previous studies have shown that, under the same feeding conditions, the dominant rumen bacterial genera in Tarim wapiti were Prevotella and Oscillospira, in contrast to cows and sheep (Qian et al. 2017). These findings highlight the substantial heterogeneity in gastrointestinal microbiota among cervids, which is influenced by factors such as genetic background, feeding structure and geographical distribution.

The Tarim wapiti is primarily found in the Tarim River and its tributaries, which are in the arid Tarim Basin of the Xinjiang Uygur Autonomous Region in China. This subspecies of Chinese deer is unique in that it is adapted to living in desertified habitats (Ababaikeri et al. 2020). The Tarim wapiti possesses digestive physiological characteristics that are similar to those of ruminants such as cows and sheep. However, the Tarim wapiti has developed a strong adaptability to forage with poor quality, low protein content and high crude fibre content due to long-term natural selection pressure under ecological adversity (Qian, 2017). Considering the diverse production characteristics and purposes of cervids, investigating the microbial community composition in the forestomach, abomasum, and small intestine of Tarim wapiti can significantly enhance our understanding of cervid gastrointestinal microbiota. Such investigations can also serve as a foundation for targeted research on nutrient metabolism in these animals in the future.

Materials and methods

Animal and feeding management

The Tarim wapiti used in this study were selected from the 31st regiment field of the Second Division of Xinjiang Production and Construction Corps in China, and the experiment was conducted on 20 December 2022. The study included three healthy adult males (200 ± 5.0 kg, 5 years old) and three healthy adult females (175 ± 5.0 kg, 5 years old). The study area, located at geographical coordinates 86°45′-87°00′E, 40°49′-40°59′N, has an altitude ranging from 820 to 1100 m above sea level and experiences a typical continental arid climate. The deer were provided with two feedings per day at 9 am and 7 pm and had ad libitum access to feed and water. The basal diets consisted of a 3:7 ratio of concentrate to roughage, and the composition and nutrient levels of these diets can be found in Table 1. The concentration of CP, Ca and P in the feed supplied was determined by reference to the AOAC method (AOAC, 1990). CP was determined by Kjeldahl nitrogen determination, Ca was determined by potassium permanganate titration, and P was determined by absorbance photometry. It is worth noting that the enclosure was diligently maintained, with proper ventilation, cleanliness and regular disinfection measures in place.

Table 1. Composition and nutritional level of diets.

Items	Value
Composition	Content (%)
forage
Corn stalks	19.5
Wheat stalks	13
Alfalfa	22.5
Cottonseed hull	15
concentrate
Corn	23.5
Soybean meal	2.5
Cotton meal	2.5
Salt	0.5
Premix^a	1
Total	100
Nutrient Levels
Digestible energy(DE)^b, MJ/kg	11.1
Crude protein (CP), %	11.51
Calcium (Ca), %	0.53
Phosphorus (P), %	0.24

^aPremix: VA 940 IU/kg, VE 20 IU/kg, minerals (mg/kg): S 200 mg/kg, Fe 24 mg/kg, Cu 8 mg/kg, Mn 40 mg/kg, Zn 40 mg/kg, I 0.3 mg/kg, Se 0.2 mg/kg, Co 0.1 mg/kg.

^bDE: DE was calculated value.

Collection of samples

The deer were slaughtered 2 h after morning feeding, and their GITs were dissected. To avoid refluxing the surrounding contents, the GIT junction was ligated. The contents of the forestomach, abomasum and small intestine were harvested. The contents of the rumen, reticulum and omasum were filtered through four layers of sterile gauze, and the contents of the abomasum and small intestine (duodenum, jejunum and ileum) were directly obtained, put into 5 mL cryotubes, rapidly frozen with liquid nitrogen, and then transferred to a −80 °C freezer. The samples were sent to Beijing Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) for sequencing analysis of the 16S rDNA V3-V4.

Analysis of the content of volatile fatty acids

The contents of each fraction were thawed at 4 °C, followed by centrifugation at 10,000 x g for 10 min. One millilitre of the supernatant was collected and combined with 0.25 mL of a 20% metaphosphoric acid solution. The mixture was thoroughly vortexed and shaken to ensure proper mixing, and then passed through a 0.22 μm aqueous filter membrane (mixed cellulose [MCE]). The quantification of VFAs (acetate, propionate, butyrate and valerate) in each fraction was performed using gas chromatography (SP7800, Beijing Jingke Ruida Technology Co., Ltd.). The parameters for the gas chromatography analysis were set as follows: column temperature = 120 °C, injector temperature = 230 °C, detector temperature = 250 °C and injection volume = 2 μL.

DNA extraction and PCR products, acquisition, quantification and qualification

The genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China, Catalog #: DP210831), and the purity and concentration of DNA were detected by 1% agarose gelelectrophoresis. The V3-V4 region of 16S rDNA was amplified using the universal primers 341 F (GTGCCAGCMGCCGCGGTAA) and 806 R (GGACTACHVGGGTWTCTAAT). All PCR reactions were carried out with 15 µL of Phusion^® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, Catalog #: pro_10), 0.2 µM of forward and reverse primers, and approximately 10 ng of template DNA. Thermal cycling consisted of initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s and elongation at 72 °C for 30 s, followed by a final elongation step at 72 °C for 5 min. The PCR products were Mix same volume of 1X loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. PCR products were mixed in equidensity ratios. Then, mixture PCR products were purified with Universal DNA Purification Kit (TianGen, Beijing, China, Catalog #: DP214).

Library preparation and sequencing

The NEB Next^® Ultra™ II FS DNA PCR-free Library Prep Kit (New England Biolabs, USA, Catalog #: E7430L) was used for library construction, and the constructed library was quantified using Qubit and qPCR. After passing qualification, the library was subjected to PE250 sequencing on a NovaSeq 6000 instrument.

Paired-end reads assembly and quality control

Each sample read was merged with FLASH (V1.2.11, http://ccb.jhu.edu/software/FLASH/) to produce Raw Tags (Magoč and Salzberg 2011). Quality filtering on the raw tags were performed using the fastp software version 0.23.1 (Institute of Applied Ecology, Chinese Academy of Sciences, China) to obtain high-quality Clean Tags (Bokulich et al. 2013). The Clean Tags were compared with the reference database (Silva database (16S), https://www.arb-silva.de/), using UCHIME Algorithm to detection and removal of chimeric sequences to ensure that effective Tags are obtained (Edgar et al. 2011).

ASVs denoise and species annotation

For the Effective Tags obtained previously, denoise was performed with DADA2 in the QIIME2 software (Version QIIME2-202006) to obtain initial ASVs, and then ASVs with abundance less than 5 were filtered out (Wang et al. 2021). Species annotation was performed using QIIME2 software. For 16S, the annotation database was Silva Database. LEfSe (LDA score = 4) was used to analyse overall differences in microbial composition.

Alpha diversity and beta diversity

Calculated the Alpha diversity indices of the samples, including observed ASVs, Shannon and chao1, using the QIIME2 software. Used R software version 3.5.3 (R Development Core Team) with the ggplot2 package and the ade4 package for NMDS based on Bray Curtis distance. Additionally, the ADONIS was used to test for differences in statistical cross validation. For function annotation analysis, we used the PICRUSt2 software version 2.1.2-b (Berkeley National Laboratory, Berkeley, USA).

Statistical analysis

The concentrations of VFAs, Shannon index, Chao1 index and inter-group differences at the phylum and genus levels in the forestomach, abomasum and small intestine were analysed using Duncan’s multiple comparison in SPSS version 21.0.0.0 (SPSS Inc., Chicago, IL), expressed with the Standard Error of the Mean (SEM). Function annotation was conducted T-test statistical analysis on the significance of differences. The p value significance threshold was set to 0.05. To identify differential VFAs, OPLS-DA analysis was used in SIMCA version 18.0.0.372 (Umetrics Inc., Sweden). Correlations between major microorganisms and VFAs in the forestomach, abomasum and small intestine were analysed using Pearson’s bivariate correlation analysis. The correlation heatmap was plotted using OriginPro 2021 version 9.8.0.200 (OriginLab lnc., North Andover, MA, USA).

Results

Concentration of VFAs in the forestomach, abomasum, and intestine

In a broader context, the rumen, reticulum and omasum show a substantial elevation in the concentration of VFAs such as acetate, propionate, butyrate and valerate, surpassing that of the small intestine. It’s worth noting that within this context, the rumen and reticulum prominently display markedly higher concentrations of acetate and propionate when compared to the omasum (p < 0.05) (Figure 1). According to the OPLS-DA plot (Figure 2), there was a large difference in the composition of VFAs in the forestomach, abomasum and small intestine of Tarim wapiti, with the forestomach forming a distinct cluster and the abomasum and small intestine forming another cluster. Upon further analysis, it was found that acetate (with a VIP value of 1.7123) was the substance with a VIP value higher than 1, indicating that it was the dominant substance in the VFAs present in the forestomach, abomasum and small intestine of Tarim wapiti (Supplementary Figure 1).

Figure 1. Changes in volatile fatty acids (VFAs) content in the forestomach, abomasum, and small intestine.

Note. Different letters represent significant differences (p < 0.05).

Figure 2. Characteristics of volatile fatty acids (VFAs) in different segments of the forestomach, abomasum, and small intestine OPLS-DA (Score plot).

Microbial diversity of the forestomach, abomasum and small intestine

It was found that a total of 626,89 high-quality effective tags were obtained for the bacterial 16S rDNA. According to the rarefaction curve of the number of individuals for ASVs, it was observed that the increase in the number of species obtained slows down when the number of original sequences is higher than 25,000. This indicates that under the sequencing depth conditions where the number of original sequences is higher than 25,000, the sequencing results can cover the majority of microorganisms (Supplementary Figure 2(a)). ASVs clustering revealed that the rumen had 1648 unique ASVs, the reticulum had 1878 unique ASVs, the omasum had 2309 unique ASVs, the abomasum had 1204 unique ASVs, the duodenum had 1752 unique ASVs, the jejunum had 1623 unique OTUs, the ileum had 1204 unique ASVs. Additionally, the number of cores ASVs was 488, accounting for 0.78% of the total number of ASVs (Supplementary Figure 2(b)).

It was observed that the abundance and diversity of bacterial communities differ between the forestomach, abomasum and small intestine. Both the Chao1 abundance index and Shannon diversity index of reticulum, omasum and abomasum bacterial communities were found to be higher than in rumen and small intestine, with the lowest values observed in the ileum (p < 0.05). However, there was no significant difference between the rumen and the duodenum, jejunum (p > 0.05) (Table 2).

Table 2. Diversity and richness of the forestomach, abomasum, and small intestinal microbiota.

Regions	Effective Tegs	ASVs	Chao1 index	Shannon index
Rumen	102222	1530.67^b	1636.41^b	8.4577^b
Reticulum	113290.5	1860.3^a	2017.68^a	9.0342^a
Omasum	112508.8	1937^a	2125.13^a	9.1365^a
Abomasum	116062.5	1977.67^a	2136.01^a	9.3024^a
Duodenum	112946.17	1365.83^b	1479.01^b	8.1319^b
Jejunum	108224	1252^b	1368.35^b	7.7086^b
Ileum	118306.33	847.5^c	956.41^c	5.319^c
SEM	2333.08	72.65	76.82	0.23
p Value	0.63	<0.01	<0.01	<0.01

Different letters represent significant differences (p < 0.05).

ASVs: Amplicon Sequence Variants.

The NMDS analysis and ADONIS based on the Bray Curtis distance indicated that the forestomach, abomasum and small intestine clustered into two distinct groups. Furthermore, there was a difference observed between the ileum and duodenum-jejunum (Figure 3, Supplementary Table 1).

Figure 3. NMDS analysis with clustering indicates variations in the microbial communities of the forestomaches, abomasum and small intestine. F represents male, M represents female (rumen n = 6, reticulum n = 6, omasum n = 5, abomasum n = 6, duodenum n = 6, jejunum n = 6 and ileum n = 6). NMDS: Non-Metric Multi-Dimensional Scaling.

Microbial abundance and composition in the forestomach, abomasum and small intestine

At the phylum level, Firmicutes and Bacteroidetes are the dominant bacterial phyla in the forestomach, abomasum and small intestine (Figure 4(a)). Bacteroidetes is the most abundance phylum in the forestomach and abomasum, with no significant difference between the forestomach and abomasum (p > 0.05), with relative abundances of 56.04%, 58.08%, 42.82% and 55.22%, respectively. Firmicutes is the second most abundant phylum in the forestomach and abomasum, with relative abundant of 35.03%, 31.73%, 44.22% and 35.79%, respectively, not significantly different between the forestomach and abomasum (p > 0.05), but significantly lower than in small intestine (p < 0.05). According to the findings, Firmicutes is the most abundant phylum in the small intestine, with its relative abundances ranging from 68.12% to 89.27% as across the transition from the duodenum to the ileum, indicating an increasing trend. Interestingly, the relative abundance in the jejunum is significantly higher than in the duodenum and ileum (p < 0.05). Based on the analysis, it was found that the relative abundance of Bacteroidetes in various parts of the small intestine did not differ significantly (p > 0.05). On the other hand, the relative abundance of Proteobacteria in the abomasum (1.43%) was significantly lower than that in the duodenum (11.49%) (p < 0.05). Additionally, there was a decreasing trend in the relative abundance of Proteobacteria from the duodenum to the ileum. According to the findings, the relative abundances of Actinobacteria were significantly higher in the duodenum and jejunum (5.02% and 7.57%, respectively) compared to the other segments. Conversely, the relative abundance of Actinobacteria in the rumen was the lowest (0.48%) (p < 0.05). Furthermore, the relative abundance of Fibrobacterota in the omasum was significantly higher than in other segments (p < 0.05), with a percentage of 1.72% (Supplementary Table 2).

Figure 4. The relative abundance of bacteria of the forestomaches, abomasum and small intestine. (a) The phylum level; (b) The genus level.

At the genus level, the dominant bacteria in the forestomach and abomasum are Rikenellaceae RC9 gut group, Prevotella and F082 (Figure 4(b)). Relative abundances of Rikenellaceae RC9 gut group in the forestomach and abomasum were 20.85%, 19.73%, 17.12% and 13.88%, respectively, with no significant differences between the forestomach and abomasum, but significantly higher than small intestine (p < 0.05). According to the analysis, Prevotella was found to be the second dominant genus in the forestomach and abomasum, with relative abundances of 8.80%, 11.81%, 5.18% and 12.16%, respectively. The relative abundance of Prevotella in all parts of the small intestine was significantly lower than in the forestomach and abomasum (p < 0.05). F082 was identified as the third most abundant genus in the forestomach and abomasum with relative abundances of 6.39%, 6.19%, 5.98% and 7.23%, respectively. No significant difference in the relative abundance of F082 was observed between the forestomach and abomasum. However, the relative abundance of F082 was significantly higher in the forestomach and abomasum than in all segments of the small intestine (p < 0.05). Based on the analysis, it was found that Clostridium sensu stricto 1, Acetitomaculum, Paeniclostridium and Romboutsia species were predominant in the small intestine. The relative abundance of Clostridium sensu stricto 1, Paeniclostridium and Romboutsia in the small intestine showed an increasing trend. Furthermore, the relative abundance of Clostridium sensu stricto 1 (25.84%), Paeniclostridium (18.08%) and Romboutsia (14.92%) in the ileum was found to be significantly higher than in other segments of the forestomach, abomasum and small intestine (p < 0.05). The relative abundance of Acetitomaculum in the duodenum and jejunum (9.33% and 13.27%, respectively) was significantly higher than in other segments (p < 0.05) (Supplementary Table 3).

Significant biomarkers were further compared in the compound stomach and small intestine of the Tarim wapiti used LEfSe analysis (Figure 5). The results showed that a total of 81 biomarkers were identified from 5 phyla, among which the rumen showed enrichment of Veillonellales Selenomonadales at the phylum level; Negation at the order level; Rikenellaceae, P 251 O5, Ruminococcaceae, Bacteroidales BS11 gut group, Selenomonadaceae, Muribaculaceae at family level; genus level, Rikenellaceae RC9 intestinal group, P 251 O5, Bacteroidetes BS11 intestinal group, Ruminococcus, Muribaculaceae, Quinella; species level, Bacteroidetes. Bacteroidota at the phylum level; Bacteroidia at the class level; Bacteroidales at the order level; Prevotellaceae at the family level, and Prevotellaceae UCG 003 at the genus level were all highly abundant in the reticulum. The omasum was significantly enriched in Patescibacteria at the phylum level; Oscillospirales, Clostridiales UCG 014, Clostridiales at the order level; Eubacterium coprostanoligenes group, Clostridiales UCG 014, Burkholderiaceae, UCG 010, Hungateiclostridiaceae at the family level; Eubacterium coprostanoligenes group, Clostridiales UCG 014, UCG 010, Ralstonia at the genus level. The omasum was significantly enriched in Patescibacteria at the phylum level; Oscillospirales, Clostridiales UCG 014, Clostridiales at the order level; Eubacterium coprostanoligenes group, Clostridiales UCG 014, Burkholderiaceae, UCG 010, Hungateiclostridiaceae at the family level; Eubacterium coprostanoligenes group, Clostridiales UCG 014, UCG 010, Ralstonia at the genus level. At the order level, the abomasum was highly enriched in Christensenellales; at the family level, F082, Christensenellaceae, Bacteroidales RF16 group; and at the genus level, Prevotella, F082, Christensenellaceae R 7 group, Bacteroidales RF16 group, NK4A214 group. At the phylum level, Duodenum was significantly enriched with Euryarchaeota; at the order level, Methanobacteria; at the family level, Oscillospiraceae, Methanobacteriaceae, Anaerovoracaceae, Alcaligenaceae; and at the genus level, Methanobrevibacter, Achromobacter, Saccharofermentans. The jejunum was significantly enriched in Actinobacteriota at the phylum level; Coriobacteriia, Actinobacteria at the order level; Lachnospirales, Coriobacteriales, Enterobacteriales at the order level; Lachnospiraceae, Enterobacteriaceae, Eggerthellaceae at the family level; Acetitomaculum, Lachnospiraceae, NK3A20 group, DNF00809 at the genus level. The ileum was significantly enriched in Firmicutes at the phylum level; Clostridia, Bacilli at the order level; Peptostreptococcales Tissierellales, Clostridiaceae, Erysipelotrichaceae at the family level; and Clostridium sensu stricto 1, Paeniclostridium, Romboutsia, Turicibacter at the genus level.

Figure 5. Bacterial lefse analysis of the forestomach, abomasum, and small intestine. (a) LDA value distribution histogram; (b) Cladogram diagram. (a) LDA value >4. (b) The classification level of the circle from the outside to the inside is in order of phyla, class, order, family, genus and species. Different colour points in the phylogenetic tree stand for bacteria which is important in each group, respectively. Yellow points indicate fungi have no significant difference among groups.

Correlation analysis of microbiota and VFAs in the forestomach, abomasum, and small intestine

The dominant bacteria in the Tarim wapiti’s compound forestomach, abomasum and small intestine were shown to be related with VFAs (Figure 6). Rikenellaceae RC9 gut group and Prevotella exhibited a positive association with VFAs, whereas Firmicutes, Proteobacteria, Clostridium sensu stricto 1, Acetitomaculum, Paeniclostridium and Romboutsia showed a negative correlation with VFAs. Notably, the bacteria that associated positively with VFA concentration were from the dominant bacteria in the forestomach and abomasum, whereas the bacteria that connected negatively with VFA level were from the main bacteria in the small intestine. Furthermore, the most prevalent bacteria were associated with propionate, whereas the least dominant bacteria were linked to valerate.

Figure 6. Analysis of the correlation between dominant bacteria and volatile fatty acids (VFAs) in the forestomach, abomasum and small intestine. *indicates significant differences between groups and **indicates highly significant differences.

Metabolic pathways of the microbiota in the forestomach, abomasum and small intestine

Carbohydrate metabolism, replication and repair, translation, membrane transport, amino acid metabolism, energy metabolism, nucleotide metabolism and lipid metabolism are the primary metabolic pathways of the microbiota in Tarim wapiti’s forestomach, abomasum, and small intestine (Supplementary Figure 3). A PCA analysis demonstrates that the forestomach, abomasum, and small intestine are divided into two distinct groups. Furthermore, there is a distinct difference between the duodenum, jejunum, and ileum (Figure 7).

Figure 7. Principal coordinate analysis (PCA) of microbial functional diversity across all samples using the relative abundances of functional pathways.

The relative abundance of functional genes involved in terpenoid and polyketide metabolism in the reticulum rose considerably across the rumen to reticulum transition (p < 0.05) (Figure 8(a)). The relative abundance of functional genes involved in membrane transport rose considerably across the shift from reticulum to omasum (p < 0.05). The relative abundance of functional genes involved in energy metabolism reduced considerably (p < 0.05) (Figure 8(b)). The relative abundance of functional genes involved in folding, sorting and degradation in the abomasum rose considerably across the transition from omasum to abomasum (p < 0.05) (Figure 8(c)). Across the transition from jejunum to ileum, there is a significant increase (p < 0.05), in the relative abundance of functional genes involved in membrane transport, lipid metabolism and cell motility in the ileum, whereas there is a significant decrease in functional genes involved in energy metabolism, nucleotide metabolism, replication, repair and translation (p < 0.05) (Figure 8(e)). Across the transition from abomasum to duodenum, the relative abundance of functional genes involved in membrane transport, signal transduction, and cell motility increased significantly (p < 0.05), whereas the relative abundance of functional genes involved in carbohydrate metabolism, glycan biosynthesis and metabolism, lipid metabolism, and the digestive system reduced significantly (p < 0.05) (Figure 8(d)). There is no modification in functional genes at the duodenum-jejunum transition (p > 0.05). The relative abundance of functional genes involved in membrane transport, lipid metabolism and cell motility increases significantly (p < 0.05) across the transition from jejunum to ileum, while functional genes involved in energy metabolism, nucleotide metabolism, replication, repair and translation decrease significantly (p < 0.05) (Figure 8(e)).

Figure 8. Analysis of the difference of KEGG metabolic pathway between segments at the second level in the stomach and small intestine; (a) rumen vs. reticulum; (b) reticulum vs. omasum; (c) omasum vs. abomasum; (d) abomasum vs. duodenum; and (e) jejunum vs. ileum. The middle shows the difference ratio of functional abundance in the 95% confidence interval, and the right value is the p-value.

Discussion

This study examined the communities of bacteria and VFA concentrations in Tarim wapiti’s forestomach, abomasum and small intestine. Results showed a significant difference in the concentrations of acetate, propionate, butyrate and valerate in the forestomach compared to the small intestine. Surprisingly, the reticulum had lower amounts of acetate and propionate than the rumen and omasum. The fluctuation in VFA concentration across the rumen to the ileum with the current investigation followed a consistent pattern found in dairy cows (Mao et al. 2015). However, the VFA concentration in all segments had been found to be lower than that seen in dairy cows (Mao et al. 2015). It is possible that the difference observed can be attributed to the lactation phase of dairy cows, which requires a higher energy intake. These results suggest that the trend in VFAs across different segments of the animal intestine is generally similar, although the specific VFA content may vary based on the energy demands of the organism. Additionally, variations in gut VFA levels between animals may be influenced by factors such as the fermentation process of feed by micro-organisms from various sections of the GIT, the rate at which VFAs are absorbed by the intestines, and the rate at which VFAs circulate within the body. Rumen fermentation produces a substantial amount of VFAs, which help keep the pH relatively low (Shabat et al. 2016). Regarding the VFAs produced by the rumen, it is worth noting that approximately 50–80% of these compounds are absorbed through the rumen epithelium, while the remaining fraction is taken up in the omasum and abomasum. In the small intestine, in addition to digesting and converting carbohydrates into glucose, there is a process where undigested starch and glucose break down through bacteria, leading to the synthesis of VFAs and the release of gas. This study suggests that the lower levels of acetate and propionate observed in the reticulum, compared to the rumen, may be explained by the effect of rumen absorption and conversion mechanisms related to VFAs, along with the fermentation rate inherent in the reticulum itself. In addition, it may also be due to the influence of differences in permeability/absorption of VFAs. The study found that among the VFAs identified within the small intestine, only the acetate concentration exhibits an upward trend, with both the jejunum and ileum displaying significantly higher levels than the duodenum. This suggests that undigested carbohydrates from the duodenum are primarily subjected to acetate conversion within the jejunum and ileum segments. The research reveals that in the dietary composition of ruminant species, an augmented ratio of concentrates leads to propionate fermentation in the rumen. In contrast, a diminished proportion of concentrates favours acetate fermentation (Shabat et al. 2016). The study showed that an increase in the acetate to propionate ratio in the rumen is in line with a diet that has a roughage-to-concentrate ratio of 3:7. Interestingly, the study also found a correlation between dominant bacterial populations and VFA levels in the forestomach and abomasum of the Tarim wapiti. However, the results were different in the small intestine, which suggests that the dominant bacterial communities in each segment of the GIT play a significant role in influencing VFA levels throughout the entire GIT. In conclusion, the study highlights the importance of considering the composition and abundance of dominant bacteria when investigating VFA levels in different segments of the GIT.

The NMDS analysis of the bacterial communities in different segments of the Tarim wapiti’s GIT revealed differences in their composition and structure. The reticulum, omasum and abomasum compartments exhibited higher microbial diversity and richness compared to the small intestine. The results presented are consistent with previous studies conducted on elk (Kim et al. 2019). Furthermore, the rumen microbial community’s diversity and richness were found to be higher than that of the ileum, which contrasts with the results obtained from a study on the Chinese roe deer (Li et al. 2014). The production of VFAs through rumen fermentation primarily maintains a low rumen pH, as highlighted by Shabat et al. (2016). The small intestine, which constitutes a significant portion of the GIT, maintains high concentrations of bile salts and digestive enzymes, which can influence bacterial growth (Wang et al. 2016). The pH and VFAs present in different segments of the GIT vary (Wang, Lee, et al. 2022; Wang, Zhang, et al. 2022). The above-mentioned research indicates that there are differences in the diversity and richness of bacterial communities in different segments of the GIT among different species, leading to lower microbe diversity and richness in the rumen and ileum of the Tarim wapiti. This could be due to the Tarim wapiti’s rumen having a lower pH, and the ileum maintaining high concentrations of digestive enzymes and bile salts. In addition, the physiological activity in different parts of the GIT could be a contributing factor to these findings (Contijoch et al. 2019).

This study discovered that Tarim wapiti have Firmicutes and Bacteroidetes as the predominant phyla in their forestomach, abomasum and small intestines. Bacteroidetes were found to be more abundant in the forestomach and abomasum, while Firmicutes were more abundant in the small intestines. In addition, the biomark of the reticulum is Bacteroidota, while the biomark of the ileum is Firmicutes. These findings are consistent with previous studies on elk (Kim et al. 2019) and Chinese Roe Deer (Li et al. 2014), which also showed a higher abundance ratio of these two phyla, especially in the small intestine. Firmicutes are known to play a critical role in breaking down cellulose, protein, and carbohydrates in the GIT of ruminants (Backhed et al. 2005). On the other hand, Bacteroidetes’s primary function is to degrade fermentable carbohydrates and polysaccharides in fibre cell walls and ferment organic compounds (Flint and Bayer 2008). The Firmicutes/Bacteroidota ratio is closely linked to fat deposition (Ley et al. 2006), as found in a study on red deer (Minich et al. 2021). This could be due to the variations in the fibrous material content within their foraging structures. Tarim wapiti exhibit a higher a bundance of Firmicutes and Bacteroidota, allowing them to effectively obtain more energy and store higher amounts of fat in low-fibre environments. It has been observed in this study that Proteobacteria were found to be abundant in the duodenum and jejunum, with the lowest relative abundance in the rumen. Elk also showed a significantly higher relative abundance of Proteobacteria in the intestine compared to the stomach, with the highest relative abundance in the duodenum (Kim et al. 2019). On the other hand, Chinese Roe Deer showed an enrichment of Proteobacteria in the rectum (Li et al. 2014). Previous research has also shown that Proteobacteria are enriched in the small and large intestine of cows, except for the rectum (Mao et al. 2015), and in the rumen, duodenum, and jejunum of Crossbred Cattle (Wang, Lee, et al. 2022; Wang, Zhang, et al. 2022). High small intestinal Proteobacteria have been associated with unclassified Enterobacteriaceae (Mao et al. 2015). Proteobacteria play important roles in carbon, nitrogen, and sulphur cycles due to their complex metabolisms (Mao et al. 2015). It seems that there is a variable distribution of Proteobacteria within the GIT of animals. Further research is needed to comprehend the distribution patterns and specific functions of Proteobacteria in the animal GIT.

In addition, the study also found that Actinobacteriota exhibited a significantly higher relative abundance in the duodenum and jejunum compared to other regions of the GIT such as the forestomach, abomasum, and ileum. Actinobacteriota, which can synthesise metabolites with several biological functions such as antibacterial, antiparasitic, antifungal, antiviral, and growth-promoting properties (Al-shaibani et al. 2021), have been found to be enriched in different segments of the GIT of different animal species. For instance, the Actinomycetes phylum exhibited an abundance of over 30% in the rumen of elk (Kim et al. 2019), whereas its abundance was considerably lower in the gastrointestinal microflora of Chinese Roe Deer (Li et al. 2014). The study on Crossbred Cattle found that Actinobacteriota had a higher relative abundance in the duodenum and identified Eggerthellaceae and Nocardiaceae within Actinobacteriota as biomarkers for the small intestine due to their ability to produce antibiotics (Wang, Lee, et al. 2022; Wang, Zhang, et al. 2022). These findings highlight the importance of Actinobacteria phylum metabolites and biomarkers in different segments of the digestive tracts across species, contributing significantly to intestinal homeostasis and overall animal health.

At the genus level, the most abundant bacteria found in the forestomach and abomasum are the Rikenellaceae RC9 gut group, Prevotella and F082. The Rikenellaceae RC9 gut group is considered the rumen biomarker while Prevotella and F082 are known as the rumen biomarkers. Prevotella can break down a variety of substances including starch, protein, xylan, and pectin, and a high abundance of Prevotella can increase the concentration of propionic acid which is beneficial for the host (Matsui et al. 2000; Thompson et al. 2015). The Rikenellaceae RC9 gut group can effectively degrade soluble polysaccharides and insoluble cellulose, while increasing the production of succinic acid and propionic acid (Zhu et al. 2021). Prevotella and Rikenellaceae RC9 were found to be the dominant genera of rumen microbes in reindeer living in the scarce natural resources of the Arctic region (Yildirim et al. 2021). Moreover, Prevotella was found to be the predominant genus of rumen fluid in Canadian cervids and white-tailed deer, surpassing the abundance observed in the solid-phase (Gruninger et al. 2014). Similarly, Prevotella were found to be dominant in the rumen fluid of Tarim wapiti (Qian et al. 2017). On the other hand, Ruminalococcus was identified as the dominant genus of rumen-dominant bacteria in wild roe deer (Wilson. 2019). In a recent study conducted by Kim et al. (2019), found that Prevotella is highly abundant in the rumen, omasum, and abomasum of elk. This finding is consistent with previous studies that have shown Rikenellaceae RC9 gut group and Prevotella to remain stable throughout the process of transition in the forestomach and abomasum, with Prevotella has a relatively lower abundance in the omasum compared to the rumen, reticulum, and abomasum, but still above 5% abundance. The function of the omasum is primarily water absorption, filtration, and transport of small food particles to the next stage, while retaining larger particles for further digestion. The decrease in the relative abundance of Prevotella in the omasum may be related to its physiological activity. These findings highlight the importance of microorganisms residing in gastric compartments other than the rumen in the process of nutrient digestion and energy supply to the organisms. It is worth noting that F082 belongs to the phylum Bacteroidetes, but its function in the forestomach and abomasum is currently unknown. It is possible that F082 may play a role similar to that of Bacteroidetes in carbohydrate decomposition and organic material fermentation. However, F082 was not identified as the dominant genus at the gastrointestinal microbial genus level in elk (Kim et al. 2019). These results suggest that the dominant bacterial genera in the deer’s GIT could be influenced by feeding structure and that unique dominant genera exist. Further investigation is required to unveil the specific function of F082 in the GIT.

This study has revealed interesting findings about the dominant bacterial genera present in the small intestine. These include Clostridium sensu stricto 1, Acetitomaculum, Paeniclostridium and Romboutsia. It was found that Acetitomaculum is the jejunum biomarker, whereas Clostridium sensu stricto 1, Paeniclostridium, and Romboutsia are the ileum biomarkers. Clostridium sensu stricto 1, which belongs to the Firmicutes, mediates the conversion of bile acids in the GIT and produces butyrate and propionate by fermenting amino acids such as threonine and serine. The metabolites produced by Clostridium are known to play crucial roles in metabolic and immune regulation of intestinal epithelium and the whole body (Pi et al. 2017). Paeniclostridium has been found to have similar characteristics to Clostridium (Sasi Jyothsna et al. 2016), and Romboutsia has been found to produce VFAs, especially butyrate (Wang, Zhang, et al. 2022). Acetitomaculum is a member of the phylum Firmicutes and is capable of synthesising acetic acid using H₂ and CO₂ produced in the rumen, as well as using monosaccharides to produce acetic acid (Hua et al. 2017; Ragsdale 2008). Acetitomaculum ruminis, which produces acetic acid autotrophically, competes with methane for H₂ by producing acetate (Le Van et al. 1998; Leedle and Greening 1988). According to Xie et al. (2021), Methanobrevibacter was found to be present in significantly higher proportions in the small intestine compared to the forestomach, abomasum, and large intestine. This is interesting as Pérez-Barbería (2017) found that deer produce lower methane emissions per unit of dry matter intake than cows. The study also reported a relatively high abundance of Clostridium and Romboutsia in the small intestine, which is similar to previous studies in elk and yaks (Kim et al. 2019, Zhang et al. 2019). However, the dominant species in different segments of the Chinese roe deer differed from previous studies (Li et al. 2014). In addition, research has shown that yaks with the Romboutsia genus have a higher adaptability to high altitude environments (Zhang et al. 2019). Wild deer also have a relatively high abundance of Clostridium in their faeces (Sun et al. 2023), while the caecum of the monogastric Mongolian horse harbours a high abundance of Clostridium (Sun et al. 2023). This indicates that different sections of the small intestine may have distinct dominant bacterial species in different animals, potentially contributing to variations in metabolic processes and energy acquisition. Lastly, the study reported a higher relative abundance of Acetitomaculum in the small intestine than in the forestomach and abomasum, suggesting its involvement in H₂ metabolism and the maintenance of small intestinal homeostasis.

The study discovered that across the reticulum to omasum transition, there was a decline in bacterial function in relation to energy metabolism and a corresponding decline at the genus level in the relative abundance of Prevotella, the dominant genus. The researchers concluded that the decrease in Prevotella might be attributed to a decline in their energy metabolic function. The ratio of Firmicutes to Bacteroidetes was strongly linked to fat deposition in previous studies (Ley et al. 2006), and was also strongly linked to winter fat deposition in red deer (Minich et al. 2021). The lipid-metabolising function of intestinal bacteria was found to increase from the jejunum to the ileum, while the energy-metabolising function decreased accordingly, and the ratio of Firmicutes to Bacteroidetes also increased accordingly. The ileum, according to the study, plays a critical role in fat deposition in the small intestine. The study discovered some correlation between dominant bacterial genera and key metabolic pathways in different segments of the forestomach, abomasum, and small intestine, indicating that the core microorganisms in each segment may have a significant influence on how the bacterial community operates in that segment.

Conclusion

Taken together, VFAs and bacterial community composition were found to differ significantly in the forestomach, abomasum, and small intestine of Tarim wapiti. The VFAs were positively correlated with the dominant bacteria in the forestomach and abomasum, but negatively correlated with the dominant bacteria in the small intestine. Additionally, certain correlations were observed between dominant bacteria species and major pathways in different segments of the forestomach, abomasum and small intestine.

These findings provide valuable insights into the microorganism and metabolite diversity in the forestomach, abomasum, and small intestine of Tarim wapiti. Understanding the characteristics of the digestive tract microbiota can help explore targeted approaches for regulating nutrient metabolism, which in turn can enhance production performance.

Ethical approval

All animal procedures were approved by the Animal Ethic Committee of Tarim University (Xinjiang, China), conducted in accordance with the Guidelines for the Care and Use of Research Animals in China (GB14925-2001).

Acknowledgements

All authors would like to thank the Science and Technology Innovation Program of the Xinjiang Production and Construction Corps, China, for financial support.

Disclosure statement

All authors declare that they have no conflict of interest to state.

Data availability statement

The data that support the findings of this study are openly available in NCBI (https://www.ncbi.nlm.nih.gov/sra/PRJNA982455).

Additional information

Funding

This work was supported by the Science and Technology Innovation Program of the Xinjiang Production and Construction Corps in China [project number 2023AB007-02].