Comparative Analysis of the Microbiota Between Rumen and Duodenum of Twin Lambs Based on Diets of Ceratoides or Alfalfa

Many environmental factors such as nutrition, habitat, and host genetics impact the components and functions within the gut microbiome (Ussar et al. 2016). The rumen of sheep envelops a complicated microbiota and acts as the primary location for fermentation of consumed feed (McCann et al. 2017), which directly impacts sheep’s health and physiological functions.

The rumen is the largest compartment of ruminants where plant cell walls and other herbage materials are degraded by intricate microbial communities predominated by bacteria (Sirohi et al. 2012). The bacteria systematically decompose plant cell materials (Flint and Bayer 2008) and break down plant biomass, serving as a link between the sheep and the nutrients absorbed by the sheep. The succeeding rumen fermentation manufactures ammonia and short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate. In a previous study, we found that diet directly impacts the microbiota structure of the rumen and affects the metabolic process in sheep muscle (Wu et al. 2020). However, the nutritional components that entered the small intestines were not analyzed.

The duodenum, the first part of the small intestines, is where absorption of nutrients begins due to its ability to receive partially degraded food from the stomach. The duodenum is the most proximal phase of degradation, and it represents the most oversized diameter, the densest villi, and the deepest part within the small intestines. The duodenum takes fluid and bile produced by the pancreas and the liver, thereby assisting the intestines in breaking fat, protein, and starch (Faichney 1969; DeGregorio et al. 1982; Lewis and Dehority 1985). Few studies have been done regarding the microbiota of the duodenum, and there is a need to analyze the metabolome of the duodenum, compare it with the microbiota of the rumen, to determine the structure of the bacterial community in the lambs based on two different feeds.

The microbial diversity of the rumen and duodenum. Based on Simpsons and Shannon indices, there was a significant difference in the rumen’s microbial diversity between the HFLP and the LFHP groups (p < 0.05). This could be the effect of the high fiber content in the HFLP diet (Table II). However, there was no significant difference in the duodenum’s microbial diversity between the two groups (Table III). The diversity of the microbiota in the HFLP group was higher than the LFHP group in the rumen. However, that pattern could not be entirely detected in the duodenum. The diversity of the microbiota in the duodenum was higher than those in the two groups’ rumen.

Phylum community distribution of the rumen and duodenum. On the phylum level, the abundance of 11 phyla of the rumen and duodenum was calculated and used to draw histograms for comparisons. A greater number of sequences obtained from the phylum class pertained to the phyla Bacteroidetes, and Firmicutes but at the duodenum’s phylum level, a larger proportion of the sequences obtained related to Firmicutes, and Bacteroidetes. The major bacteria in the rumen’s phylum level were Bacteriodetes, but were replaced with Firmicutes in the duodenum. The major bacteria were higher in the LFHP group than the HFLP group for the rumen and duodenum (Fig. 1 and 2).

Fig. 1.

Fig. 2.

Genus community distribution of the rumen and duodenum. A detailed examination of the relative abundance of bacterial OTUs showed that the two different feeds have a varied influence on both the rumen and duodenum’s microbiota. According to the 16s rRNA gene of the rumen and duodenum bacteria sequences, 1,230 OTUs and 2,373 OTUs among eight rumen and duodenum samples were identified. In the duodenum, 143 OTUs were significantly different between the two groups.

Ninety-two non-identical genera were designated from the sequences at the genus level. The genera were among almost all specimens. Prevotella was abundant in the rumen of the LFHP group compared to the HFLP group (Fig. 3a). Unclassified Lachnospiraceae were prevalent in the LFHP group’s duodenum compared to the HFLP group (Fig. 3b). The high protein in the LFHP feed increases the genera Prevotella and unclassified Lachnospiraceae of the rumen and duodenum.

Fig. 3.

Comparison between the microbiota of ceratoides (HFLP) and alfalfa (LFHP) feeds, and theirFirmicutes/Bacteriodetes(F:B) ratio in the rumen and duodenum. The F:B ratio in the rumen microbiota of the twin lambs fed on the LFHP pellets was 0.104, and that of the HFLP pellets was 0.275 (Fig. 4a and 4b). The F:B ratio in the duodenum microbiota of the twin lambs fed on the LFHP pellets was 6.419, and that of the HFLP pellets was 5.356 (Fig. 4c and 4d). There was a change in the F:B ratios in the rumen and duodenum even under the different feeds.

Fig. 4.

Differential expression metabolites between two groups. After the data was filtered, 3,696 stable metabolic features were detected. A partial least squares discriminant analysis (PLS-DA) was performed between the two groups to identify metabolic differences in duodenum of twin sheep fed different diets (Fig. 5). The results showed that 407 significantly different metabolites (p-value ≤0.05) were screened between the two groups. In the HFLP group, 273 of the metabolites showed a lower expression, and 134 of the metabolites showed a higher expression. Tandem mass spectrometry (MS/MS) was used to detect 16 significantly different metabolites indicated in the heat map (Fig. 6). The HFLP group increased 7 of the metabolites while that of the LFHP group influenced 9 of the metabolites. Some of the metabolites discussed are adenosine, taurine, L-alanine, and nicotinic acid (Fig. 7). It was detected that there was a significant difference between the HFLP diet and the LFHP diet (p-value ≤ 0.05).

Fig. 5.

Fig. 6.

Fig. 7.

The correlation analysis indicated that the abundance of 5,6-dihydrouracil correlated positively with adenosine, and negatively correlated with 9(R)-HODE and acrylic acid. Adenosine was observed to be positively correlated with 5,6-dihydrouracil, and correlated negatively with 9(R)-HODE and acrylic acid. 9(R)-HODE had a positive correlation with acrylic acid, and correlated negatively with 5,6-dihydrouracil and adenosine. Also, acrylic acid was seen to be correlated positively with 9(R)-HODE but negatively correlated with 5,6-dihydrouracil, and adenosine (Fig. 8).

Fig. 8.

Relationship between duodenal microbiota and metabolites. Correlation analysis between the microbiota and metabolites was carried out to find the possible coexistence. 79 correlations were related positively (|rho| > 0.8, p-value ≤ 0.05) while 194 correlations were related negatively (|rho| > 0.8, p-value ≤ 0.05) between the OTUs and the metabolites. Prevotella and 1-aminocyclohexanecarboxylic acid had the greatest positive correlations (r = 0.84, p-value ≤ 0.05). Methanobrevibacter and 3-deshydroxysappanol trimethyl ether were detected to have the greatest negative correlations (r = –0.79, p-value ≤ 0.05) between the bacteria and metabolites.

The bacterium Candidatus saccharibacteria (OTU31, OTU970) is positively correlated with the metabolites 9(R)-HODE and acrylic acid and correlated negatively with 5,6-dihydrouracil, adenosine, and L-alanine. The bacterium Cyanobacteria sreptophyta (OUT217) was found to be correlated positively with 9(R)-HODE and acrylic acid but correlated negatively with 5,6-dihydrouracil, adenosine, and L-alanine. The bacteria Firmicutes blautia, Firmicutes Firmicutes unclassified, and Firmicutes Eubacterium sp. C2 (OTU224, OTU769, OTU798) which all belong to phylum Firmicutes were positively correlated with 9(R)-HODE and acrylic acid but negatively correlated with 5,6-dihydrouracil, adenosine, and L-alanine. The bacterium Planctomycees planctomycetaceae (OTU1882) correlated positively with 5,6-dihydrouracil, adenosine, and L-alanine but correlated negatively with 9(R)-HODE and acrylic acid (Fig. 9).

Fig. 9.

Many former researchers on ruminants only concentrate on the rumen microbiomes. However, we decided to compare the rumen and duodenum microbiomes using two feed types and the duodenal metabolism to investigate the relationship between microbiota and metabolome because metabolites can influence the meat quality and health of the host (Xu et al. 2013; Muroya et al. 2019).

The biodiversity of the rumen and duodenum microbiota. The biodiversity of the rumen was higher when fed with ceratoides (HFLP) than when fed with alfalfa pellets (LFHP) (Table II). The outcome of this work is compatible with our previous research, which reveals that the microbiota in the lambs’ rumen fed a high fiber diet (HFLP) were more diverse than those fed a low fiber diet (LFHP) (Wu et al. 2020). A study shows that Firmicutes and Bacteroidetes are the major predominant phyla of microorganisms in the gut community of terrestrial animals (Qin et al. 2010). In this research, the most predominant phyla in the rumen’s microbiota of the twin lambs were Bacteroidetes and Firmicutes (Fig. 1 and 2), which are more associated with the breakdown of carbohydrates and proteins. Results of this kind have been revealed in earlier reports (Hook et al. 2011). Bacteroidetes aids in the degradation of starch in the rumen (Stevenson and Weimer 2007).

The LFHP diet influenced the genus Prevotella and the phylum Bacteroides of the rumen (Fig. 3). Results of this kind have been detected in earlier research (Zhang et al. 2014). The genus Prevotella plays a significant role in breaking down dietary protein to ammonia, which can reduce the utilization of dietary amino acids. The degradation of peptides in the rumen is associated with the breakdown approach in which dietary protein is disintegrated into ammonia, thereby leading to an inexpedient utilization of dietary amino acids (Walker et al. 2005).

There was no significant difference between the two diets in the duodenum in this study (Table III). Regardless of diet, amino acid profiles of the duodenal digesta were similar due to the presence of forage (Merchen et al. 1986). In this study, Firmicutes was relatively abundant in the duodenum’s phylum level, which confirms a research study where Firmicutes were the predominant phylum among all the bacterial groups across the GIT besides those within the omasum and abomasum, in which Bacteroidetes were more prevalent (Wang et al. 2017). The Lachnospiraceae was found to be the most abundant genus in the duodenum (Fig. 3). This finding agrees with a research study that revealed that other Firmicutes members showing a great abundance in lamb’s gut were Lachnospiraceae (Palomba et al. 2017).

The Firmicutes/Bacteroidetes ratio (F:B) in the rumen and duodenum’s microbiota. The microbiota’s F:B ratio was reversed between the rumen and the duodenum due to differences in the organs and functions. The most potent organ, which degrades and converts plant materials to SCFAs in the ruminants, is the rumen (Wang et al. 2020). It possesses the complex microbiota that plays a vital role in the fermentation of feed and energy metabolism, and the SCFAs provide more than 70% of the energy to guarantee host growth and reproduction performance (Flint et al. 2007). Several researchers suggested that bacteria detected in intestinal contents as they go through the abomasum could come from lysed cells (Waghorn et al. 1990; Koenig et al. 1997; Hristov 2007), and the role of the duodenal microbiota in terms of its function in feed degradation is likely to be different from that of the ruminal bacterial community.

A study revealed that including dried distillers’ grains with solubles (DDGS) at the detriment of corn bran decreased the flow of bacteria from the rumen (Castillo-Lopez et al. 2014). The DDGS consists of about 31% crude protein, 34% neutral detergent fiber, 12% fat, and 5% starch (Paz et al. 2013). Also, the rumen’s microbiota clustered differently compared to that of the duodenum showing different bacterial diversity between ruminal bacteria and the duodenal digesta. Therefore, including the DDGS in the feeds would increase the flow of saturated fatty acids to the duodenum and cause a shift in the rumen and duodenum’s bacterial diversity (Castillo-Lopez et al. 2014).

The small intestine, which comprises the duodenum, is a long and coiled tube where the remaining degradable activities occur. Villi that line the small intestine are the main site where nutrients are absorbed and are distributed to the whole body. Amino acids, fatty acids, and sugars which are the end products of digestion, are absorbed from the small intestines, enters the lymph, and distributed (National Research Council 2007). Another study revealed that when fat was injected into the duodenum of lambs, it was absorbed quickly, but when introduced to the rumen, absorption was slow and took several days (Heath and Morris 1962).

The feed delivered plays a crucial function in the F:B ratio (Ramirez Ramirez et al. 2012). The F:B ratio of the microbiota appreciably changed in the rumen when the twin lambs consumed either LFHP or HFLP feeds (5.356 and 6.419), respectively (Fig. 4a and 4b). There was also a change in the F:B ratio of microbiota in the duodenum when the twin lambs consumed either LFHP or HFLP feeds (0.275 and 0.104), respectively (Fig. 4c and 4d).

The more significant F:B ratio in fecal specimens is related to an increase in human weight (Ley et al. 2006), and a study found that the frequency of particular microbial phylotypes could be affected within the offspring of farm animals such as cattle due to the sire breed when utilizing disparate feeds (Hernandez-Sanabria et al. 2013). Also, the changes of the F:B ratios in this current research agrees with a previous study that analyzed the F:B ratio in mice and humans, where changes in the GIT were demonstrated to affect obesity and the capability of the host to harvest energy (Krajmalnik-Brown et al. 2012). It implies that the shift in the F:B ratio in this study could result from the different kinds of feed.

The relationship between the two feed (LFHP and HFLP) groups and the metabolites. Adenosine can influence meat quality. Apart from influencing the components of the gut microbiota, the type of diet can also regulate metabolic homeostasis in twin lambs. In the presence of low protein, meat quality improves in the HFLP group (Fig. 7a). A study summarized that Tibetan sheep meat was preferred to Small-tailed Han sheep meat even though variations between the breeds were not much; however, meat quality was enhanced in the two breeds with the growth of the nutritional energy level when a low-protein feed was given (Jiao et al. 2020). The presence of adenosine in this study could influence the health and regulate the sheep’s immune system. It reduces the production of tumor necrosis factor (TNF), induces the manufacture of Nitric Oxide (NO), and plays a vital role in maintaining tissue perfusion (Adanin et al. 2002).

Taurine was influenced by the HFLP feed (Fig. 7b). Taurine present in this study is vital when inspecting the relationship between taurine and palatability. A study mentioned that ribose 5-phosphate, and pyrrolidone carboxylic acid or taurine were natural antecedents of 4-hydroxy-5-methyl-3(2H)-furanone, which is a taste part removed from beef broth and has a caramel-like and burnt chicory smell (Tonsbeek et al. 1968; Weenen et al. 2005). The abundance of taurine in lambs and much more in beef could also enhance the beef’s nutritional value apart from contributing to flavor (Purchas et al. 2004). Taurine is important for meat quality, and increasing the concentration of taurine in mutton could be a future breeding objective for Sunit sheep.

The metabolite L-alanine, which was influenced by HFLP diet (Fig. 7c) could benefit the host by reducing tuberculosis. Tuberculosis is caused by Mycobacterium tuberculosis, and it is a major health issue globally. A study discovered that exogenous L-alanine can lead to the manufacturing of reactive oxygen species in Mycobacterium smegmatis by accelerating the tricarboxylic cid cycle and/or primary metabolism synergizing with fluoroquinolones, which, in the long run, results in the destruction of M. smegmatis (Zhen et al. 2020).

The LFHP diet could increase nicotinic acid in the twin lambs (Fig. 7d). A study showed that replacing dietary protein with non-protein nitrogen depresses nicotinic acid (Buziassy and Tribe 1960). Another study revealed that both nicotinamide and insulin-induced hypoglycemia reductions in free fatty acid enhanced growth hormones released in dairy cows, and each of these cases provided a possible function for glucose and free fatty acid in modulating the growth hormone-releasing factor, which stimulates the release of growth hormones in ruminants (Reynaert et al. 1975; Sartin et al. 1988).

The joint analysis of metabolites and microbiota in the duodenum. The bacteria, which all belong to phylum Firmicutes (OTU224, OTU769, and OTU798) correlated negatively with adenosine (Fig. 9). The bacteria Planctomycetes planctomycetaceae (OTU1882) correlated positively with adenosine and L-alanine. Early research states that Planctomycetes contain a strong proline- and cysteine-rich proteins envelope and not a peptidoglycan cell wall (Liesack et al. 1986). Also, Candidatus saccharibacteria (OTU31, OTU970), and Cyanobacteria sreptophyta (OUT217) correlated negatively with adenosine and L-alanine. Recently, whole genomes of Saccharibacteria, acquired via metagenomics, reported that a few members ferment metabolites, glucose, and various sugars and produce lactate (Albertsen et al. 2013). Cyanobacteria has various unique roles that include the ability to restore nitrogen, synthesize vitamin B and K21, syntrophically manufacture hydrogen, and obligate anaerobic fermentation (Di Rienzi et al 2013).

Based on the relationship between the bacteria and metabolites, Methanobrevibacter can produce methane in the gut. Methanogenic archaea represented by Methanobrevibacter ruminantium produce ruminant’s methane and is found in ruminants fed on varied kinds of feeds worldwide (Leahy et al. 2010).

This study shows how 16S rRNA sequencing combined with metabolome analysis may be used in discovering new and significant influences on the functions of a microbe inside the host. The results showed that the diet could directly affect the diversity of rumens’ microbiota but not the microbiota in duodenum. There was a shift in the F:B ratio in the rumen and duodenum of the twin lambs even under the different feeds. We found that some bacteria had a relationship with the metabolites. In summary, these findings could provide knowledge of how the diet affects the microbiota of the rumen and duodenum, lay a theoretical basis for controlling feed intake, and determine the relationship between the duodenum’s microbiota and metabolism.