Fermentation parameters involving GP and nutrient disappearance rates were investigated in this study when the FB was supplemented to artificial rumen fluid in the experiment of in vitro fermentation. Generally, GP plays an important role in rumen fermentation, and it is positively correlated with nutrient degradation rate22. Our present study shows that the 48-h GP of fast- and slow-degradable fraction and theoretical GP demonstrated a significant increase under the FB supplements of 1:1000 to 1:250. Particularly, at the two doses of 1:1000 and 1:500, the GP was the greatest among all doses of FB, suggesting that FB may promote the degradation of nutrients by microorganisms, which is consistent with our results of high disappearance rates of CP and NDF at the two doses of 1:1000 and 1:500 in this study. However, the GP of slow-degradable fraction and theoretical GP were not affected by the highest dose of FB (1: 250). The reports from Bachmann et al.23 indicated that low or high level of prebiotics might reduce GP of in vitro fermentation, and our present results also suggest that only appropriate doses of FB could promote the degradation of nutrients.
The effects of FB on fermentation parameters (pH, NH3-N, and MCP) in both in vitro and in vivo test were also examined in this work. The NH3-N is a main nitrogen source of MCP synthesis for ruminants, and reflects the balance between synthesis and degradation of proteins in the substrate. Moreover, MCP in rumen also affects the utilization of ruminal NH3-N by microorganisms, and more MCP production contributes to the increase of utilization efficiency of nitrogen24, which is generally related to microbial diversity in rumen. Our data in present study demonstrate a significant decrease for NH3-N concentration and a significant increase for MCP concentration when the FB of 1:500 was added to drinking water, which could be explained by the changes of microbial diversity in rumen or jejunum. From venn diagram and alpha-diversity index, whether in rumen or jejunum, the FB supplements of 1:500 to 1:1000 significantly improved diversity of bacterial community, revealing that the unique OTU number increased by 42.3–91.5% in rumen and 39.1–54.1% in jejunum. The change of bacterial community could promote the transformation of NH3-N to MCP and meanwhile consume more NH3-N, which is in agreement with Ghorbani et al.25. Also, our present results of NH3-N and MCP in in vivo experiment are consistent with those in fermentation experiment in vitro. In in vitro experiment, different levels of FB reduced the NH3-N content in artificial rumen fluid; particularly for the FB doses between 1:500 and 1:1000, the NH3-N decreased by 8.58–9.21% while the MCP increased by 16.22–18.92%, when compared to the control. These results suggest that probiotics or other unknown active ingredients in FB may have some positive effects. Previous studies on probiotics (e.g., Bacillus) also showed a decrease for NH3-N content and an increase for MCP in rumen or intestine26, with which our results are in agreement.
A panel of VFA parameters in rumen of lambs offered FB supplements were also measured in this study. The contents of isobutyric acid and propionic acid were increased by the FB supplements. Published literatures have demonstrated that isobutyric acid might accelerate the reproduction of rumen cellulolytic bacteria, resulting in the enhancement of nutrients digestion, paticularly cellulose27,28. In this case, NDF degradation rate was elevated by 1:500 or 1:1000 FB offered in drinking water, which suggests that FB would prefer to promote the degradation of cellulose to some degree. Propionic acid, as an important VFA in rumen, has been proved in recent studies that its augment would promote more energy to translate into body weight in fattening animals to bring more benefit in weight gain29. Moreover, in this experiment, whether in vivo or in vitro, A/P ratio in rumen liquid demonstrates a decrease due to the addition of FB, which suggests that rumen fermentation pattern may have a change toward the pathway of propionic acid production. This pattern is usually deemed as a high-efficient fermentation mode of energy utilization in animals or microorganisms. The decrease of A/P here may be explained that our FB contains many bioactive components such as polysaccharide, saponin and flavonoid, which could promote the digestion and absorption of nutrients and improve energy utilization. Early studies have demonstrated that polysaccharide, saponin, or flavone supplements in the diet improved the growth performance involving weight gain and feed conversion rate in animals30–32. Moreover, the bioactive substances would stimulate the development of rumen and alter its microstructure to help digestion33. It is well known that rumen development is very important for ruminants, and a well-developed rumen contributes to feed conversion and energy utilization34. In this study, under microscope, the rumen microstructure was influenced by FB supplements, and the increases of papilla length, mucosal thickness, muscle thickness, and papilla superficial area were observed. In general, papilla length and its superficial area are important indexes to evaluate rumen morphology; particularly, papilla superficial area may be directly associated with the chance of chyme touching rumen, which demonstrates that more papilla superficial area would help for nutrient absorption in rumen35. In addition, jejunum, as a part of small intestine, has strong decomposing and absorbing function on nutrients36. The FB promoted the jejunum development of lambs in this experiment, and its microstructural parameters including villus height, muscle thickness, villus superficial area, and villus height/crypt depth were improved, which would increase the area for nutrients touching jejunum to promote the absorption of nutrients37. Previous studies suggested that the development of gastrointestinal tract was also associated with feed additives, gastrointestinal microecology, and diet composition38. In our experiment, the FB contained some saponin, which is extracted mainly from alfalfa, and saponin could promote the reproduction of beneficial bacteria in gastrointestinal tract, which have been reported by Tan et al.39, because it may modulate microenvironment suitable for gastrointestinal microorganisms, and promote the development of rumen and jejunum40.
Majority of published studies have reported that maintaining balance of gastrointestinal microorganism is helpful to protect animal health and improve its digestibility41–43. The FB in this study may enrich a variety of bioactive ingredients (alfalfa flavonoid, alfalfa saponin, and astragalus polysaccharide, etc.), as well as some probiotics such as lactobacillus. Many studies on probiotics improving microbial community have been reported in past few decades44,45. It is generally believed that probiotics enters gastrointestinal tract via dietary addition, and competes, inhibits, and coexists with inherent gastrointestine-colonizing bacteria, eventually forming a stable and healthy gastrointestinal environment46. In this study, three methods including venn diagram, alpha-diversity, and beta-diversity were used to analyze the effects of FB on microbial community in gastrointestinal tract of fattening lambs. The FB supplements in drinking water significantly improved the diversity of microbial community in rumen and jejunum, particularly in rumen. As mentioned above, the unique OTU in rumen increased by 91.5% when the FB of 1:500 was provided in drinking water for lambs, and the PD_whole_tree and shannon index in rumen had an increased tendency for FB groups, compared with the control group. The reasons may be related to flavonoids contained in the FB. Flavonoid have some positive effects on microbial community in digestive tract, e.g., improving microbial diversity, increasing beneficial bacteria (e.g., Bifidobacteria and Lactobacillus) number, and inhibiting reproduction of harmful bacteria by destroying cell membrane and slowing down its metabolism47,48. Similar studies also showed that dietary flavonoid supplements could effectively inhibit the reproduction of Escherichia coli in the gut and enhance the abundance of Bifidobacteria49. Additionally, the microbial composition and abundance of FB-offered lambs at phylum and genus levels were also investigated in this work. Two dominant bacterial phyla in rumen were found, including Bacteroidota and Firmicutes, which agrees with previous results50,51. Bacteroidota plays an important role in decomposing polysaccharides, while Firmicutes can increase the activity of gene-coding enzymes, promote rumen development, and improve absorption capacity of starch and cellulose52. The results of this experiment show that FB of 1:500 in drinking water could increase by 70.75% in the abundance of phylum Firmicutes in rumen and decrease the abundance of phylum Bacteroidota compared to the control. However, two rumen bacteria subordinate to the phylum Bacteroidota, including Rikenellaceae_RC9_gut_group (dominant bacterial genus) and Muribaculaceae (non-dominant bacterial genus), demonstrated significant increases in bacterial abundance, even the Muribaculaceae abundance in 1:500 group was 2.9 times greater than that in the control. The work from David et al.53 indicated that Rikenellaceae_RC9_gut_group mainly exert the effects in promoting the digestion and absorption of carbohydrate. which is known as a main energy source in ruminants and can be converted into VFA to provide more energy. Though the Muribaculaceae is not the dominant bacteria in rumen, it has some functional diversity in decomposing complex carbohydrates; however, little relevant information is available54. Thus, the FB supplements in drinking water in our study could promote the degradation of carbohydrates in rumen under synergistic action of bacterial population. From analysis of microbial composition in jejunum, Firmicutes, as first dominant bacterial phylum, was not affected in abundance by FB supplements. Euryarchaeota, second dominant bacterial phylum, is traditionally called methanogenic bacteria because it is mainly involved in methane production. The abundances of phylum Euryarchaeota and Methanobrevibacter (first dominant bacteria genus) subordinate to phylum Euryarchaeota were lowered by 1:500 addition of FB. Results from Djemai et al.55 indicated that the genus Methanobrevibacter mainly participates in methane production under anaerobic conditions. The decrease in the abundance of methanogenic bacteria in our data may be due to the fact that flavonoids from alfalfa, Platycladus orientalis, and Astragali Radix in FB inhibit the activity of methanogenic bacteria56, leading to the reduction of methane production, and improve the energy utilization for ruminants. A recent study also suggested that saponin in alfalfa could reduce the methane-producing bacteria count or its activity, thereby reducing methane concentration57. In addition, the addition of 1:500 FB increased the phylum Actinomycetes abundance in jejunum. Evidence has demonstrated that Actinomycetes takes part in the degradation of organic matter and nutrients absorption, and reduces the occurrence of diarrhea58. The Aeriscardovia abundance in jejunum, belonging to the phylum Actinomycetes, increased when 1:500 FB was provided to lambs by the addition in drinking water. This genus Aeriscardovia subordinate to the family Bifidobacteriaceae has been proved for these positive functions, e.g., inhibiting harmful bacteria growth, maintaining balanced microenvironment in gastrointestinal tract, and improving immunity59. The reason for this change in our study is associated with polysaccharides in FB. Published work from Shang et al.60 has demonstrated that this bioactive ingredient can promote the growth of Bifidobacteria. Therefore, from all results of our two experiments (in vivo and in vitro), the positive effects involving digestive tract development and microbial community caused by FB supplements would be comprehensive responses to FB.