The ruminal fluid pH was measured with a Sanxin MP523-04 digital pH meter (Shanghai Sanxin Instrumentation, Inc., Shanghai, China) according to the manufacturer’s guidelines.

The analysis of volatile fatty acids (VFA) was carried out using an Agilent 7,890 A gas chromatograph (Agilent Technologies, Santa Clara, California, United States) and a 50 m (internal diameter 0.32 mm) CP-Wax Chrompack silica-fused capillary column (Varian, Palo Alto, California, United States). The initial and final oven temperatures were set at 65 and 195°C, respectively, with helium used as the carrier gas. The detector and injector temperatures were carefully calibrated to 250°C, and the injector volume was precisely configured to 1 μL. The samples were centrifuged at 10,000 × g at 4°C for 10 min. The centrifuged sample was then transferred to another 2 mL EP tube, and 0.2 mL of metaphosphoric acid was added. The mixture was refrigerated for approximately 3 h, then removed and centrifuged again at 10,000 × g at 4°C for 10 min to obtain the supernatant. A 1 mL of the supernatant sample was placed into the GC beaker and processed by gas chromatography.

The NH₃-N concentration was measured using the Chaney and Marbach (26) method, with a Cary Series UV–Vis Spectrophotometer connected to a computer running Cary Series analysis software (Agilent Technologies, California, United States). In detail, the samples were centrifuged at 4,000 × g for 10 min at 4°C, and 0.2 mL of supernatant was combined with 1.8 mL of distilled water and 8 mL of 0.2 M hydrochloric acid in 15-mL centrifuge tubes. From each 15-mL tube, 0.4 mL was transferred to a 10-mL tube, and 2 mL of each solution A and B were added. Lastly, analyzed by a Cary Series UV–Vis Spectrophotometer. The measured extinction values were substituted into the regression formula, and the results were multiplied by a dilution factor to determine the final NH₃-N concentration in the sample.

The ANKOM^RF gas production system is designed to measure the gas pressure in psi. The gas production for each bottle was measured and recorded over time. The data was then converted into moles of gas using the ‘ideal’ gas law and further converted into milliliters (mL) of gas using Avogadro’s law.

The ideal gas law:

where: n—is the quantity of gas generated, measured in moles (mol).

Avogadro’s law states that, under standard conditions, 1 mole will fill 22.4 L at 273.15°K and 101.325 kPa (1 psi = 6.894757293 kilopascal). Therefore, the following formula can be used to convert a gas mole measurement to milliliters (ANKOM manual):

Sequencing, library preparations, and taxonomic analysis were performed by Shanghai Personal Biotechnology Co. Ltd. (Shanghai, China). A total of 216 ruminal fluid samples produced 30,196,306 original sequences, with 19,625,098 (90,857 sequences per sample) identified as high-quality bacterial sequences. The 16S ribosomal RNA (rRNA) gene was amplified using the polymerase chain reaction (PCR) method. Barcoded tags and the widely used bacterial primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) were used in the amplification process (27). The V4 region of the gene was amplified from genomic DNA and the resulting amplicons were prepared for sequencing using Illumina’s MiSeq technology (28).

Based on the distribution of OTU in different samples, the levels of alpha diversity in each sample were assessed. An association network was constructed for each sample, and the topological index was calculated to identify the key species. The composition and distribution of each sample at phylum, class, order, family, genus, and species levels were analyzed visually and statistically using QIIME2 in the R programming language. This analysis involved removing singleton data points and statistically summarizing all the information in a table. To fully evaluate the richness of the microbial community alpha diversity, the Chao1 (29) index, as well as the Shannon (30) and Simpson (31) indices, were used and analyzed. Additionally, Principal Coordinates Analysis (PCoA) of beta diversity was conducted to compare the distance between the two samples.

For rumen bacteria community composition analyses, a total of 216 samples of ruminal fluid were used, with four replications per fermentation time. In diet 1, supplementation with YC, OA, and their interactions significantly increased the Chao1 value, an indicator of bacterial richness (Figure 3A). Specifically, the OA (p < 0.001, at 12 h) and YC and OA interaction (p < 0.05, at 48 h) increased the Chao1 values. YC, OA, and their interactions also increased the Shannon (Figure 3B) and Simpson (Figure 3C) diversity indices (p < 0.001) at 12 h. In diet 2, the Chao1 index results showed that YC and OA supplementation increased bacterial richness and abundance (Figure 3D). The YC and OA interactions (p < 0.001) showed a higher Chao1 index value. However, the Chao1 index value of the YC alone supplemented treatments was significantly lower than that of the control. Supplementation with YC and OA interaction also altered the Shannon (Figure 3E) and Simpson (Figure 3F) diversity indices in this diet. Lower YC and lower OA supplementation significantly increased the Shannon and Simpson indices at 12 h. Additional results on bacterial alpha diversity indices are also shown in Supplementary Figures 1, 2 for diets 1 and 2, respectively. The β-diversity principal coordinate analysis (PCoA) also showed that in each type of supplement, the bacterial community composition was closer to each other at 12 and 48 h but scattered at 0 h in both diets (Figure 4). This PCoA result showed that there is a community composition variation between the control and the YC, OA, and their interaction-supplemented treatments in both diets. However, there is no significant community variation between the lower and higher levels of each supplement. The YC, OA, and their interaction-supplemented treatments bacterial community composition were closer to each other without a significant difference caused by the level of YC and OA.

Bacteroidetes, Firmicutes, and Actinobacteria were the most dominant phyla in both diets. As shown in Table 5 and Figure 5A, in diet 1, supplementation with OA affected the composition and abundance of rumen bacterial phyla. The abundance of Bacteroidetes was significantly higher in the control treatment at 12 h. However, at 48 h, OA supplementation significantly increased the abundance of Bacteroidetes (p < 0.001). Neither YC supplementation alone nor YC and OA interaction had an impact on the abundance of Firmicutes. However, their abundance was reduced in treatments supplemented with OA (p < 0.001). The YC and OA interactions increased Actinobacteria phylum abundance (p < 0.001). The addition of lower levels of YC and OA significantly increased the abundance of the Spirochaetes and Verrucomicrobia phyla.

Adding YC and OA to diet 2 also increased the number of Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria, which are the most dominant phyla (p < 0.001; Table 6; Figure 5B). Specifically, treatments supplemented with OA alone showed a higher relative abundance of the Bacteroidetes phylum (p < 0.001). The lower levels of YC and OA supplementation significantly increased the abundance of the Bacteroidetes phylum (p < 0.001). The relative abundance of Firmicutes was also significantly increased with the addition of YC at both higher and lower levels (p < 0.001).

Prevotella, Selenomonas, Butyrivibrio, Succiniclasticum, and Ruminococcaceae_Ruminococcus were the dominant genera identified in both diets. The results of the bacterial genus composition in diet 1 are shown in Table 7; Figure 5C, and Supplementary Table 1. Hence, YC, OA, and their interaction increased the Prevotella genus abundance at 48 h (p < 0.001). The supplemented treatments also showed a significantly higher abundance of the Selenomonas genus at 48 h. However, the Butyrivibiro genus was higher in the control treatment. In diet 2, the Prevotella genus was found to have a higher abundance in the supplemented experimental treatments than in the control (p < 0.001; Table 8; Figure 5D; Supplementary Table 2). Selenomonas and Succiniclasticum were also the dominant genera, which were highly increased in the supplemented treatments (p < 0.001). The Butyrivibiro genus was higher in the control treatment. However, this genus was significantly increased in higher YC supplemented treatment at 12 h. As shown in Figure 5, the dominant bacterial genera were highly distributed in the supplemented treatments.

In ruminant nutrition, the optimal ruminal pH is an indicator of the rumen’s performance and the overall health of the animal (38). In modern profit-driven sheep production systems, it is common for ruminal pH to decrease and for subacute ruminal acidosis (SARA) to occur. Therefore, it is necessary to buffer and maintain an optimal ruminal pH in an intensive sheep production system. In both diets, YC and OA supplementation significantly increased the pH levels at different fermentation times. Similarly, reports have shown that YC might have a buffering effect on the rumen (39, 40). Nevertheless, studies examining the impact of OA have shown that it does not have a statistically significant influence on rumen pH (41). Therefore, supplementing with YC and OA may help to stabilize the rumen environment. This increase in ruminal pH may imply an improvement in fiber digestion, protein degradation, microbial activities, nutrient absorption, and overall rumen health.

Ruminants have a special stomach physiology that enables them to utilize different forms of nitrogen as protein sources. Ammonia nitrogen in the rumen is derived from the degradation of dietary protein and nonprotein nitrogen sources. It serves as an indicator of rumen internal performance, being continuously produced in the rumen, and is essential for microbes to synthesize microbial protein (42). In both diets, YC and OA interactions significantly increased NH₃-N concentrations at different fermentation times. However, supplementing with either YC or OA separately reduced NH₃-N concentrations in both diets. Similarly, Habeeb (43) found that YC supplements could lead to reduced NH₃-N. On the other hand, in lambs fed orchard grass, the addition of YC did not affect rumen NH₃-N (44). Studies have reported that YC supplementation reduces NH₃-N concentration in the rumen (11, 12, 45). This may be because YC supplementation depresses ammonia-producing microbes (46). The current finding, however, suggests that combining YC and OA supplements could have a positive effect on increasing NH₃-N levels, ultimately leading to an increase in microbial protein synthesis in the rumen. Moreover, supplementing YC and OA together might reduce the inhibitory effects of OA on NH₃-N in the rumen, which could be due to the deamination and proteolysis effects of OA (41). The higher ammonia nitrogen concentration resulted in increased microbial protein synthesis, improved nitrogen utilization, and enhanced dietary protein degradation.

Volatile fatty acids are the main energy sources and precursors for the production of animal products. They are produced through the bioconversion of diets in the rumen by anaerobic digestion (47). In both diets, supplementation with OA at lower and higher levels as well as the interaction between YC and OA significantly increased the production of acetic acid. This suggests that OA supplementation alone had a greater impact on acetic acid production than YC and their interactions. This indicates that OA may facilitate the degradation of fiber and increase nutrient availability for the host. However, at 24 and 48 h, the control produced more acetic acid. Supplementation with YC and OA showed significantly higher TVFA production compared to the control. Individual supplementation of YC and OA had a greater impact on TVFA production than their interactions. Although there is a lack of reports on the effects of OA, many researchers have reported that YC supplementation has positive effects on increasing acetic, propionic, and butyric acids (43, 48). Therefore, the combination of YC and OA might have a promising effect on boosting essential volatile fatty acid production. Furthermore, YC and OA supplementation significantly increased propionic acid production in both diets. However, the acetic acid to propionic acid ratio was significantly lower in the supplemented treatments in both diets. A study reported that YC supplementation could significantly increase propionic and TVFA production, but reduce acetic acid, resulting in a lower acetic to propionic acid ratio (49). Hence, YC and OA supplementation may enhance VFA production in the rumen by improving rumen functions. Increased volatile fatty acid production may lead to improved energy yield, enhanced rumen efficiency, better nutrient absorption, maintained pH levels, efficient fiber digestion, and increased microbial activities.

The rumen ecosystem contains diverse microbes, predominantly bacteria (50). Factors that affect the diversity of bacterial species include diet, dietary supplements, host species, and environmental factors (51). Species diversity can be measured using different alpha diversity indices, such as the Chao1 index and the Shannon and Simpson diversity indices, which quantify the richness of species in a community (52). In diet 1, the addition of OA alone, as well as the interaction of YC and OA, increased species diversity. This aligns with a previous study that found an increase in the richness and diversity of rumen microbiota when organic acids were added to high-grain diets (53). However, this partially contradicts an other study that found oxalic acid reduced the microbial diversity in sheep, but the diversity gradually returned to normal (41). YC and OA supplementation also affected the Shannon and Simpson diversity indices of the bacterial community. Treatments supplemented with YC and OA interactions showed significantly higher Shannon diversity index values. In diet 2, the YC and OA interactions significantly increased the Chao1 index values, indicating higher bacterial species richness. However, YC alone showed significantly lower Chao1 values. Supplementing YC and OA together significantly increased the Shannon and Simpson indices. This implies that YC and OA may have a synergistic effect as a supplement to enhance rumen bacterial richness and diversity, contributing to the overall stability and resilience of the rumen ecosystem. A diverse bacterial community can help the animal cope with different stresses and disturbances by maintaining various functions in the body. Furthermore, diverse bacterial communities may help the animal withstand different diseases and environmental impacts.

The composition of ruminal bacterial phyla varies across different parts of the rumen, however, Bacteroidetes and Firmicutes are the most dominant phyla (54). This result showed that Bacteroidetes comprised 45.06%, Firmicutes 37.39%, and Actinobacteria 11.70% of the total bacterial composition. Similarly, a study found that Bacteroidetes (40.95%) and Firmicutes (36.36%) are dominant phyla in sheep rumen (55). The main fiber fermenters such as Prevotella, Butyrivibrio, and Pseudobutyrivibrio are members of the phyla Bacteroidetes and Firmicutes (56), which are also efficient hemicellulose fermenters (50). In the first experiment (diet 1), the relative abundance of Bacteroidetes was higher in the control at 12 h; however, at 48 h, OA supplementation significantly increased the abundance, possibly due to the bacteria acclimatizing to oxalic acid. The abundance of Firmicutes was not affected. However, supplementation with OA reduced the relative abundance of Firmicutes. The interaction between YC and OA increased the abundance of the Actinobacteria phylum, which contains the crucial probiotic Bifidobacterium genus for gut health (7). Additionally, supplementation with YC and OA interaction significantly increased the abundance of the Spirochaetes and Verrucomicrobia phyla. The phyla Spirochaetes and Verrucomicrobia have been identified as possible producers and facilitators of enzymes involved in the degradation of complex polysaccharides such as xyloglucans, peptidoglycans, and pectin (57). In the second experiment (diet 2), supplementation of OA increased the abundance of the Bacteroidetes phylum. The YC and OA interaction also significantly increased the composition of this phylum. Both low and high levels of YC supplementation increased the abundance of Firmicutes. Furthermore, lower level of YC and higher level of OA, and lower levels of both YC and OA supplementation, also increased the abundance of the Firmicutes phylum. The supplementation of YC and OA also increased abundance of the Actinobacteria phylum. These results indicate that the interaction of YC and OA enhanced the composition of the rumen bacterial community at the phylum level. Overall, these findings suggest that the supplementation of YC and OA may have a promising effect on increasing the abundance of beneficial bacterial phyla which are primary fermenters of cellulose and hemicellulose (50, 56).

The dominant genera identified in the recent in vitro experiment were Prevotella, Selenomonas, Butyrivibrio, Succiniclasticum, Ruminococcaceae_Ruminococcus, Shuttleworthia, and Olsenella. In both diets, YC and OA supplemented treatments increased the abundance of the Prevotella genus. Individual supplementation with YC and OA also significantly increased the abundance of this genus. The Selenomonas and Succiniclasticum genera were dominant and showed a significant increase in the supplemented treatments. However, the Butyrivibrio genus was higher in the controls than in the supplemented treatments at 12 and 48 h in both diets. Prevotella strains have been found to ferment hemicellulose but exhibit differences in fermentation end products (58). It contains the Prevotella ruminicola species which plays a role in nitrogen metabolism and is also a fibrolytic bacteria that possesses carbohydrate esterases, facilitating hemicellulose degradation (59). The Butyrivibrio genus can also degrade hemicellulose (arabinoxylans) and pectin and produce butyrate (60). In both diets, the supplementation also showed a higher Selenomonas genus at 48 h. It contains the Selenomonas bovis species, which could ferment various sugars and produce acetate, propionate, and CO₂ as end products (61, 62). Studies have reported that Selenomonas enhances the proliferation of the microbial community in the rumen (63). Additionally, it encompasses several species that actively participate in the process of nitrate and nitrite reduction inside the rumen (64) and could facilitate propionic acid producing bacteria (65).

This in vitro experiment showed that adding YC and OA to hemicellulosic diets significantly altered the composition of beneficial rumen bacterial genera. This could be attributed to the creation of a favorable environment for bacterial growth and performance by the supplemented ingredients. As the pH results indicated, YC and OA supplementation increased the pH level of the ruminal fluid. Additionally, the ammonia nitrogen concentration increased in these experiments, suggesting that bacteria were able to obtain sufficient microbial protein, thereby facilitating their proliferation in the rumen. Hence, this may explain the significant increase in rumen bacteria at the genus and phylum taxonomic levels.

The correlation between the composition of ruminal bacteria and rumen fermentation parameters provides valuable insights into their roles in the rumen. The dominant genera that were highly abundant in the supplemented groups showed a significant and positive correlation with nutrient disappearance. Specifically, Prevotella, Selenomonas, and RFN20 were among the genera that were greatly increased with YC and OA supplementation and showed significant medium positive correlations with IVDMD in diet 1. Similarly, a report showed that Prevotella was positively correlated with digestibility (66). Another study also reported a positive correlation between Selenomonas and DM digestibility (67). In diet 2, Olsenella, Desulfovibrio, and Bifidobacterium showed a significant positive association with the IVNDFD. The IVCPD showed a significant medium positive correlation with Succiniclasticum and Desulfovibrio genera. The dominant genera also showed a positive correlation with the rumen fermentation parameters. The Succiniclasticum, Treponema, Desulfovibrio, and Sharpea genera showed a highly significant strong positive correlation with pH. Hence, these genera might be responsible for increased ruminal pH by facilitating fiber digestion. Succiniclasticum dextrinosolvens and Succinimonas amylolytica are prominent species that play a significant role in starch digestion and are responsible for succinate production. This succinate can then be transformed into propionate through the activity of Selenomonas ruminantium (68). Treponema enhances cellulose and hemicellulose breakdown (69). NH₃-N had a significant and strong positive correlation with the Olsenella, Bulleidia, and Oscillospira genera. However, it showed a significantly strong negative correlation with the Prevotella, Shuttleworthia, and Bifidobacterium genera. This correlation analysis showed that changes in the composition of the bacterial community may be responsible for the improved disappearance of nutrients and the production of rumen fermentation products including NH₃-N concentration and volatile fatty acids.