Fermented Total Mixed Ration Alters Rumen Fermentation Parameters and Microbiota in Dairy Cows

Simple Summary Fermented total mixed ration (FTMR) is an effective method of preserving high-moisture byproducts. However, few studies have investigated the effect of feeding FTMR on the rumen microbiota of dairy cows. This study aimed to determine changes and interactions of ruminal microbiota and chemical parameters in dairy cows fed FTMR. Compared with unfermented total mixed ration (TMR), FTMR improved bacterial and fungal richness and diversity. Meanwhile, the ruminal acetate and NH3-N concentration were increased. The prediction of the metabolic function of the flora showed that FTMR could increase lipid and amino acid metabolism. Abstract This study aimed to determine changes and interactions of ruminal microbiota and chemical parameters in dairy cows fed FTMR. Twelve multiparous Holstein dairy cows (Body weight = 616 ± 13.4 kg; day in milk = 106 ± 7.55 d; and parity = 2.31 ± 0.49; mean ± standard deviation) were divided randomly into two treatments depending on the day in milk, milk production, and parity. The two treatments were: (1) total mixed ration (TMR) and (2) FTMR. Illumina MiSeq sequencing was used to explore the changes in the ruminal microbiota. The results revealed that the bacterial and fungal diversity of the FTMR group were significantly higher than the TMR group. The predominant microbiota phyla in the bacteria and fungi showed significant differences between TMR and FTMR, as follows: Verrucomicrobia (p = 0.03) and Tenericutes (p = 0.01), Ascomycota (p = 0.04) and Basidiomycota (p = 0.04). The dominant bacterial genera in the bacteria, fungi, protozoan, and archaea that showed significant differences between TMR and FTMR were Unclassified_Bacteroidales (p = 0.02), Unclassified_RFP12 (p = 0.03), Candida (p = 0.0005), Bullera (p = 0.002), Cryptococcus (p = 0.007), and Ostracodinium (p = 0.01). LefSe analysis was performed to reveal the biomarker genera of the rumen microbiota community (bacteria, fungi, protozoan, and archaea) in the TMR and FTMR were the genera Shuttleworthia, Ruminococcus, Cryptococcus, Mycosphaerella, Bullera, Candida, and Ostracodinium. NH3-N concentration (p < 0.0001), total VFA concentration (p = 0.003), and molar proportion in total VFA of acetate (p = 0.01) were higher for the cows fed FTMR compared with the cows fed the TMR. Several bacterial genera showed significant correlations with rumen fermentation parameters. The genus Unclassified_Bacteroidales and Bullera were positively correlated with total volatile fatty acids (VFA) and acetate, whereas Candida and Ostracodinium showed negative correlations. Meanwhile, propionate was positively correlated with Candida and negatively correlated with Bullera. The PICRUSt functional profile prediction indicated that the xenobiotics biodegradation and metabolism, the lipid, amino acid, terpenoids, and polyketides metabolisms of the FTMR group were significantly higher than that of the TMR group. The results imply that FTMR can increase lipid and amino acid metabolism, and modulate the rumen microbiome and improve ruminal fermentation.


Introduction
Ruminants cannot effectively degrade feedstuffs independent of the vast ensemble of ruminal microbes containing bacteria, anaerobic fungi, ciliate protozoa, and archaea [1]. These microbial populations cooperate and contribute significantly to the breakdown of feed ingredients and the formation of volatile fatty acids, lactate, amino acids, lipids, and hydrogen, which are necessary for the maintenance, growth, and productivity of ruminants [2]. Regulation and optimization of the ruminal microbiota for a wide range of feeding conditions is a requirement for improving rumen function and feed efficiency [3][4][5].
Fermented total mixed ration (FTMR), which is prepared by mixing wet byproducts with dry feeds, has gradually become an effective method of preserving high-moisture byproducts [6]. The benefits of FTMR include (1) improving the population of lactic acid bacteria and concentration of lactic acid resulting in the silage pH to inhibit the growth of mold and yeast and achieving the objective of long storage time [7], (2) convenience for shortdistance transportation [8], and (3) the possibility for probiotics to be delivered via silage [9]. A study has reported that additives such as lactic acid bacteria or enzymes improved the fermentation quality of FTMR [10][11][12]. Lactobacillus buchneri or Lactobacillus plantarum can improve fermentation quality and the aerobic stability of silage [10,13]. Previous studies have shown that combining inoculations with enzymes improves silage fermentation quality by degrading fiber to water-soluble carbohydrates (WSC) for lactic acid bacteria (LAB) fermentation [12,14]. In addition, feeding FTMR in livestock has been evaluated, with the results indicating that FTMR increases nutrition digestibility and volatile fatty acid content compared to non-fermented total mixed ration (TMR) [15,16]. Some studies have reported that silage-inoculated LAB can improve animal performance by increasing milk yield and feed efficiency [17,18]. Previous studies indicated that the host's feed composition and feed intake pattern affect the rumen microbial composition [19][20][21]. Weinberg, Muck [22] demonstrated that silage inoculant LAB survived in rumen fluid in vitro. Some studies have reported silage inoculated LAB changed the ruminal bacterial community [23,24]. Different studies have shown interrelations between production variables, such as feed efficiency, milk production and composition, and the rumen microbiota [25,26]. However, there is little information about the interrelations between the rumen microbiota and rumen fermentation by feeding FTMR in dairy cows. We, therefore, hypothesized that the rumen microflora and chemical parameters would differ between FTMR and TMR. This research aimed to determine the effect of feeding FTMR on rumen fermentation parameters and microbiota in dairy cows to provide a better reference for the application of FTMR in dairy cows.

Animals, Experimental, and Design Diets
The animal use and care described in this study has been approved by the Animal Care and Use Committee of the Northeast Agricultural University (protocol number NEAUEC 20200236). The feeding experiment was conducted at the Songhuajiang Dairy Farm (Harbin, China). Twelve multiparous Holstein dairy cows (body weight = 616 ± 13.4 kg and day in milk (DIM) = 106 ± 7.55 d; and parity = 2.31 ± 0.49; mean ± standard deviation) were divided randomly into 2 treatments according to parity, DIM, and milk yield. The two treatments were (1) TMR (n = 6); (2) FTMR (n = 6). The experiment was conducted over 12 wk, of which the first 2 wk were used for adaptation. Cows were housed in separate stall barns that provided free access to drinking water, were fed twice daily (0630 and 1830 h) at 105% ad libitum intake, and milked twice daily at 0600 and 1800 h. The ration ingredient composition is shown in Table 1. Two diets were formulated to be isonitrogenous with a 50:50 ratio of forage to concentrate (dry matter basis) and to meet the dairy' s requirements for nutrition [27]. All feed ingredients were mixed with a horizontal feed mixer, and lactic acid bacteria (LAB, a combination of Lactobacillus plantarum and Lactobacillus buchneri applied at a ratio of 1:1) and fibrolytic enzymes (EN, XS Biotechnology Co., Ltd., Beijing, China; 10,000 U/g) were added to the FTMR. Additives were dissolved in water and sprayed evenly over the mixture with a sprayer. The FTMR packed in polyethylene stretch film with a density of 800 kg dry matter (DM)/m 3 were kept at ambient temperature at 16-30°C for 60 days. Samples were collected after 60 days by opening the packs and testing the chemical composition of the FTMR.

Rumen Sample Collection and Measurements
Rumen fluid samples (approximately 200 mL) were collected by an oral stomach tube at the end of the feeding period (12 wk) approximately 3 h after the morning feeding [28]. We discarded the first 100-200 mL of fluid collected through the stomach tube to minimize the possibility that the tube would be contaminated with saliva before collecting the rumen fluid sample. Each rumen sample weighed 5 mL, which was rapidly frozen in liquid nitrogen and kept at −80 • C for later DNA extraction. The pH was measured immediately with a pH meter (PB-10; Sartorius Co., Göttingen, Germany). The samples were centrifuged under 10,000× g, 4 • C, 20 min, and the supernatant was separated for the further analysis of NH 3 -N and VFA. NH 3 -N concentration was measured by an alkaline phenol hypochlorite method described in 143 Solorzano. The VFA concentration was decided by gas chromatography (GC-8A; Shimadzu Corp., Kyoto, Japan) as described by Hao, Gao [29].

Sequencing Data Processing
Using the MiSeq platform, the sequences were processed via the opensource software pipeline QIIME (version 1.8.0) [32]. Briefly, (1) the reads with an average quality score of no less than 25 and with a length of 220-250 nt were retained; (2) only sequences with a 10 bp or greater overlap were assembled using FLASH v1.2.7 based on their overlapping sequences; (3) raw reads were quality filtered under specific filtering conditions based on QIIME's quality control procedures (v1.8.0) to obtain high-quality clean tags. Sequences less than 160 bp in length or homopolymers longer than 8 bp were removed. Sequences were organized into operational taxonomic units (OTUs) following 97% identity using UCLUST [33], and by using USEARCH, chimeric sequences were identified and eliminated (v5.2.236, http://www.drive5.com/usearch/ accessed on 16 December 2022). The most abundant sequence in each OTU from specific libraries (bacteria, fungi, protozoa, and archaea) was selected as the "representative sequence" with an alignment against the SILVA bacterial database (version 119) [34], NCBI-nt protozoa database [35], Unite fungi ITS database (version 7.0) [36], and SILVA archaea database utilizing PYNAST [32], respectively, with the default parameters set by QIIME. Alpha diversity indices (Chao1 and Shannon) were calculated by QIIME from sparse samples using the abundance and diversity indices of the bacterial communities. Beta diversity was calculated using principal component analysis (PCA). Linear discriminant analysis (LDA) effect size (LefSe) analysis was performed on the Galaxy online analysis platform to reveal the significant ranking of abundant modules in TMR and FTMR groups [37]. A scale-effect threshold of 4.0 for the logarithmic LDA score was used to identify functional biomarkers.

Statistical Analyses
Statistical analysis of the data was performed by one-way analysis of variance (ANOVA) using the general linear model (GLM) of the SAS procedure (version 9.3, SAS Institute Inc., Cary, NC, USA). Pearson correlation coefficients were generated using the R software (http://www.r-project.org accessed on 16 December 2022). Significant correlations were declared at p ≤ 0.05.

Rumen Fermentation Parameter
See Table 2 for the results of the rumen fermentation parameter. The total VFA content of the FTMR group was higher than that of the TMR group. Compared with cows fed TMR, cows fed FTMR had a higher molar percentage of acetate in total VFA (p = 0.001). The molar ratio of butyrate was lower (p = 0.03) in the cows fed FTMR than in those fed the TMR. Neither the molar ratio of propionate nor the proportion of acetate to propionate was significantly different between TMR and FTMR (p = 0.19 and p = 0.12, respectively).

Sequence, Diversity, Species Richness, and Composition of Bacterial Microbiota
A total of 367,333 high-quality bacterial sequences were obtained from 6 TMR and 6 FTMR rumen samples, with an average of 27284-33184 (Mean = 30611 ± 484) sequences per sample. In total, 11,772 OTUs were identified, of which 2694 OTUs were found in two treatment groups ( Figure 1A). The Good's coverage in this study was close to 99%. Within sample groups, core bacterial communities are defined by the taxa shared by all individuals within those groups. As shown in Figure 2A, the observed species (OTUs) in TMR and FTMR. Cows fed FTMR demonstrated a higher species (p < 0.01) than cows fed TMR. As sequencing depth increases, the rarefaction curve at 97% similarity reveals an increase in OTU numbers. The ends of the sparsity curve tended to be flat with the increase in sequencing data ( Figure 2B).   The x-axis shows the observed species (OTUs), and the y-axis shows different groups (between TMR and FTMR). The OTU similarity threshold of 97% was considered. Boxes represent the interquartile range (IQR) between the first and third quartiles (25th and 75th percentile, respectively), and whiskers indicate the extremes of the data. * p < 0.05 and ** p < 0.01 (Student's t-test). Bacterial (B), anaerobic fungi (D), protozoan (F), and archaea (H) rarefaction curves of OTUs. The x-axis shows the number of valid sequences per sample, and the y-axis shows the observed species (OTUs). Each curve in the graph indicates a different sample and is shown in a different color. The longer the curve, the higher the sequencing depth and the more OTUs are observed. The flatter the curve, the more the sequencing results reflect the diversity contained in the current sample. TMR, total mixed ration; FTMR, fermented total mixed ration.
Analysis of the alpha diversity (Chao1 and Shannon) ( Figure 3A,B) showed that FTMR-fed cows had higher Chao 1 and Shannon values (p < 0.05 and p < 0.01, respectively) than TMR-fed cows ( Figure 3A,B). PCA analyses showed that rumen bacterial x-axis shows the observed species (OTUs), and the y-axis shows different groups (between TMR and FTMR). The OTU similarity threshold of 97% was considered. Boxes represent the interquartile range (IQR) between the first and third quartiles (25th and 75th percentile, respectively), and whiskers indicate the extremes of the data. ** p < 0.01 (Student's t-test). Bacterial (B), anaerobic fungi (D), protozoan (F), and archaea (H) rarefaction curves of OTUs. The x-axis shows the number of valid sequences per sample, and the y-axis shows the observed species (OTUs). Each curve in the graph indicates a different sample and is shown in a different color. The longer the curve, the higher the sequencing depth and the more OTUs are observed. The flatter the curve, the more the sequencing results reflect the diversity contained in the current sample. TMR, total mixed ration; FTMR, fermented total mixed ration.
Analysis of the alpha diversity (Chao1 and Shannon) ( Figure 3A,B) showed that FTMRfed cows had higher Chao 1 and Shannon values (p < 0.05 and p < 0.01, respectively) than TMR-fed cows ( Figure 3A,B). PCA analyses showed that rumen bacterial communities differed between TMR and FTMR ( Figure 4A). TMR and FTMR can be largely separated at the OTU level. Boxes indicate the interquartile range (IQR) between the first and third quartiles (25th and 75th percentile, respectively), and whiskers indicate the extremes of the data. Boxes with the different asterisk above their whiskers are significantly different (* p < 0.05 and ** p < 0.01, respectively) among treatments. TMR, total mixed ration; FTMR, fermentation total mixed ration. In total, 17 different bacterial phyla were recognized, and the top 10 phyla were identified based on the relative abundance of rumen bacteria present in Figure 5A. The relative abundance of bacterial phyla higher than 1% was considered predominant. Among these phyla, the predominant bacteria in the TMR and FTMR firstly with Firmicutes, followed by Bacteroidetes, Verrucomicrobia, Spirochaetes, Proteobacteria, Cyanobacteria, and Tenericutes. An analysis of the phyla showing significant differences between TMR and FTMR were Verrucomicrobia (p = 0.03) and Tenericutes (p = 0.01) ( Table 3). By analyzing the microbiota composition, 143 bacterial taxa were identified at the genus level, and 53 genera were observed in all samples. The relative abundance of the top 10 genera of rumen bacteria is displayed in Figure 5B. The bacterial genera with relative abundance >1% were considered to be dominant. Among these genera, the dominant bacteria in the TMR and FTMR were Prevotella, Unclassified_Clostridiales, Unclassified_Ruminococcaceae, Unclassified_Bacteroidales, Succiniclasticum, Ruminococcus, and Unclassified_Lachnospiraceae. The results showed significant genera differences between TMR and FTMR were Unclassified_Bacteroidales (p = 0.02) ( Table 3). In total, 17 different bacterial phyla were recognized, and the top 10 phyla were identified based on the relative abundance of rumen bacteria present in Figure 5A. The relative abundance of bacterial phyla higher than 1% was considered predominant. Among these phyla, the predominant bacteria in the TMR and FTMR firstly with Firmicutes, followed by Bacteroidetes, Verrucomicrobia, Spirochaetes, Proteobacteria, Cyanobacteria, and Tenericutes. An analysis of the phyla showing significant differences between TMR and FTMR were Verrucomicrobia (p = 0.03) and Tenericutes (p = 0.01) ( Table 3). By analyzing the microbiota composition, 143 bacterial taxa were identified at the genus level, and 53 genera were observed in all samples. The relative abundance of the top 10 genera of rumen bacteria is displayed in Figure 5B. The bacterial genera with relative abundance >1% were considered to be dominant. Among these genera, the dominant bacteria in the TMR and FTMR were Prevotella, Unclassified_Clostridiales, Unclassified_Ruminococcaceae, Unclassi-fied_Bacteroidales, Succiniclasticum, Ruminococcus, and Unclassified_Lachnospiraceae. The results showed significant genera differences between TMR and FTMR were Unclassi-fied_Bacteroidales (p = 0.02) ( Table 3). An analysis of LefSe was conducted to uncover the rumen bacteria community's biomarker genera in the TMR and FTMR in Figure 6A,B. The TMR group's most differentially abundant bacterial taxa belonged to the genera Shuttleworthia, whereas the genera Ruminococcus was more abundant in the FTMR group. The genera Shuttleworthia and Ruminococcus were the most differentiated among communities, with an absolute LDA score factor of four. other species were combined as "others". TMR, total mixed ration; FTMR, fermentation total mixed ration. Table 3. The diversity of main rumen microbiota that significantly changed in the phyla and genera level between the TMR and FTMR in dairy cows. (accounting for ≥1% of the total sequences in the abundance of top 10 genera in at least one of the samples).

Item
Phyla and Genera Level An analysis of LefSe was conducted to uncover the rumen bacteria community's biomarker genera in the TMR and FTMR in Figure 6A,B. The TMR group's most differentially abundant bacterial taxa belonged to the genera Shuttleworthia, whereas the genera Ruminococcus was more abundant in the FTMR group. The genera Shuttleworthia and Ruminococcus were the most differentiated among communities, with an absolute LDA score factor of four.  , and protozoa (F). Significant differences are defined as p < 0.05 and LDA score > 4.0. The histogram length represents the LDA score, i.e., the degree of influence of the species with the significant differences between the different groups. TMR, total mixed ration; FTMR, fermentation total mixed ration.

Sequence, Diversity, Species Richness, and Composition of Anaerobic Fungal Microbiota
A total of 586,464 high-quality rumen fungi sequences were acquired from six TMR, and six FTMR rumen samples, with 40,207-58,811 (Mean = 48,872 ± 1670) analyzed sequences (Mean length = 238.9 bp) in each sample. There were 1283 OTUs identified, among which 205 OTUs were found in two treatment groups ( Figure 1B). The Good's coverage in this study was close to 99%. Figure 2C shows the observed species (OTU) in TMR and FTMR, and the number of species in the TMR was lower than in the FTMR. FTMR-fed cows demonstrated a higher observed species (p < 0.01) than cows fed TMR. As sequencing depth increases, the rarefaction curve at 97% similarity reveals an increase in OTU numbers. The ends of the sparsity curve tended to be flat with the increase in sequencing data ( Figure 2D).
Analysis of the alpha diversity (Chao1 and Shannon) ( Figure 3C,D) and Beta diversity (PCA) ( Figure 4B) of the rumen fungi revealed variance in the microbial community. FTMRfed cows demonstrated higher Chao 1 and Shannon values than those TMR-fed cows. The PCA results demonstrated the diversity of the rumen fungi community and showed that TMR and FTMR could substantially be distinguished from one another at the OTU level.
In total, seven rumen fungal phyla were confirmed across all the samples. The relative abundance of all shared phyla in rumen fungi is displayed in Figure 5C. The relative abundance of fungal phyla higher than 1% was considered predominant. Among these phyla, the dominant rumen fungi in the TMR and FTMR were Ascomycota and Basidiomycota. An analysis of the phyla showing significant differences between TMR and FTMR were Ascomycota (p = 0.04) and Basidiomycota (p = 0.04) ( Table 3). Through analysis of microbiota composition, this study identified 244 fungal taxa and 100 genera observed across all samples. The relative abundance of the shared top 10 genera in rumen fungi is displayed in Figure 5D. The relative abundance of fungal genera with >1% was considered predominant. Among these genera, the dominant fungi in the TMR and FTMR were Candida, Pichia, Mycosphaerella, Trichosporon, Bullera, Ruminococcus, and Cryptococcus. A significant difference was found between TMR and FTMR as follows, Candida (p = 0.0005), Bullera (p = 0.002), and Cryptococcus (p = 0.007) ( Table 3).
LefSe analysis was conducted to uncover the biomarker genera of the rumen anaerobic fungi community in the TMR and FTMR in Figure 6C,D. The largest difference of fungi in the TMR group belonged to the genera Candida. Cryptococcus, Mycosphaerella, and Bullera were more abundant than that in the FTMR group. The genera Cryptococcus, Mycosphaerella, Bullera, and Candida, were the most differentiated among communities, with an absolute LDA score factor of 4.

Sequence, Diversity, Species Richness, and Composition of Ciliate Protozoal Microbiota
In total, 370,861 high-quality rumen protozoan sequences were obtained from 6 TMR and 6 FTMR rumen samples, and 27,043-35,796 (Mean = 30,905 ± 858) sequences (Mean length = 350.1 bp) were analyzed in each sample. A total of 2994 OTUs were identified, of which 1073 OTUs were found in two treatment groups ( Figure 1C). Good's coverage in this study was close to 99%. The core rumen protozoa communities are the bacterial population common to all group members. Figure 2E showed the species (OTUs) observed in the TMR and FTMR (OTU). The cows fed FTMR had no significant differences in observed species (p > 0.05) to cows fed TMR. As sequencing depth increases, the rarefaction curve at 97% similarity reveals an increase in OTU numbers. The ends of the sparsity curve tended to be flat with the increase in sequencing data ( Figure 2F).
Analysis of the alpha diversity (Chao1 and Shannon) ( Figure 3E,F) and Beta diversity (PCA) ( Figure 4C) of the rumen protozoan revealed variance in the microbial community. There were no significant differences in Chao 1 and Shannon values between cows fed FTMR and those fed TMR (p > 0.05 and p > 0.05, respectively). PCA analyses showed that the rumen protozoan community differed between TMR and FTMR. TMR and FTMR can be largely separated at the OTU level.
Rumen protozoal phyla were not identified across all the samples ( Figure 5E). By analyzing the microbiota composition, 13 protozoan taxa were identified, and 10 genera were observed across all samples. The relative abundance of shared the top 10 genera in rumen protozoan is displayed in Figure 5F. The relative abundance of protozoal genera higher than 1% was considered predominant. Among these genera, the dominant protozoan in the TMR and FTMR were Entodinium, Ostracodinium, and Ophryoscolex. A significant difference was found between TMR and FTMR in the abundances of Ostracodinium (p = 0.01) ( Table 3).
LefSe analysis was conducted to uncover the biomarker genera of the rumen ciliate protozoan community in the TMR and FTMR in Figure 6E,F. The TMR group's most differentially abundant ciliate protozoan taxa belonged to the genera Ostracodinium. No rumen ciliate protozoan community was not observed in the FTMR group. The genera Ostracodinium was the most differentiated among communities, with an absolute LDA score factor of 4.

Sequence, Diversity, Species Richness, and Composition of Archaeal Microbiota
A total of 389140 high-quality sequences in rumen archaea were acquired from six TMR and six FTMR rumen samples, and 27,065-37,010 sequences (Mean = 32,428 ± 999) were analyzed (Mean length = 287.3 bp) in each sample. There were 2059 OTUs identified, among which 712 OTUs were found in two treatment groups ( Figure 1D). The Good's coverage in this study was close to 99%. The taxa that all individuals shared were considered core rumen archaea communities within each sample group. The observed species (OTUs) in TMR and FTMR are shown in Figure 2G. The cows fed FTMR had no significant differences in observed species (p > 0.05) to cows fed TMR. As sequencing depth increases, the rarefaction curve at 97% similarity reveals an increase in OTU numbers. The ends of the sparsity curve tended to be flat with the increase in sequencing data ( Figure 2H).
Analysis of the alpha diversity (Chao1 and Shannon) ( Figure 3G,H) and Beta diversity (PCA) ( Figure 4D) of the rumen archaea revealed variance of the microbial community. There were no significant differences in Chao 1 and Shannon values between cows fed FTMR and those fed TMR (p > 0.05 and p > 0.05, respectively). PCA analyses showed that the rumen archaea community differed between TMR and FTMR. TMR and FTMR can be largely separated at the OTU level.
In total, seven rumen archaeal phyla were confirmed across all samples. The relative abundance of shared phyla in rumen archaea is displayed in Figure 5G. The relative abundance of archaeal phyla higher than 1% was considered predominant. Among these phyla, the dominant rumen archaea in the TMR and FTMR were Euryarchaeota and Firmicutes. There were 45 archaeal taxa identified at the genus level, and 16 genera were observed in all samples. The relative abundance of shared the top 10 genera in rumen archaea is displayed in Figure 5H. The phyla with greater than 1% relative abundance were classified as dominant phyla. Among these genera, the dominant archaea in the TMR and FTMR were Methanobrevibacter and Unclassified_Methanosphaera. An analysis of the phyla showing significant differences between TMR and FTMR was Methanobrevibacter (p = 0.046) ( Table 3). LefSe analysis revealed the biomarker genera for the rumen archaeal community in the TMR, and FTMR was not shown in the rumen.

The Correlations between the Relative Abundance of Predominant Ruminal Microbiota and Rumen Fermentation Parameter
A correlation analysis was performed to examine the relationship between the predominant ruminal microbiota and rumen fermentation parameters (Table 4). In rumen bacteria, the relative abundance of Unclassified_Bacteroidales had a positive association (p < 0.05) with total VFA and acetate molar proportion. In rumen fungi, total VFA, acetate, and propionate molar proportion were positively correlated with (p < 0.05) the relative abundance of Bullera, whereas evenness showed a negative correlation with (p < 0.05) the relative abundance of Candida. The relative abundance of Cryptococcus was positively related to (p < 0.05) the NH 3 -N and acetate molar proportion. In rumen protozoa, the relative abundance of Ostracodinium was negatively correlated with (p < 0.05) NH 3 -N, total VFA, and acetate molar proportion. In rumen archaea, the relative abundance of Methanobrevibacter and butyrate molar proportion displayed a negative correlation. Only the significantly affected ruminal microbiota (at the genera level) with a mean relative abundance >1% in the top 10 genera were considered in the correlation. * Significant (p ≤ 0.05) Pearson correlation coefficient.

Prediction of Different Treatment Functions of Rumen Bacteria
The functional gene composition between FTMR and TMR was inferred using the PICRUSt software by comparing the species composition information from 16S sequencing data. Differential analysis of KEGG metabolic pathways was used to analyze the differences and changes in the functional genes of microbial communities in the metabolic pathways between different groups, thereby evaluating metabolic changes in different rumen samples in response to environmental changes. In the present study, at KEGG level 2, a total of six KEGG metabolic pathways enriched significant differences between FTMR and TMR (p < 0.05) (Figure 7). In comparison with the TMR group, the xenobiotics biodegradation and metabolism, lipid metabolism, amino acid metabolism, and metabolism of terpenoids and polyketides of the FTMR group were higher (p < 0.05) (Figure 7). However, compared to the FTMR group, the biosynthesis of other secondary metabolites and energy metabolism of the TMR group were higher (p < 0.05), indicating that FTMR increases lipid and amino acid metabolism. of terpenoids and polyketides of the FTMR group were higher (p < 0.05) (Figure 7). However, compared to the FTMR group, the biosynthesis of other secondary metabolites and energy metabolism of the TMR group were higher (p < 0.05), indicating that FTMR increases lipid and amino acid metabolism. The middle shows the difference between the proportions of functional abundance in the 95% confidence interval, and the value at the rightmost is the p-value. p < 0.05 represents the significant difference. TMR, total mixed ration; FTMR, fermentation total mixed ration.

Rumen Fermentation Parameter
The current study looked at the interactions between rumen fermentation parameters and ruminal microbiota when TMR or FTMR were fed to animals. The result of this study was were in accordance with Cao, Takahashi [15], who also saw higher concentrations of NH3-N when FTMR was fed to sheep compared to non-fermented TMR. Ruminal NH3-N is the major source of N for microbial growth [38][39][40]. Cao, Takahashi [15] observed higher digestible energy in sheep that were fed fermented TMR compared with those fed nonfermented TMR. Thus, cows fed FTMR diets may have increased microbial crude protein synthesis and thus affected milk protein content [41,42]. However, this hypothesis needs to be tested by further studies. The total VFA concentration of the FTMR diet was The middle shows the difference between the proportions of functional abundance in the 95% confidence interval, and the value at the rightmost is the p-value. p < 0.05 represents the significant difference. TMR, total mixed ration; FTMR, fermentation total mixed ration.

Rumen Fermentation Parameter
The current study looked at the interactions between rumen fermentation parameters and ruminal microbiota when TMR or FTMR were fed to animals. The result of this study was were in accordance with Cao, Takahashi [15], who also saw higher concentrations of NH 3 -N when FTMR was fed to sheep compared to non-fermented TMR. Ruminal NH 3 -N is the major source of N for microbial growth [38][39][40]. Cao, Takahashi [15] observed higher digestible energy in sheep that were fed fermented TMR compared with those fed nonfermented TMR. Thus, cows fed FTMR diets may have increased microbial crude protein synthesis and thus affected milk protein content [41,42]. However, this hypothesis needs to be tested by further studies. The total VFA concentration of the FTMR diet was higher than that of the TMR diet. In this study, the total VFA concentration agreed with Cao, Takahashi [15] and Miyaji, Inoue [43]. This suggests that the consumption of smallmolecule carbohydrates can increase the VFA concentration. Although the increased VFA concentration in cows fed the FTMR diet, the difference in pH between the two diets was not significant. FTMR promoted the absorption of VFA by rumen epithelium [15]. The acetic acid concentration in cows fed the FTMR diet was due to improved in situ degradation of neutral detergent fiber (NDF) and silage processing in this study. Dairy cows fed easily fermentable carbohydrates also show a high propionic acid concentration, which agrees with the previous results [15]. In addition, ruminal lactate-utilizing bacteria play a key role in maintaining normal rumen fermentation, reducing the accumulation of lactate, and maintaining a normal rumen environment by converting lactate into propionate [15,44]. The ratio of acetate to propionate in both diets was far greater than 2.5, considered the threshold for milk fat reduction [45].

Diversity, Species Richness, and Composition of Bacterial, Anaerobic Fungal, Ciliate Protozoal, and Archaeal Microbiota
In this study, our results showed that cows fed FTMR positively affected bacterial richness (e.g., Chao1 value) and diversity (e.g., Shannon index). This was possibly due to this study confirming that providing more readily fermentable carbohydrates decreases bacterial richness and diversity. In the present study, regardless of which diet, Firmicutes and Bacteroidetes were the most dominant bacterial phylum in the cow rumen, confirmed by many former researchers [46][47][48]. The relative abundance of Verrucomicrobia significantly increased in the cows fed FTMR. This was observed in the rumen microbiota of goats after 4 weeks of medium non-fiber carbohydrates feeding [49]. Previous research indicated that Verrucomicrobia plays an important role in acquiring immune tolerance in mice gut [50,51]. However, further studies are needed to investigate the role of Verrucomicrobia in the dairy gut.
In this study, Prevotella, Unclassified_Clostridiales, Unclassified_Ruminococcaceae, Unclas-sified_Bacteroidales, Succiniclasticum, Ruminococcus, and Unclassified_Lachnospiraceae were the major bacteria, which were recognized by most past studies [48,[52][53][54]. The relative abundance of Unclassified_Bacteroidales significantly increased the cows fed FTMR over those fed TMR. LefSe analysis showed that biomarkers between the TMR and FTMR groups on the genus level constitute Shuttleworthia and Ruminococcus showed significant differences.
In the current study, feeding FTMR to cows positively affected the fungal abundance and diversity, probably because Ascomycota and Basidiomycota were the most dominant fungal phylum in cow rumen, whereas previous studies have reported similar findings [5,30]. Possibly because of the primers used for sequencing [30], several ruminal fungi were uncommon in former research [55,56]. Most of these phyla had been detected from marine samples (Jones et al., 2015) and were seldom found in ruminants. In the rumen ecosystem, it has yet to be discovered what metabolic and functional significance. Therefore, the reasons for decreasing Ascomycota and increasing Basidiomycota in FTMR-fed animals still need to be clarified. The differences in Candida, Bullera, and Cryptococcus genera between FTMR and TMR groups were judged to be significant. LefSe analysis showed that biomarkers indicating significant differences between the TMR and FTMR groups at the genus level included Cryptococcus, Mycosphaerella, Bullera, and Candida.
Entodinium, Ostracodinium, and Ophryoscolex were the most abundant bacterial community in the present study, which was similar to the previous studies [30,55,57,58]. Zhang, Shi [30] and Henderson, Cox [47] observed that Ostracodinium as the predominant bacteria in the rumen decreased linearly as the percentage of dietary concentrate level increased. Sandra, Henning [56] found that Ostracodinium was the major genera in ruminants fed silage. However, a relatively low abundance of Ostracodinium was ob-served in the FTMR group. LefSe analysis showed that biomarkers indicating significant differences between the TMR and FTMR groups at the genus level included Ostracodinium. Ostracodinium has a large capacity to degrade cellulose [58], explaining its relatively low abundance in FTMR, where there was a lower fiber content because of the addition of lactobacillus and fibrolytic enzyme [12]. Moreover, a previous study found that the total VFA and NH 3 -N concentrations were increased when sheep were fed an FTMR involved in whole grain rice and rice bran [15]. The relatively low abundance of Ostracodinium can be associated with the increased fermentation rate of FTMR and the concomitant decrease in pH [59,60].
In this study, Euryarchaeota, Methanobrevibacter, and Unclassified_Methanosphaera were the most predominant archaeal phylum and genus in the rumen, which was found in most previous studies [30,47]. In accordance with a comparative study [61,62], Methanobrevibacter was hydrogenotrophic and produced CH 4 by utilizing hydrogen or formate in ruminants. Total VFA (Table 2) in the rumen possibly resulted in the relatively high abundance of Methanobrevibacter in the FTMR group. At the same time, the relatively high abundance of Methanobrevibacter in the FTMR group revealed that the cows fed FTMR possibly increased CH 4 content, which was different from the observation of Cao, Takahashi [15]. Cao, Takahashi [15] observed that FTMR containing whole-crop rice and rice bran decreased ruminal methane emission, possibly because lactate-utilizing bacteria convert lactic acid or pyruvic acid to propionic acid in sheep. However, no increase in propionic acid and lactate-utilizing bacteria was observed in this study.

Correlations between the Relative Abundance of Predominant Ruminal Microbiota and Rumen Fermentation Parameter
The study found that Unclassified-Bacteroidales were positively correlated with acetate, an important end product of rumen metabolism. Zhu et al. [63] found that an increase in the abundance of acetate producing microbiota led to an increase in acetate content. Therefore, this result indicated that Unclassified-Bacteroidales enabled acetate production. Candida and Bullera belong to the yeast; however, we observed an opposite relationship with total VFA, acetate, and propionate. These results agree with the relative abundance of Candida and Bullera as well as total VFA, acetate, and propionate. Ostracodinium was negatively correlated with NH 3 -N, total VFA, and acetate. Moreover, the relative abundance of Ostracodinium in the FTMR group was significantly lower than in the TMR group. A previous study showed that Ostracodinium was not detected when fed a high-concentrate diet [60]. These results indicated that FTMR contained more soluble carbohydrates and thus promoted more VFA production from rumen fermentation. Methanobrevibacter was negatively correlated with butyrate. Methanobrevibacter is a major intestinal methanogenic archaeon that can grow on H 2 /CO 2 or formate. Meanwhile, some microbes can convert H 2 and CO 2 to butyrate in the gut [64]. Overall, the interactive analysis provided novel insights to elucidate the symbiotic relationships between rumen microbiota and metabolism.

Function Prediction of Rumen Bacteria
PICRUSt was used to understand the metabolic functions of rumen bacteria further. PICRUSt analysis indicated that comparing two dietary treatments simultaneously; we investigated the functional genes required for biodegradation and metabolism of xenobiotics, lipid metabolism, amino acid metabolism, and metabolism of terpenoids and polyketides of the FTMR group was significantly higher than that of the TMR group. This suggests that FTMR can increase lipid and amino acid metabolism. In addition, studies show that the lipid metabolism and amino acid metabolism of the high-feed efficiency group were higher than those of the low-feed efficiency group [65]. Meanwhile, a meta-analysis of 23 in vivo studies also demonstrated an improvement in feed efficiency, fat, and protein-corrected milk following supplementation with Agolin oil during periods longer than one month [66]. In this study, the lipid metabolism and amino acid metabolism of the FTMR group were significantly higher than that of the TMR group, which indicated that FTMR could increase feed efficiency. The improvement in feed efficiency will reduce feed resource consumption and lower feeding costs [67]. Therefore, feeding FTMR may be a better alternative strategy to improve the performance of lactating cows. Although the relatively high abundance of Methanobrevibacter in the FTMR group may have increased CH4 content, the higher feed efficiency may have mitigated methane emissions.

Conclusions
In summary, through high-throughput sequencing, the present study investigates impacts of FTMR on ruminal microbial communities. Compared with unfermented TMR, FTMR improved bacterial and fungal richness and diversity. The relative abundances of Unclassified_Bacteroidales, Bullera, and Cryptococcus were significantly increased, whereas Ostracodinium decreased in FTMR compared to TMR. Meanwhile, the ruminal acetate and NH 3 -N concentration were increased. Our results also showed positive correlations of total VFA, acetate with Unclassified Bacteroidales and Bullera, but negative correlations with Candida and Ostracodinium; propionate was positively correlated with Candida and negatively with Bullera. The PICRUSt functional profile prediction showed that the FTMR could increase lipid and amino acid metabolism. However, further experiments are required to gain a deeper understanding of the mechanisms involved.
Author Contributions: J.S. was responsible for formal analysis, investigation, methodology, software, visualization, Writing-original draft. H.Z. was responsible for investigation, methodology, software, visualization. Y.M. was responsible for investigation, methodology, software, visualization. Y.Z. was responsible for funding acquisition, methodology, project administration, resources, supervision, validation, writing-review and editing. G.Z. was responsible for conceptualization, data curation, methodology, project administration, supervision, validation, writing-review and editing. L.W. was responsible for methodology, visualization, writing-original draft. All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript.
Funding: This study was funded by the earmarked fund for CARS36, Heilongjiang Province Natural Science Foundation for Joint Guide Project (LH2022C030), the Young Talents" Project of Northeast Agricultural University (2021), and the Key Science and Technology Project of Henan (No. 222102110391).

Institutional Review Board Statement:
The animal study protocol was approved by y the Animal Ethics Committee of the Northeast Agricultural University (protocol number NEAUEC 20200236).

Data Availability Statement:
The Illumina sequencing raw data for our samples have been submitted to the NCBI Sequence Read Archive (SRA) under accession number: PRJNA870710. All raw sequence data in sequencing data processing were deposited to NCBI Sequence Read Archive under accession number SRP089832.

Conflicts of Interest:
The authors declare no conflict of interest.