pH in rumen fluid
After six weeks of HCD feeding, eight cows showed SARA-positive, which characterized as rumen fluid pH at 5.775 ± 0.10 (means ± SEM) (pH value < 5.8 was sustained for more than 3 h at different time periods in the cows fed with HCD for 6 weeks). The remained half animals showed SARA-negative, which featured as rumen fluid pH at 6.32 ± 0.09 (means ± SEM, Table 1).
Comparation of the rumen bacterial microbiota between SARA-resistant and SARA-prone cow prior to HCD feeding
Rumen fluid samples were gathered from eight Holstein dairy cows on day 0 (at the day beginning of HCD feeding) and on week six (six weeks after HCD feeding) to detect the bacterial community of rumen fluid using the V4 region of the bacterial 16S ribosomal RNA (rRNA) gene amplified PCR. Of the pyrosequencing reads that passed the quality control tests, total 2,809,712 reads, with an average of 87,830 reads per samples in rumen fluid. A species accumulation boxplot of 32 samples indicated that sampling depth had sufficient sequences to characterize the majority of bacterial communities (see supplementary fig. S1). To explore whether the susceptibility of SARA was associated the rumen microbiota in cows, we analyzed the bacterial community in rumen fluid from SARA-resistant and SARA-prone cows before feeding with HCD. Chao 1, and ace analysis showed that the bacterial richness of SARA-resistant were higher than in the SARA-prone cows (Fig. 1A-B). Shannon index and Simpson’s index of the SARA-resistant and SARA-prone cows showed no significant difference in bacterial community diversity between the two groups (Fig. 1C-D).
At the phylum level the results indicated that phyla Proteobacteria, Firmicutes, Bacteroidete, Tenericutes, and Spirochaetes were the most abundant among rumen fluid from different treatment group cows (Fig. 1E). At the genus level, the relative abundance of Stenotrophomonas, unidentified_Ruminococcaceae, Mycoplasma, unidentified_Prevotellaceae, unidentified_Rikenellacea, Succiniclasticum, Succinivibrio, and unidentified_Bacteroidales were the eight most prevalent genera in rumen fluid from different treatment group cows (Fig. 1F). In addition, t-test analysis was used to identify differences in bacterial genera of rumen fluid between SARA-resistant and SARA-prone group cows, and the data showed unidentified_Spirochaetaceae and Anaeroplasma were significant higher in SARA-prone group than those in SARA-resistant group cows (Fig. 1G).
Ven diagram was used to estimate whether a unique bacterial microbiota was associated with the development of SARA. As shown in Figure. 2A-B, most genera were shared between SARA-resistant and SARA-prone group cows. Core genera accounted for 66.92% of all rumen bacteria in SARA-resistant group and 75.48% in SARA-prone group cow (Fig. 2B). Since most bacterial genera were shared between SARA-resistant and SARA-prone group cows, it is likely that changes in bacterial relative abundance are more important for the susceptibility of SARA than unique differences in bacterial communities. Furthermore, the LEfSe was used to provide biomarkers at the genus-level with a linear discriminant analyses (LDA) also detected that unidentified_Spirochaetaceae and Anaeroplasma genus were enriched in SARA-prone cows (Fig. 2C-D). These results suggested that the relative abundance of unidentified_Spirochaetaceae and Anaeroplasma in rumen maybe associated with the susceptibility of SARA in cows.
The changes of rumen bacterial microbiota in SARA-resistance and SARA-prone cows before and after feeding with HCD
The α-diversity of the rumen bacterial community showed that microbiota richness had reduced in SARA-resistance before and after feeding with HCD for six weeks (Fig. 1A-B). In addition, the difference of rumen microbiota in SARA-resistance cows before and after feeding with HCD identified that the Saccharofermentans genus were increased in HCD feeding cows (Fig. 3A and 3D-E).
Furthermore, the richness and diversity of rumen fluid bacterial community from SARA-positive cows was significantly declined after feeding with HCD (SARA-prone vs SARA-positive cows) (Fig. 1A-D). Comparation of phyla and genus levels showed that Proteobacteria phyla and the genus of Stenotrophomonas, Sphingomonas, Delftia were significantly increased, while the relative abundance of phyla Fimicutes, Bacteroidetes, Spirochaetes, Fibrobacteres, and Kiritimatiellaeota, and the genus of unidentified_Ruminococcaceae, unidentified_Rikenellaceae, unidentified_Lachnospiraceae, unidentified_Bacteroidales,Succiniclasticum, unidentified_Spirochaetaceae, Saccharofermentans, Anaeroplasma, Papillibacter, Fibrobacter, and Anaerovorax in rumen fluid were significantly decreased in SARA-positive cows before feeding with HCD when it compared to post HCD feeding (Fig 3A-C). Moreover, LEfSe analysis showed that genus Stenotrophomonas, which belongs to family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria, and phyla Proteobacteria, was identified as biomarkers in group SARA-positive cows (Fig. 3F-G).
Comparation of the rumen bacterial microbiota between SARA-resistant and SARA-prone cow after feeding with HCD
In order to compare the rumen bacterial microbiota between SARA-negative and SARA-positive cows, we analysis the rumen microbiota of SARA-resistance and SARA-prone cows after feeding with HCD. Chao 1, ace, simpson and shannon index showed that the bacterial community richness and diversity of rumen microbiota from cows of SARA-negative were higher than that in SARA-positive cows (Fig. 1A-D).
The difference of rumen bacteria in SARA-negative and SARA-positive cows were further detected at phylum and genus levels. The results showed that the relative abundance of Proteobacteria phyla, Stenotrophomonas and Sphingomonas genus were increased, while the relative abundance of Bacteroidetes and Firmicutes phyla and unidentified_Lachnospiraceae genus were reduced in rumen fluid from SARA-positive compared to the SARA-negative cows (Fig. 4A-B). Furthermore, LEfSe analysis to identity the biomarkers between SARA-negative and SARA-positive cows showed the phyla Proteobacteria, class Gammaproteobacteria, order Xanthomonadales, family Xanthomonadaceae, and genus Stenotrophomonas were significantly enriched in SARA-positive group cows (Fig. 4C-D).
Comparation of the rumen fungal microbiota between SARA-resistant and SARA-prone cow before feeding with HCD
For V4 amplicons, a total 2,922,553 reads for all 32 rumen samples were obtained by high-throughput sequencing (HTS), and with the average number of reads per rumen samples was 91,329. A species accumulation boxplot for a satisfactory sequencing depth to analyze the core microbiome (see supplementary Fig. S2). Comparation of α-diversity between SARA-resistance and SARA-prone cows showed that the ruminal fungi richness has no significant difference (Fig. 5A-B), while ruminal fungi diversity was obviously higher in SARA-prone cows compared to the SARA-resistance cows (Fig. 5C-D).
Further analyzed indicated that ruminal fungi population was dominated by Ascomycota, Neocallimastigomycota, Basidiomycota, and Mortierellimycota phylum among different treatment groups (Fig. 5E). Moreover, the relative abundance of Meyerozyma, Byssochlamys, Fusarium, Candia, Sarocladium, Penicillium, Wallemia, Orpinomyces, Acermonium, Aspergullus, Talaromyces, Plectosphaerella, Cladosporium, Alternaria, Passalora, Phoma, Papilootrema, Stephononectria, and Pseudeurotium were the top 20 genu in the rumen fluid of different treatment group cows (Fig. 5F). In addition, among these fungi, the abundance of Saroclasium was higher, while the abundance of Aspergillus, and Papiliotrema were lower in SARA-prone cows than those in SARA-resistance cows (Fig. 5G).
Venn showed that most fungal genera were shared between SARA-resistant and SARA-prone cows (Fig. 6A-B). Of the OTUs, about 69.57% in SARA-resistance cows, and 60.72% in SARA-prone cows obtained from the core fungal microbiota (Fig. 6B). Furthermore, the LEfSe was to identify the abundance of fungi taxa with sequences in rumen fluid between SARA-resistance and SARA-prone cows. The results showed that the Aspergillus genus, Caecomyces genus, Papiliotrema_Laurentii species, and Alternaria_alternata species were enriched in SARA-resistance group cows, while the Komagataella_pastoris, Sarocladium_zeae, and Fusarium_oxysporum were enriched in SARA-prone group cows (Fig. 6C-D). These results suggested that Papiliotrema_Laurentii, Alternaria_alternata, Komagataella_pastoris, Sarocladium_zeae, and Fusarium_oxysporum in rumen maybe associated with the susceptibility of SARA in cows.
The changes of rumen fungal microbiota in SARA-resistance and SARA-prone cows before and after feeding with HCD
Comparison of rumen fungi richness and diversity among SARA-resistance, SARA-prone, SARA-positive, and SARA-negative cows demonstrated that feeding with HCD for 6 weeks significantly reduced the rumen fungi richness and diversity both in SARA-resistance and SARA-prone cows (Fig. 1A-D). The differences of rumen fungal based on t-test and LEfSe analysis showed that Ascomycota phylum, Penicillium genus, Sarocladium genus, Sarocladium_zeae species, Meyerozyma genus, Meyerozyma_caribbica species, Fusarium genus, Fusarium_oxysporum species were increased, while Wallemia genus, Wallemia_sebi species, Plectosphaerella genus, Acremonium genus, Papillotrema genus reduced in both SARA-resistance and SARA-prone cows (Fig. 7A-H).
Comparation of the rumen fungal microbiota between SARA-resistant and SARA-prone cow after feeding with HCD
The estimators of fungi community richness and diversity of rumen fluid in SARA-positive cows were obviously reduced compared to the SARA-negative cows after feeding with HCD for six weeks (Fig. 1A-D). Moreover, t-test and LEfSe both indicated that the levels of genus of Fusarium was reduced, while the relative abundance of Pseudeurotium was increased in SARA-negative cows compared to the SARA-positive cows. In addition, the Cephalotrichum_nanum, Colletotrichum_gloeosporioides, Blumeria_graminis, Scopulariopsis_brumptii, Penicillium_citrinum, Monographella_nivalis, and Talaromyces_funiculosus species were both enriched in SARA-negative cows compared to the SARA-positive cows (Fig. 8A-C).