In order to conduct the SARA challenge, the dietary fibre content was reduced and that of starch increased. Hence, it was expected that this challenge would reduce abundances of fibrolytic and pH sensitive bacteria and increase those of amylolytic and pH tolerant bacteria, as well increase those of bacteria that utilize intermediates of starch fermentation [1, 2, 4]. In a parallel study , we showed that, across SCFP treatments, the SARA challenge increased the duration of the pH below 5.6 and reduced the acetate to propionate ratio in the rumen from 11.1 to 311.1 min/d, and from 3.07 to 1.74, respectively. The threshold used for SARA that was used in our study was a rumen pH depression below 5.6 of 180 min/d [1, 2]. Based on this threshold, SARA was induced successfully. This threshold was developed on the assumption that microbial enzymes and the growth of microorganisms in the rumen are sensitive to this pH depression, and that a more severe rumen pH depression increase the concentrations of serum amyloid A (SAA) in peripheral blood plasma and lipopolysaccharide endotoxin (LPS) in rumen fluid [1, 4]. In agreement, the parallel study also showed that, in the absence of SCFP supplementation, the SARA challenge increased the concentrations of SAA from 72.5 to 209.9 μg/mL and that of rumen LPS from 15,389 to 123,296 EU/mL. This endotoxin is shed by gram-negative bacteria, and can cause inflammation and disruption of the barrier function of the rumen epithelium [1, 4]. Hence, this increase in rumen LPS is additional evidence of the adverse effects of the SARA challenge on rumen bacteria.
The observed reductions in the microbial richness and diversity in the rumen during the SARA challenge agree with earlier research that shows that SARA compromises the functionality of the rumen microbiota [3, 10, 11]. Also, in agreement with previous studies [3, 10, 11], our gene sequencing data demonstrated that Bacteroidetes, Firmicutes and Proteobacteria were the dominant ruminal phyla. Members of the Bacteroidetes phyla are considered more efficient fermenters of complex polysaccharides, compared to Firmicutes, and thus, represent the primary degraders for these compounds in the rumen . As such the reduction in the relative abundance of Bacteroidetes during the SARA challenge may be undesirable. Reductions in this abundance of Bacteroidetes and increases in the relative abundances of Firmicutes and Proteobacteria due to excessive grain feeding and grain-based SARA challenges have been reported previously [3, 13]. However, in contrast to earlier studies, our gene sequencing data did not show that the SARA challenge increased the relative abundances of Firmicutes [3, 10, 14] and Proteobacteria . However, in contrast with our findings and using a similar sequencing technology, Fernando et al.  observed that switching cows from a low grain to a high grain diet increased the relative abundance of Bacteroidetes, but did not affect that of Proteobacteria. These discrepancies among studies may result from differences in the amount of dietary grain, and therefore starch, in the control and high grain treatments, as large differences between these treatments may result in larger responses. Responses to such increases also vary among cows. This was shown by Khafipour et al. , who demonstrated that a large increase in grain feeding increased the relative abundance of Firmicutes in the rumen in cows that responded severely to a grain-based SARA challenge, but not in cows that responded moderately to this challenge.
The ordination plots of the microbiome beta-diversity based on 16S rRNA gene sequencing and shotgun metagenomics also demonstrated that the SARA challenge affected the microbiological beta-diversity of the rumen.
The effects of the SARA challenge on the abundances of bacterial phyla determined with shotgun metagenomics differed from that determined by 16S rRNA gene sequencing. In contrast to the 16S rRNA gene sequencing data, the metagenomic data did not show effects of the SARA challenge, the SCFP treatment and their interaction on the abundances of Bacteroidetes or Proteobacteria. These discrepancies between methods may be due to differences in sequencing targets, biases and coverage, including unequal amplification of species’ 16S rRNA genes, insufficient depth of metagenomic sequencing for the identification of low abundance species, and insufficient taxa-specific microbial reference genome sequences between the techniques [16,17,18]. As a result, Shah et al.  recommended that, until sufficient reference sequences are available, metagenomic shotgun analysis needs to be combined with 16S rRNA gene sequencing to obtain sufficient accuracy on the effects of dietary treatments on the composition of gut microbiota.
In the rumen, Bacteroidales and Prevotella were the most abundant taxa within Bacteroidetes and several genera within Ruminococcaceae were the most abundant genera within Firmicutes, which agrees with previous studies [3, 10, 13]. The proportion of the rumen genera affected by the SARA challenge was higher in our study than that in earlier studies, and there is no agreement among studies on which genera are the most affected by a SARA challenge [10, 11]. Reasons for this discrepancy may be that the rumen pH depression in our study was greater than in previous studies, and that the response of rumen genera to grain challenge is highly variable. Nevertheless, our study and earlier studies agree that the number of genera affected by SARA challenges is limited.
Our study, as well as earlier studies using qPCR, agree that SARA challenges affect the populations of many species of rumen bacteria. Whereas in agreement with previous studies [3, 10, 13], the SARA challenge increased the populations of starch and sugar utilizing bacteria, this challenge only reduced the populations of fibrolytic bacteria in the absence of SCFP. The reduction in the populations of fibrolytic bacteria in the absence of SCFP agrees with earlier studies [3, 13, 15]. Hence, at the species level, the changes in the size of these populations appear to reflect changes in available substrates. However, as rumen bacteria compete for substrates and share functionality, assessing the populations of only a selected few species of rumen bacteria may not provide a comprehensive overview of changes in the species composition of rumen microbiota [10, 11, 19].
The analysis of the relative abundances of metabolic functions by shotgun metagenomic sequencing showed that the SARA challenge increased or tended to increase protein metabolism (P = 0.08), and metabolism of aromatic compounds (P = 0.04). The challenge tended to decrease nitrogen metabolism (P < 0.1) and increased sulphur metabolism (P < 0.05) in the absence of SCFP supplementation. In the rumen, nitrogen metabolism primarily involves microbial proteolytic activity [20, 21]. The challenge only decreased phosphorus metabolism (P < 0.05) when SCFP were supplemented. This activity of proteolytic microbes depends on the chemical structure of dietary proteins, the rumen pH, and the predominant proteolytic species of bacteria present in the rumen . Thus, the influence of the SARA challenge on nitrogen metabolism in the rumen may be a consequence of changes in the abundance of proteolytic bacteria, which may be driven by a reduction of the rumen pH .
The impact of the reductions in the abundances of Paenibacillaceae and Spirochaetaceae by the SARA challenge is unclear. The family Spirochaetaceae is associated with diseases in ruminants with the rumen and feces being considered a reservoir for these microbes . The family Paenibacillaceae are organoheterotrophic, and utilize carbohydrates as well as amino acids .
The reductions of the abundances of the genes coding for the enzymes MCM and FDH that resulted from the SARA challenge may impair the functionality of rumen microbiota. The enzyme FDH catalyzes the oxidation of formate to CO2, and has been found in bacteria  and hydrogenotrophic methanogenic archaea . As a result, many species of these archaea utilize formate instead of H2. A reduced expression of FDH may, therefore, enhance H2 utilization, and, lessen the decline in pH during a SARA challenge. In contrast, MCM catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA, and is therefore, part of key metabolic pathways . In humans, it is the first vitamin B12-depedent enzyme, and a deprivation of its activity results in metabolic acidosis and methylmalonic acidemia . Little is known about the role of this enzyme in ruminants, but due to its critical role in metabolism, a reduced expression of the MCM gene may be considered adverse.
The SARA challenges resulted in many changes in the composition and predicted functionality of the rumen microbiota. However, it is difficult to conclude if these changes are normal adaptations to a higher grain diet, or if these changes affect the health of dairy cows adversely. However, as SARA challenges reduce feed intake, fibre digestion, and milk fat production, and increase in bacterial endotoxins in rumen digesta and blood plasma, as well as markers of inflammation [1, 4, 10], it may be assumed that, overall, these changes of the rumen microbiota are adverse.
In both the in vitro and the in vivo trial, the qPCR data demonstrated that SCFP increased the populations of major fibrolytic and amylolytic bacteria, suggesting that the supplementation of SCFP is beneficial to the utilization of carbohydrates in the rumen . In the in vivo trial, the populations of F. succinongenes and R. flavefaciens increased more under the depressed pH than under the high pH, which is beneficial as the populations of these bacteria are reduced by high grain feeding [3, 10, 13]. However, the increases in F. succinogenes, and M. elsdenii observed in vitro, were not evident in vivo. These discrepancies may have been due to the higher concentrations of cellulolytic bacteria and lower protozoal concentrations in the in vitro study. Despite the similarity of the qPCR method that was used, our in vivo qPCR results differ from those of Mullins et al. , as these authors did not observe effects of SCFP supplementation on the populations of selected species and genera of rumen microbiota. These authors suggested that the lack of these effects may be due to the incomplete coverage of the rumen microbiota by their qPCR analysis, and that the functionality, rather than the taxonomic composition of rumen microbiota, was altered by the SCFP supplementation. Other reasons for this discrepancy may include differences in the main grain source between the studies. Whereas dry-rolled barley grain was used in our study, high moisture corn was the main grain source in the earlier study. The higher rumen degradability of high moisture corn compared to dry-rolled barley grain may have resulted in differences in the rumen environment between these studies. This discrepancy highlights that factors other than the dietary starch content affect the taxonomic composition of rumen microbiota as determined by qPCR.
Our in vivo qPCR data also showed that SCFP supplementation increased the populations of Bifidobacterium spp. and ciliate protozoa. Bifidobacterium possess unique pathways to ferment non-structural carbohydrates . Hence, an increase in the population of these bacteria will enhance rumen fermentation, especially when high-grain diets are fed. Protozoa stabilize the rumen environment by modulating bacterial metabolism and reducing the rate of fermentation of starch . Hence, the attenuation of the SARA-induced reduction of the population of ciliate protozoa in the rumen due to supplementation with SCFP benefits rumen function during high grain feeding.
Many results of our study showed that the impacts of the SARA challenge on the composition and functionality of rumen microbiota were attenuated by the supplementation with SCFP. These effects of SCFP include limiting the SARA-induced reductions in the richness and diversity and the changes in the beta-diversity of rumen microbiota. Members of the rumen microbiota vary in their preferred substrates and functionality . Hence, a larger richness and diversity of rumen microbiota allows more efficient use of resources under different conditions, including during nutritional challenges [2, 3, 29]. Hence, the limitation of the SARA-related drop of the richness and diversity of the rumen microbiota due to SCFP supplementation is beneficial to the health and production of the host cows.
Despite of the SARA-mitigating effects of SCFP, including effects on the abundances of several species of rumen bacteria, this supplementation only reduced the impact of SARA on one bacterial phylum, i.e. Bacteroidites, and one bacterial genus, i.e. unclassified Bacteriodales. Also, the abundance of only one genus, i.e. other Firmicutes, tended to be increased by SCFP supplementation. This raises the question why SCFP supplementation has so many more effects on the species than on the phylum and genus levels. This may, as suggested by Mullins et al.  be due to an incomplete coverage of the rumen microbiota in the qPCR, analysis, but other differences between qPCR and 16S rRNA sequencing may also play a role. Hence, it would be beneficial to confirm the qPCR results with 16S rRNA sequencing, but unfortunately the latter technique is currently not sufficient accurate for taxa identification at the species level.
Our results at the genus level, also contrasts recent findings that reported a decline in Prevotella and an increase in Butyrivibrio with SCFP supplementation in calves . Yet, despite the proteolytic activity of Prevotella and butyrate producing activity of Butyrivibrio, no differences in the rumen concentrations of VFA were observed due to SCFP supplementation in their study and in our parallel study . The discrepancy could possibly be driven by differing primers between the two sequencing studies (V1-V3 compared to V3, respectively), animal age and basal diet, as the calves were still receiving a milk-based diet in the study of Xiao et al. .
At the microbial functionality level, our shotgun metagenomic analysis showed that, in the absence of SCFP, the potential metabolisms of phosphorus and sulphur increased and that of nitrogen tended to decrease during the SARA challenge. This decrease in nitrogen metabolism may be related to a reduced proteolytic activity from rumen microbiota [31, 32]. The effects of increases in phosphorus and sulphur metabolism in the rumen translated to changes in functionalities at the level of the host cow. However, as most changes in functionalities resulting from the SARA affect the production and health of cows negatively, it may be assumed that these changes in microbiome functionalities are adverse, and that the SCFP mitigating effects on these functionalities are, therefore, beneficial.
Supplementation with SCFP only increased the abundances of genes encoding for FDH and MCM during the SARA challenge. Increased expression of FDH may, therefore, enhance H2 utilization, and, potentially lessen the decline in pH during a SARA challenge . Little is known about the role of MCM in ruminants, but due to its critical role in metabolism, an increased expression of the MCM gene may be considered beneficial to cows .
The reductions in the abundances of Paenibacillaceae and Spirochaetaceae caused by the SARA challenge were also attenuated by SCFP supplementation. The impact of the SARA-mitigating effects of SCFP on the abundances of these families is unclear. Supplementation with SCFP also reduced the abundances of Methanobacteria and unclassified Euryarchaeota during the SARA challenge, but increased these abundances in the absence of SARA. As these taxa include methanogens , their reduction has the potential to enhance the energetic efficiency of ruminal fermentation.