In recent years, the microorganisms residing in the gut of multicellular organisms, termed gut microbiome, have been shown to play an important role in their host's physiology [1], [2]. In mice and humans, a link has been demonstrated between the gut microbiota and the physiological features of energy-harvesting abilities, with genetically predisposed obese mice exhibiting a different ratio of the phyla Firmicutes to Bacteroidetes [3], [4]. The transfer of microbiota from obese mice to lean mice resulted in significant physiological changes in the latter, related to tissue adiposity, suggesting a causative effect of the microbiota on its host's physiology. The conclusion was that the “obese” microbiome has an increased capacity to harvest energy from the diet [4]. Recently, gnotobiotic mice colonized with obese human microbiota exhibited increased adiposity, but when these same mice were exposed to lean human microbiota transplanted in the same manner, the obese microbiota was invaded by bacterial components of the lean microbiota, causing a decrease in the obese phenotype [5].

The bovine rumen houses a complex microbiota that is responsible for cattle's ability to convert indigestible plant mass into energy [6]. This ability is of tremendous importance for mankind, as domesticated animals are a crucial intermediate between light energy harvested via photosynthesis and the production of digestible compounds such as milk and meat [7], [8]. The rumen functions as a pregastric anaerobic fermentation chamber inhabited by a highly dense microbial community composed of microorganisms from all domains of life, 95% of which are bacteria [9]. Fermentation products of rumen microbial activity—mainly volatile fatty acids—serve as a major source of energy for the animal [7], [8]. These fermentation products have a direct effect on the animal's physiological parameters, such as milk composition [10]. Using denaturing-gradient gel electrophoresis (DGGE), a connection between volatile fatty acid composition and rumen bacteria was suggested, by examining microbial differences between efficient and inefficient cows [11]. High inter-animal variation exists in the stability of the rumen microbiome [9], [12], but intra-animal variation is quite low [13]. Therefore, it is tempting to speculate that these inter-microbiome variations might be linked to the physiological parameters of their individual hosts. In the present study, by analyzing the whole rumen bacterial communities of 15 dairy cows—previously sequenced in our lab, and comparing them to the cows' production parameters and milk composition, we explored the possible link between bacterial components of the rumen microbiome and the physiological parameters of the animal host during lactation.

Our aim in this study was to determine whether there are any correlations between the bacterial community residing in the cow rumen and the physiology of the individual cow hosts. We analyzed 15 lactating dairy cows, whose ruminal bacterial communities had been previously pyrosequenced [12], under a high-energy diet (Table S1). Their rumen fluid was sampled during lactation and their physiological parameters were recorded and calculated. These included milk yield, milk content (carbohydrate, protein, and fat), pH, dry matter intake (DMI) and RFI, which serves to evaluate the animal's feed efficiency (Table S2). After quality-filtering based on length (<200 bp) and quality of the reads, we obtained 141,344 reads averaging 338 bp each (Table S3). Overall, 17 phyla were detected, but only 7 were found in all cows (Figure 1A). The three dominant phyla observed, in agreement with all studies of mammalian gut microbiota, were Bacteroidetes, Firmicutes and Proteobacteria, as previously described and reported in other mammalian gut studies [2], [12], [26]. However, there was a large variation in the abundance of the two main phyla—Bacteroidetes and Firmicutes—between the different animals [13]. Although Bacteroidetes was more abundant in most of the samples, some exhibited a higher percentage of Firmicutes compensating for a lower abundance of Bacteroidetes (Figure 1B). The ratio of Firmicutes to Bacteroidetes has been shown to affect energy harvesting and body fat in humans and mice [3], [4]. We therefore examined whether cattle physiological parameters correlate with a change in this ratio. The Firmicutes-to-Bacteroidetes ratio was found to be strongly correlated with daily milk-fat yield (Pearson R = 0.72, P = 2×10⁻³) (Figure 2). This finding mirrors that in mice, where a decreased amount of Bacteroidetes in the microbiota was correlated with increased fat in the blood and tissue [4].

We then compared the physiological parameters with the microbiota at the genus level. To confirm adequate sequencing depth for these analyses, we generated rarefaction curves for each sample as a function of the number of observed OTUs (OTU≥94%, defined as genus level) and found our coverage to be sufficient for further analyses at the genus level (Figure S1). Overall, 151 genera were detected in our samples. We focused our analysis on the more abundant taxa, and only genera that were in at least half of the samples at over 0.1% of the microbiota in at least one animal were included in the analysis. Therefore, only 42 genera were compared to the physiological parameters. These included those found to be part of the core community in a previous study, i.e., shared by all of the animals sampled, and accounting for over 90% of the overall rumen bacterial OTUs [12]. Our assumption was that these 42 genera represent important components of the healthy rumen ecosystem, and would therefore be more likely to reveal a connection between host physiology and the bacterial community residing in its rumen. A correlation matrix was created to evaluate each of these genera with each physiological parameter (Figure 3). Prevotella, the most abundant genus in the samples (up to 72% in some samples), showed a significantly negative correlation (Pearson R = −0.69, P = 5×10⁻³) with milk-fat yield, explaining most of the Bacteriodetes' negative correlation to this parameter, as well as its correlation with Firmicutes-to-Bacteroidetes ratio. Firmicutes, on the other hand, was composed of many lower-abundance genera, only a fraction of which compensated for the decreasing abundance of Prevotella in the samples. One species of Prevotella, P. bryantii, has been associated with probiotic activities: cows inoculated with P. bryantii strain 25A had decreased lactate production [27]. That same study also showed an increase in milk fat during the weeks following inoculation. This effect, however, was caused by the inoculation of that one specific strain and did not reflect a general modulation by the genus Prevotella. Analysis of the genera from the phylum Firmicutes revealed that 9 out of the 23 genera analyzed (Figures 4 and S2) were more abundant in samples with low levels of Prevotella, and 5 of these were correlated with milk-fat yield; most of these belonged to the order Clostridiales—the genus Eubacterium (Pearson R = 0.62, P = 0.012) and the family Lachnospiraceae (Pearson R = 0.62, P = 0.014) (Figure 3), and some belonged to the class Negativicutes, only recently defined as such, and formerly members of the Clostridia, such as the genus Dialister (Pearson R = 0.64, P = 0.009). Some of the genera belonging to Firmicutes were of relatively similar abundance between the samples regardless of the abundance of Prevotella, whereas others were found in higher abundance in samples with a low abundance of Prevotella. Two genera, Dialister and Lactobacillus, were almost nonexistent in samples with over 50% Prevotella, whereas they were present in all samples with less than 50% Prevotella (Figures 4 and S2). Genera belonging to other phyla also showed a correlation with milk-fat yield, such as the genus Desulfovibrio, belonging to the Proteobacteria. From the phylum Actinobacteria, both Bifidobacterium and Lactobacillus, widely used as probiotics, also showed a positive correlation to milk-fat yield, along with the genus Bulleidia, belonging to the Firmicutes. Whereas the correlations between the microbiota and milk-fat yield were found to be the strongest, we also detected both negative and positive correlations with other parameters related to the host's physiology and milk composition, including milk lactose and protein contents. With respect to host physiology, some bacteria were correlated with the variation in ruminal pH between cows, such as the genus Rosburia (Pearson R = −0.5, P = 0.06). One study reported that members of this taxon are affected by changes in pH [28], with optimal growth under slightly acidic conditions. Those authors suggested that this genus is affected by either pH or competitors that emerge at more neutral pH values. Interestingly, significant positive correlations were observed between four genera, all belonging to the order Coriobacteriales, and milk-lactose content. These were Atopobium and Adlercreutzia, and two unknown genera belonging to the order Coriobacteriales, one of them also positively correlating with average milk yield (Pearson R = 0.57 P = 0.027) (Figure 3). The fact that these taxa are phylogenetically related suggests that they share functions that affect the host's physiology in a similar manner. In addition, both Mitsuokella and Desulfovibrio were positively correlated with milk-lactose yield (Pearson R = 0.59 for both genera). No significant correlation was detected between the bacterial community and RFI; however, a positive, albeit nonsignificant correlation (Pearson R = 0.51, P = 0.055) was detected between an unclassified genus from the putative order RF39, found in all of the animals sampled, and RFI. This genus, although little studied, is found in many gut environments [12], [29], including the rumen, hinting at a potentially crucial role in the gut of many species. Nevertheless, additional sampling is required to determine whether this taxon is associated with feed efficiency in cattle.

Overall, our findings in this study show remarkable similarities with those in other mammalian host systems regarding their interaction with the gut microbiome. This, along with our previous study examining rumen colonization—which also revealed mechanistic similarities between different organisms and their digestive strategies [30]—suggests an underlying mechanism of acquisition and energy utilization that may be common to many of the studied gut systems, regardless of the apparent phylogenetic distances between the hosts.

Our current study suggests a connection between the physiological parameters of dairy cattle and their resident rumen bacteria and reveals potential candidate taxa that may prove useful for future inoculation studies. Additional work is needed to evaluate the causative relationships between the host and gut microbiota in cattle.