Date Published: November 29, 2018
Publisher: Public Library of Science
Author(s): Filippo Biscarini, Fiorentina Palazzo, Federica Castellani, Giulia Masetti, Lisa Grotta, Angelo Cichelli, Giuseppe Martino, Juan J Loor.
The rumen microbiome is fundamental for the productivity and health of dairy cattle and diet is known to influence the rumen microbiota composition. In this study, grape-pomace, a natural source of polyphenols, and copper sulfate were provided as feed supplementation in 15 Holstein-Friesian calves, including 5 controls. After 75 days of supplementation, genomic DNA was extracted from the rumen liquor and prepared for 16S rRNA-gene sequencing to characterize the composition of the rumen microbiota. From this, the rumen metagenome was predicted to obtain the associated gene functions and metabolic pathways in a cost-effective manner. Results showed that feed supplementations did alter the rumen microbiome of calves. Copper and grape-pomace increased the diversity of the rumen microbiota: the Shannon’s and Fisher’s alpha indices were significantly different across groups (p-values 0.045 and 0.039), and Bray-Curtis distances could separate grape-pomace calves from the other two groups. Differentially abundant taxa were identified: in particular, an uncultured Bacteroidales UCG-001 genus and OTUs from genus Sarcina were the most differentially abundant in pomace-supplemented calves compared to controls (p-values 0.003 and 0.0002, respectively). Enriched taxonomies such as Ruminiclostridium and Eubacterium sp., whose functions are related to degradation of the grape- pomace constituents (e.g. flavonoids or xyloglucan) have been described (p-values 0.027/0.028 and 0.040/0.022 in Pomace vs Copper and Controls, respectively). The most abundant predicted metagenomic genes belonged to the arginine and proline metabolism and the two- component (sensor/responder) regulatory system, which were increased in the supplemented groups. Interestingly, the lipopolysaccharide biosynthetic pathway was decreased in the two supplemented groups, possibly as a result of antimicrobial effects. Methanogenic taxa also responded to the feed supplementation, and methane metabolism in the rumen was the second most different pathway (up-regulated by feed supplementations) between experimental groups.
The rumen microbiota is a preeminent microbial community in the gastrointestinal tract of ruminants. This finely regulated ecosystem is what makes it possible for ruminants to digest fibrous plant material (inedible for other livestock), use it as source of energy and other metabolites, and transform it into high-quality food. In dairy cattle, the rumen microbiome plays a key role in milk production , well-being and health of the animals [2, 3]. The development of culture-independent high-throughput next-generation sequencing techniques provids a breakthrough in the characterization and analysis of microbiomes , with the rumen microbiome being no exception . In particular, 16S rRNA gene sequencing  is a powerful technique to identify and quantify (in relative terms) the taxonomic composition of the rumen microbial population . From metataxonomics results, the associated metagenome and related metabolic functions can be predicted, based on relative abundances and using a database of microbial genes functional annotations [8, 9]. The variability of the rumen microbiota across animals and over time has been investigated in a number of studies e.g. [10–13]. The diet is known to alter the composition of the rumen microbiota [14, 15]. Specific feed supplements have been the object of experimental trials on the rumen microbiome: these include canola , probiotic bacteria , organic acids [18, 19]. Mostly, feed supplementations had the objective of counteracting the effects of the high-energy diets typical of dairy cows on the rumen pH, the composition of the rumen microbiota, and the health of the animals. Grape-pomace is the solid residue from grape processing for wine production. It has high content of tannins and polyphenols e.g. [20, 21], which are known to exert an antioxidant activity and were previously shown to reduce rumen methane emissions in late-lactation dairy cows milk-fat yield . Coppers is an essential trace element in the diet of livestock, and was shown to alter the gastrointestinal microbial composition of lactating cows . It is therefore of interest to further investigate the role of these two feed supplements on the rumen microbiome composition and function.
The characterization of the rumen microbiota and its functional profile from 15 calves fed different dietary supplementations has been presented here. The sequencing of the V3-V4 variable regions of the rRNA gene (16S subunit) appeared to be adequate as shown by the asymptotic plateauing of both sequence- and sample-based rarefaction curves (Fig 1). The sensitivity analysis of sequence quality filtering (comparisons of Phred quality score thresholds: Phred > 3 vs Phred > 19) indicated an overall robustness of results from the closed-reference OTU picking approach; however, a stricter quality filtering (Phred > 19) is likely to remove most poor quality sequences and, consequently, most spurious OTUs.
The sequencing of the 16S rRNA marker gene constitutes an extraordinary advancement in the genetic analysis of microbial communities. Coupling metataxonomics with metagenome prediction gives insights into the genes and metabolic pathways associated with a microbiome, and is a very powerful technique for the functional profiling of microbial communities. Here, it was applied to the profiling of the rumen microbiome in dairy calves fed differentially supplemented diets. Copper and grape-pomace feed supplementations appeared to alter the rumen microbiome, both in terms of species diversity and gene functions. Results were in line with previous findings in both ruminal, human and murine microbiota. The addition of grape-pomace, in particular, seemed to modify the rumen microbial population, with an apparent effect also on methanogenic bacteria and methane metabolism in the rumen. It needs be emphasized though, that Archaea and methanogenic taxa were not specifically targeted by the sequencing approach employed in this study, and results should therefore be considered as indicative.