Differently Pre-treated Alfalfa Silages Affect the in vitro Ruminal Microbiota Composition

Abstract

Alfalfa (Medicago sativa L.) silage (AS) is an important feedstuff in ruminant nutrition. However, its high non-protein nitrogen content often leads to poor ruminal nitrogen retention. Various pre-ensiling treatments differing with respect to dry matter concentrations, wilting intensities and sucrose addition have been previously shown to improve the quality and true protein preservation of AS, and have substantial effects on in vitro ruminal fermentation of the resulting silages. However, it is unknown how these pre-ensiling treatments affect the ruminal microbiota composition, and whether alterations in the microbiota explain previously observed differences in ruminal fermentation. Therefore, during AS incubation in a rumen simulation system, liquid and solid phases were sampled 2 and 7 days after first incubating AS, representing an early (ET) and late (LT) time point, respectively. Subsequently, DNA was extracted and qPCR (bacteria, archaea, and anaerobic fungi) and prokaryotic 16S rRNA gene amplicon sequence analyses were performed. At the ET, high dry matter concentration and sucrose addition increased concentrations of archaea in the liquid phase (P = 0.001) and anaerobic fungi in the solid phase (P < 0.001). At the LT, only sucrose addition increased archaeal concentration in the liquid phase (P = 0.014) and anaerobic fungal concentration in the solid phase (P < 0.001). Bacterial concentrations were not affected by pre-ensiling treatments. The prokaryotic phylogenetic diversity index decreased in the liquid phase from ET to LT (P = 0.034), whereas the solid phase was not affected (P = 0.060). This is suggestive of a general adaption of the microbiota to the soluble metabolites released from the incubated AS, particularly regarding the sucrose-treated AS. Redundancy analysis of the sequence data at the genus level indicated that sucrose addition (P = 0.001), time point (P = 0.001), and their interaction (P = 0.001) affected microbial community composition in both phases. In summary, of the pre-ensiling treatments tested sucrose addition had the largest effect on the microbiota, and together with sampling time point affected microbiota composition in both phases of the rumen simulation system. Thus, microbiota composition analysis helped to understand the ruminal fermentation patterns, but could not fully explain them.

Pretreatment of Rapeseed Meal Increases Its Recalcitrant Fiber Fermentation and Alters the Microbial Community in an in vitro Model of Swine Large Intestine

“…The relative abundances of Ruminococcaceae NK4A214 group, Ruminococcaceae UCG-002, Ruminococcaceae UCG-005, Roseburia, Bifidobacterium, and Christensenellaceae R-7 group were significantly increased in ALK, PECT1, PECT2, and CELL (Figure 4). Research has shown that genera of family Ruminococcaceae contain major (hemi)cellulolytic and pectinolytic species (Pettipher and Latham, 1979;Nyonyo et al, 2014;Hartinger et al, 2019a). Thus, microbes from family Ruminococcaceae play an important role in degrading (hemi)cellulose and pectin in their activity against recalcitrant fiber (Shinkai and Kobayashi, 2007;Flint et al, 2008).…”

The Present Role and New Potentials of Anaerobic Fungi in Ruminant Nutrition

“…Indeed, cell wall degradation by AF can be higher than by bacteria under certain in vitro conditions [ 25 , 26 ] but quantifying the exact fungal contribution to fiber breakdown in the rumen is difficult. However, several in vitro and in vivo studies demonstrated the association between AF and an improved fiber degradability [ 25 , 26 , 27 , 28 ], which is also suggested by recent in vitro data from Ma et al [ 26 ]. These authors investigated the impact of co-culturing methanogens with either AF or bacteria on in vitro degradation of rice and wheat straw, observing higher dry matter and fiber degradation in the presence of AF, the extent of which was similar to lignocellulose breakdown by whole ruminal content.…”

Omics and imaging combinatorial approach reveals butyrate-induced inflammatory effects in the zebrafish gut

“…Amplicon libraries of the V4 region of the 16S rRNA were generated from the cDNA synthetized from single gut and water samples, using barcoded and modified F515-806R primers [ 86 ]. The PCRs were performed in triplicate, purified, and quantified as previously described [ 29 ]. Purified PCR amplicons were pooled in an equimolar mix and sent for library preparation and sequencing using the Illumina NovaSeq 6000 S2 PE150 XP technology at Eurofins Genomics Germany GmbH (Eurofins Genomic, Ebersberg, Germany).…”