Nitrogenous emissions from ruminant livestock production are of increasing public concern and, together with methane, contribute to environmental pollution. The main cause of nitrogen-(N)-containing emissions is the inadequate provision of N to ruminants, leading to an excess of ammonia in the rumen, which is subsequently excreted. Depending on the size and molecular structure, various bacterial, protozoal and fungal species are involved in the ruminal breakdown of nitrogenous compounds (NC). Decelerating ruminal NC degradation by controlling the abundance and activity of proteolytic and deaminating microorganisms, but without reducing cellulolytic processes, is a promising strategy to decrease N emissions along with increasing N utilization by ruminants. Different dietary options, including among others the treatment of feedstuffs with heat or the application of diverse feed additives, as well as vaccination against rumen microorganisms or their enzymes have been evaluated. Thereby, reduced productions of microbial metabolites, e.g. ammonia, and increased microbial N flows give evidence for an improved N retention. However, linkage between these findings and alterations in the rumen microbiota composition, particularly NC-degrading microbes, remains sparse and contradictory findings confound the exact evaluation of these manipulating strategies, thus emphasizing the need for comprehensive research. The demand for increased sustainability in ruminant livestock production requests to apply attention to microbial N utilization efficiency and this will require a better understanding of underlying metabolic processes as well as composition and interactions of ruminal NC-degrading microorganisms.

Pre-ensiling treatments can significantly influence the composition of lucerne (Medicago sativa L.) silages (LS). Besides dry matter (DM) content and availability of water-soluble carbohydrates (WSC), wilting intensity may exert a strong impact on the crude protein (CP; nitrogen [N] × 6.25) fractions. The present study aimed to evaluate the effects of DM level, wilting intensity, and sucrose addition on N compounds and fermentation products in LS. Pure lucerne stand (cultivar Plato) was wilted with either high or low intensity to DM contents of 250 and 350 g kg−1, respectively, and ensiled with or without the addition of sucrose. Non-protein-N (NPN) concentration in LS was affected by all pre-ensiling treatments and with 699 g kg−1 CP, NPN was lowest in high-intensity wilted high-DM LS with sucrose addition. No effects were observed on in vitro-estimated concentrations of utilizable CP at the duodenum, a precursor to metabolizable protein. Sucrose addition and higher DM level decreased acetic acid and ammonia-N concentration in the silages. Therefore, the present study demonstrated the beneficial manipulation of CP fractions in LS by high-intensity wilting to higher DM contents and that the provision of WSC may be necessary for sufficient silage fermentation and protein preservation.

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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.