Protein supply to ruminants relies mainly on the flow of microbial crude protein (MCP) from the rumen, which is commonly assumed to primarily depend on energy supply. This study evaluated this assumption with recent data and tested if ruminally fermented organic matter (FOM) was a better predictor of MCP flow than total-tract digestible organic matter (DOM) and if more variables could improve the prediction of MCP flow. A previously published data set was extended by additional studies resulting in a data set of 139 studies including 407 treatment means, typical to Central European rations. Either DOM or FOM had to be reported and estimates of MCP were all based on gastrointestinal measurements. Dietary treatments were restricted to a maximum concentrate proportion of 0.6 and a CP concentration from 10% to 20% of dietary dry matter (DM). Treatments with more than 1% of animal by-products were excluded. Mixed models with "study" as random effect were used to test the ratios between MCP and DOM or FOM. Dietary characteristics including DM intake, OM digestibility (OMD), OM fermentability (OMF), and ruminal N balance and dietary concentrations of CP, rumen-degraded CP (RDP), rumen-undegraded CP (RUP), neutral detergent fibre, starch, and ether extract were evaluated as additional variables to improve prediction accuracy. Regression of MCP flow against DOM (n = 324) or FOM (n = 349) revealed estimates of 139 g +/- 36 g and 164 g +/- 40 g of MCP for each kg of DOM or FOM, respectively. The best-fitting mixed model estimating MCP/DOM [g/kg] was 312-2.75 OMD - 0.229 RUP + 0.290 RDP whereas MCP/FOM [g/kg] may be calculated as 294-2.80 OMF + 0.401 RDP, with OMD and OMF in [%] and RDP and RUP in [g/kg DM]. Microbial CP flow [g/d] was most accurately described as 1896 + 110 DOM - 23.7 OMD - 0.164 RUP + 0.153 RDP, with DOM in [kg], OMD in [%] and RDP and RUP in [g/d]. Predictions based on DOM were at least as precise as FOM-based predictions and comparison with published models revealed that the presented models were of similar accuracy as the ones from literature.

The increasing demand for a reduction of animal experiments when studying rumen fermentation has led to the development of various in vitro techniques, such as the rumen-simulation technique (RUSITEC) system that is prominently applied in European ruminant research. Yet, comparability with the in vivo situation is rather less explored with a sparse data basis. Therefore, the present study aimed to directly compare the fermentation characteristics and degradability of two common forages, i.e., grass silage (GS) and maize silage (MS), by parallel application of a coupled in vivo-in situ approach in rumen-cannulated sheep as well as the in vitro RUSITEC system. Both forages were incubated in the RUSITEC system as well as fed to rumen-cannulated sheep in six independent runs of 20 days in total with 14 days of adaptation and 6 days of sampling. The degradability coefficients of dry matter, organic matter and acid detergent fibre were affected by the method (each P<0.05), while neutral detergent fibre (aNDFom) degradability was not different between RUSITEC and in situ measurements (P = 0.10). Likewise, Pearson correlation coefficients confirmed the comparability of in vitro and in situ values for aNDFom degradability, being 0.54 (P = 0.04) and 0.78 (P = 0.02) for GS and MS, respectively. Regarding the fermentation profile, total volatile fatty acid (VFA) concentrations were much higher in vitro than in vivo (P < 0.01), likely due to the missing absorptive capacity of the RUSITEC system. A comparison of absolute fermentation values between methods appears not feasible. However, the order of individual VFA proportions was similar between in vivo and in vitro and the correlations for both total and individual VFA further supported this congruency, especially for MS. The in vitro data appeared well comparable to the data from the coupled in vivo-in situ approach, especially for MS, with a high reproducibility in both methods. Therefore, the RUSITEC system may represent a sufficient replacement for laborious in vivo and in situ measurements when assessing nutrient degradability and general fermentation characteristics of feedstuffs. Adjustments in in situ incubation times as well as the frequently requested standardization of the operation of the RUSITEC system may further increase the significance of this in vitro method in the future. Likewise, further research on diurnal fermentation patterns is encouraged to substantiate the present findings.

Studies on ruminally cannulated animals are expensive and time consuming. Furthermore they are limited to a few facilities and animal welfare issues make reduced numbers of animal experiments desirable. One alternative is to simulate rumen fermentation using long-term continuous systems, allowing investigation of a large number of treatments in a relatively short period of time. Lack of standardisation of continuous culture systems impairs the comparison of research data. Therefore, the focus of this review is to compile and compare technical and other methodical aspects of the semi-continuous culture system ‘rumen simulation technique’ (RUSITEC). Crucial or non-standardised characteristics are highlighted, standardisation of procedures is suggested and limits of standardisation are considered. The literature search yielded information from 93 studies published between 1977 and 2019 and comprised data from 111 individual technical variants and test protocols. Not only the development of semi-continuous culture systems, but also technical design and details and handling of the RUSITEC system were considered. Procedural steps which were evaluated encompassed choice and feeding of donor animals, type of feeds or rations, sampling time of inoculum and, further, the technical structure and procedure of the RUSITEC systems with a motor, pump, vessels and buffer solution. Moreover, the choice of incubation bags with regard to pore size and material, the incubation time, the experimental run period and the selection of experimental feed have an impact on the results. To improve the comparability of studies, specified technical standards will reduce the variability within and between different models of the RUSITEC system. It does not appear advisable to restrict the species of the donor animal to cattle or sheep, yet the feeding of donor animals, the sampling time of inoculum, the preparation and the starter volume of rumen fluid should be specified in guidelines. Moreover, technical measures such as pore size of incubation bags, particle size of incubated feedstuffs and artificial saliva composition, as well as flow rate, need to be standardised. In contrast to standardised batch culture-based systems like the Hohenheim gas test, there are no uniform guidelines for the application of RUSITEC systems. Finally, we propose standardisations to reduce variability in the design of simulation systems, variability in the rumen inoculum and to improve the comparability of RUSITEC research data.

Rumen microorganisms turn small N-containing compounds into amino acids (AA) and contribute considerably to the supply of AA absorbed from the small intestine. Previous studies summarized the literature on microbial AA patterns, most recently in 2017 (Sok et al. Journal of Dairy Science, 100, 5241-5249). The present study intended to identify the microbial AA pattern typical when feeding Central European diets and a maximum proportion of concentrate (PCO; dry matter (DM) basis) of 0.60. Data sets were created from the literature for liquid (LAB)- and particle (PAB)-associated bacteria, total bacteria and protozoa, including 16, 9, 27 and 8 studies and 36, 21, 60 and 18 diets respectively. Because the only differences detected between LAB and PAB were slightly higher Phe and lower Thr percentages in PAB (p < 0.05), results for bacteria were pooled. A further data set evaluated AA-N (AAN) as a proportion of total N in microbial fractions and a final data set estimated protozoal contributions to total microbial N (TMN) flow to the duodenum, which were used to calculate weighted TMN AA patterns. Protozoa showed higher Lys, Asp, Glu, Ile and Phe and lower Ala, Arg, Gly, Met, Ser, Thr and Val proportions than bacteria (p < 0.05). The AAN percentage of total N in bacteria and protozoa showed large, unexplained variations, averaging 79.0% and 70.6% (p > 0.05) respectively. Estimation of protozoal contribution to TMN resulted in a cattle-specific mixed model including PCO and DM intake (DMI) per unit of metabolic body size (kg0.75 ) as fixed effects (RMSE = 3.77). With moderate PCO and DMI between 80 and 180 g/kg0.75 , which corresponds to a DMI of approximately 10 to 25 kg in a cow with 650 kg body weight, protozoal contribution ranged between 9% and 26% of TMN. Within this range, the estimated protozoal contribution to TMN resulted in minor effects on the total microbial AA pattern.

The three pre-ensiling treatments dry matter (DM) concentration, wilting intensity and sucrose addition have recently been shown to affect the chemical composition of lucerne (Medicago sativa L.) silages (LS) with highest true protein preservation and improved fermentation quality when lucerne was wilted with high intensity to 35% DM concentration and ensiled with the addition of sucrose. Provided that ruminants prefer these LS to those produced under other pre-ensiling conditions, an improved provision of dietary energy and nutrients may be achieved. However, information on how such pre-ensiling treatments influence the feed choice and DM intake (DMI) of ruminants is lacking and thus, feed choice trials with ten Saanen-type wethers were conducted. The LS were tested in two separate trials split by the factor sucrose addition. In both trials, the animals first passed an adaptation period receiving each differently pre-treated LS (n = 4) and lucerne hay that served as a control for one day to gain forage-specific post-ingestive responses. Subsequently, each possible two-way combination of forages was offered to the animals to determine the forage preference, i.e. 30-min and 3-h DMI. The DM concentration affected the feed intakes at both time points with greater preference for 35% DM LS than for 25% DM LS and a higher consumption was observed for sucrose-treated LS than for untreated LS when related to control lucerne hay. In contrast, wilting intensity showed no influence on DMI. The higher intake of high DM or sucrose-treated LS was likely related to the superior silage quality as indicated by high negative correlations of DMI and non-protein nitrogen, ammonia-nitrogen, acetic acid, butyric acid and gamma-aminobutyric acid concentrations.

The successful ensiling of lucerne (Medicago sativaL.) depends on a rapid acidification in the silo and consequently relies on a sufficient proliferation of, particularly homofermentative, lactic acid bacteria. Similarly, growth of spoilage bacteria, such as enterobacteria and clostridia, must be suppressed and silage additives are therefore frequently applied to promote favourable conditions during ensiling. Three silage additives or soil were applied during lucerne ensiling and investigated for their effects on silage quality characteristics and abundances of total bacteria as well as the bacterial key playersLactobacillusspp., homofermentativeLact.plantarum, heterofermentativeLact.buchneri,Clostridiumspp. andEnterobacteriaceaeafter 30 days of storage. Inoculation with viableLact.plantarumresulted in highest concentration of this species and excellent silage quality, i.e. high lactic acid concentration coupled with low acetic acid and ammonia-nitrogen concentrations. A sodium nitrite and hexamine-based additive did not support growth of lactic acid bacteria, which was also apparent by higher pH and low lactic acid concentration. No effect of treatments was found on spoilage-related enterobacteria and clostridia, even not when adding soil to lucerne to increase initial clostridial contamination. However, soil treatment resulted in increased ammonia-nitrogen and acetic acid concentrations. Consequently, among the bacterial key players, lactic acid bacteria concentrations were related to silage quality. Regarding spoilage bacteria, however, alterations in silage quality characteristics were not reflected in the abundances of enterobacteria and clostridia. Future investigations should underpin the present findings and help to understand how silage additives affect microbial key players and silage fermentation.

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.

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.

The present study investigated the in vitro ruminal fermentation of alfalfa silages (AS) that had been produced using different pre-ensiling treatments, i.e., by changing the wilting intensity and dry matter concentration, and adding sucrose, and therefore differed in silage quality and their N fractions. The data were obtained using an in vitro rumen-simulation technique (Rusitec) system, in which the AS were incubated isonitrogenously in quadruplicate. Samples were take n after 2 days (first time point) and 7 days (second time point) of AS incubation, and a variety of fermentation characteristics as well as the degradability of fiber fractions and organic matter were determined. Sucrose addition substantially raised the propionate concentration during both sampling time points from an average of 17.8 to 29.7 mmol/L (P < 0.001), which might be explained by microbial utilization of residual sugars and lactate from the AS. The extraordinary high concentrations of isovalerate and ammonia-N with all AS point to enhanced deamination activity. At the second time point, the n-butyrate concentration increased during the incubation of high-intensity wilted AS (P = 0.007), which might have been caused by the higher hemi-cellulose degradability that was also observed for these silages (P = 0.002). However, the organic matter degradability decreased (P = 0.035), indicating a lower degradability of other feed fractions. The gas production (P < 0.001) and degradability of organic matter (P = 0.002) and fiber fractions (P < 0.001) decreased from first to second time point, whereas the concentrations of ammonia-N (P = 0.004), acetate (P < 0.001), and isovalerate (P < 0.001) increased. Thus, it seemed that alterations in the Rusitec system and the microbial community occurred, yet it is unclear why the acetate concentration increased, whereas the fiber degradability decreased. The beneficial effects of combining all three pre-ensiling treatments on silage quality, i.e., higher acidification and increased true protein preservation, were not fully transferred to the in vitro ruminal fermentation system, and comprehensive research on pre-ensiling treatments will pave the way for an optimized ruminal N utilization from AS in the future.

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.

The objective of the present study was to evaluate if grass silages with high contents of free amino acids (FAA) lead to enhanced amino acid catabolism compared with grass silages with low contents of FAA when fermented in a rumen simulation system (Rusitec). Eight grass silages with contents of FAA lower than 300 mmol/kg dry matter (DM; GS-LOW) and eight grass silages with contents higher than 300 mmol/kg DM (GS-HIGH) were tested in vitro for their effects on selected variables of nitrogen metabolism. Ammonia concentration, production of the branched-chain volatile fatty acids (BCVFA) isobutyric and isovaleric acid, concentration of FAA in fermenter fluid, and the content of bacterial protein in fermenter fluid were measured daily from days 1 to 28 (ammonia, BCVFA), 3 and 6 to 28 (bacterial protein), and days 7, 8, 11 to 13, 16 to 21, and 26 to 28 (FAA), respectively. Subsequent to an initial 9-day adaptation period during which hay (10.5 g DM) and concentrate (3.0 g DM) were added to fermenters, grass silages (10.5 g DM) were individually incubated in the fermenters for a period of 10 days (experimental period). The experimental period was followed by the recovery period during which the additions of hay and concentrate were equal to those of the adaptation period. Addition of GS-HIGH increased the concentration of ammonia (P<0.05) and production of isobutyric (P<0.05) and isovaleric (P<0.05) acid in contrast to GS-LOW. The concentration of FAA, as well as the content of bacterial protein in fermenter fluid, showed no differences after addition of GS-LOW or GS-HIGH (P>0.05). The calculated efficiency of nitrogen fixation in bacterial protein was expressed as units of bacterial protein per g nitrogen input with the silages and was lower or tended to be lower, respectively, with GS-HIGH on 5 days of the experimental period (P<0.1). Increased concentration of ammonia and production of BCVFA indicate that amino acid catabolism was increased by additions of GS-HIGH compared to GS-LOW whereas efficiency of nitrogen fixation in bacterial protein seemed to be decreased. The observed effects may be due to higher metabolic activity of amino acid-fermenting bacteria. (C) 2015 Elsevier B.V. All rights reserved.