Browsing by Subject "Rumen"

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Degradation of crude protein and starch of corn and wheat grains in the rumen
(2016) Seifried, Natascha; Rodehutscord, Markus
The major objectives of the present thesis were to characterize the ruminal crude protein (CP) and starch degradation of different genotypes of corn and wheat grains and to predict the effective degradation (ED) of CP and starch with easily measurable characteristics. The in situ method is the standard technique to study the ruminal degradation of feeds in many feed evaluation systems. This technique was originally applied to study forages and it was therefore necessary to clarify methodical details related to the measurements of in situ starch degradation from cereal grains. Two in vitro and one in situ approach were conducted to study the loss of secondary starch particles from bags with different pore sizes used for the in situ incubation of different cereal grains. In the first in vitro study ground wheat was incubated in bags (pore size: 50 µm) over different time spans in a modified rumen simulation technique. Bag residues and fermenter fluids were analyzed for their starch content. In the second in vitro study ground wheat, barley, and corn were incubated with bags of pore sizes of 50, 30 (except corn), 20, and 6 µm. In the in situ study ground wheat, barley, corn, and oats were rumen incubated over different time spans using bags with pore sizes of 50, 20, and 6 µm. The starch content of the grains and bag residues was analyzed enzymatically and the degradation characteristics of starch were calculated for each grain type and pore size. It was shown for the first time that incubating wheat and barley in bags with 50 and 30 µm pore size lead to a substantial amount of secondary starch particle losses during incubation process in vitro. These losses were not detectable when the grains were incubated with bags having pore sizes of 20 and 6 µm. Independent of the bags’ pore size no secondary starch particle losses were found by the incubation of corn. Thus corn can be studied in situ even with bags with 50 µm pore size. Oats showed very high washout losses with all pore sizes tested in the present thesis and therefore none of them is suitable to study the starch degradation measurements of oats. Because of methodical problems of gas accumulation in bags having pore sizes < 50 µm, no recommendations can be provided for the in situ evaluation of wheat and barley. Further research is necessary to solve these problems. In the second and third study of the present thesis ruminal in situ degradation of 20 corn grain genotypes and 20 wheat grain genotypes was measured in three lactating Jersey cows. In both experiments the same techniques were used to characterize the ruminal degradation of CP and starch. Ground grains (2 mm) were rumen incubated in bags (50 µm pore size) over different time spans. Grains and bag residues were analyzed for their CP and starch content. The degradation parameters and the ED were calculated for dry matter (DM), CP, and starch. Gas production (GP) of ground grains (1 mm) was recorded after incubation over different time spans in buffered ruminal fluid and fitted to an exponential equation to determine GP parameters. To predict ED of CP and starch correlations with physical and chemical characteristics and in vitro measurements were evaluated and stepwise multiple linear regression analyses were applied. The in situ parameters (soluble fraction, potential degradable fraction, and degradation rate) varied widely between genotypes of corn and wheat grains. The ED of DM, CP, and starch showed a high variation for corn grain genotypes. Due to the high degradation rates, the ED of wheat grains were similar between genotypes. The GP rate was in good agreement with the in situ values for corn grains, whereas no systematic relationship between both methods was observed for wheat grains. Evaluation of correlation analysis showed significant relationships between calculated ED of CP and several amino acids (AA) for both grain types. This indicates that the protein composition of the grains influences CP degradation in the rumen. Similar relationships were found between the same AA and ED of starch of corn grains which highlights the impact of the protein composition on ruminal starch degradation for this grain type. For both grain types, the ED of starch and CP could be predicted accurately from physical and chemical characteristics alone or in combination with GP measurements. Thus, the equations presented in the present thesis can be used to obtain rapid and cost effective information on ruminal degradation of CP and starch for corn and wheat grains. The results of the present thesis show that there is considerable variation of ruminal CP and starch degradation from different genotypes of corn and also – albeit to a lesser extent – for wheat grains. Differences in ED of starch should be taken into account when formulating rations containing significant amounts of corn and wheat grains. In the case of corn grains differences in ED of CP should also be accounted for.
Influence of tropical supplemental feeds on the composition and activity of rumen microorganisms, quantified by oligonucleotide probes
(2001) Muetzel, Stefan; Becker, Klaus
This study was undertaken to evaluate the applicability of oligonucleotide probes to unravel the population structure of the rumen flora in vitro. At first a RNA extraction and cell lysis method for rumen fluid samples was optimised. However when tannin containing plants were present in the samples the method failed to recover microbial RNA. The comparison of two rumen fluid sampling sites for inoculation revealed a higher in vitro gas production from samples inoculated with rumen fluid from the feed mat compared to the liquid phase. The higher gas production was not explained by a higher digestibility of the substrates. Changes in the population structure, population density and the kinetic of the fermentation might be responsible for the observed differences. This experiment showed that for interpretation of the results, population structure data have to complemented with metabolic parameters. In a supplementation experiment it was demonstrated that amount, but also the efficiency of the microbial biomass production was positively affected. Comparison of the population structure of the cell wall degrading consortium and cellulase activity revealed that Fibrobacter was mainly responsible for the expression of this enzymatic activity. Such a comparison is a new strategy which will lead to a better understanding of the complex fermentation processes in the rumen. The analysis of the population structure of the cell wall degrading organisms showed a competition for substrate or attachment sites between Fibrobacter and Ruminococcus albus which was independent of the substrate incubated, the time of sampling and the origin of the inoculum.
Investigations on the effects of forage source and feed particle size on ruminal fermentation and microbial protein synthesis in vitro
(2012) Hildebrand, Bastian; Rodehutscord, Markus
The synthesis of microbial protein in the rumen has a major impact on protein- and amino acid supply in ruminants. The amount and amino acid composition of the protein that enters the small intestine primarily depends on diet formulation. In the present studies the effects of maize silage (MS) and grass silage (GS) on ruminal fermentation and microbial protein synthesis were investigated, considering methodical aspects of in vitro studies, particularly grinding of feed samples. In the first experimental series five mixed diets with different proportions of MS and GS (100:0, 79:21, 52:48, 24:76 and 0:100) and a constant proportion of soybean meal (11%) were used. The content of crude protein (CP) and fibre fractions increased, whereas the content of organic matter (OM) and starch decreased with increasing proportion of GS in the diet. It was hypothesised that a combination of MS and GS can benefit microbial growth and thus fermentation of nutrient fractions to a higher extent than using only one forage source separately. It was also to be investigated how changes in diet composition affect the amino acid profile of microbial protein. A well standardised semi-continuous rumen simulation technique (RUSITEC) was used, which is a commonly accepted experimental model in investigations on ruminal fermentation. Changes in fermentation characteristics, as a result of changing the MS-to-GS ratio, were tested for linear and quadratic effects in order to identify possible associative effects. Prior to the in vitro incubation, feedstuffs were dried and ground. It was aimed to investigate in which way fermentation in the RUSITEC system is influenced by mean feed particle size. Therefore two milling screen sizes (MSS; 1 vs. 4 mm) were used in all diets and results on fermentation characteristic were tested for possible interactions of forage source and MSS. One incubation period lasted for 13 days (6 days adaption period, 7 days sampling period), and each treatment was tested in at least three replicates. Ruminal digesta, obtained from rumen-fistulated wether sheep, was used as the inoculum for starting the incubation. Diets were fed once daily to the RUSITEC system, and nylon feed bags remained for 48 h inside the fermentation vessel. A buffer solution, containing 15NH4Cl, was infused continuously into the vessel and the respective effluent was analysed for short chain fatty acids (SCFA) and NH3-N. Solid- and liquid- associated microbial fractions were isolated from the feed residues, the liquid inside the vessel and the effluent by differential centrifugation. The flow of microbial CP was quantified on the basis of N and 15N balances. The feed residues were analysed for crude nutrients and detergent fibre fractions and the respective degradation rates were calculated. OS and CP in the feed residues were corrected for the contribution of solid-associated microbes. The degradation of OM and fibre fractions, as well as amounts of NH3-N increased linearly with stepwise replacement of MS by GS. Degradation of CP was unaffected by diet composition, as well as total SCFA production. The degradation of OM and CP was higher in coarse milled (4 mm-MSS) than in fine milled (1 mm-MSS) treatments, accompanied by higher amounts of NH3-N and total SCFA. An improvement of growth conditions for some microbial groups, e.g. anaerobe fungi, was discussed. The amount of microbial CP increased linearly by the stepwise replacement of MS by GS, and was higher at 4 mm-MSS than at 1 mm-MSS. The amount of available N was assumed to advance microbial growth in the RUSITEC system. Efficiency of microbial CP synthesis was improved from 29 to 43 mg microbial N per g degraded OM by increasing the proportion of GS in the diet, but was unaffected by MSS. The N content and the profiles of amino acids of the three microbial fractions, as well as the ratio of solid- to liquid-associated microbes were affected by diet composition and MSS. Interactions of forage source and MSS were rare. However, the results indicated interactions between dietary factors and origin of microbial isolate on characteristics of microbial protein synthesis. In order to provide additional information on the nutritional value, the mixed diets were evaluated by two further methods. The total tract digestibility of crude nutrients was determined in wether sheep. The content of metabolisable energy was similar between diets and averaged 11.5 MJ per kg dry matter. The in vitro gas production was measured within 93 h by using a modified Hohenheim gas production test, providing information on kinetics and extent of ruminal fermentation. Cumulative gas production decreased with increasing proportion of GS in the diet. A negative effect of coarse milling on fermentation in the Hohenheim gas production test was confirmed. Across all diets gas production was delayed at 4 mm-MSS compared to 1 mm-MSS. The results from both approaches supported the findings of the RUSITEC study that a stepwise replacement of MS by GS led to a linear response in degradation of nutrients. As indicated by the gas production data, positive associative effects might only occur in the first hours after starting an incubation. When mixed diets are used effects cannot be clearly related to individual diet ingredients. Moreover, in the mixed diets interactions between soybean meal inclusion and forage source or feed particle size cannot be excluded. Therefore pure silages were incubated separately in the RUSITEC system in the second experimental series and three milling screens of different size were used (1, 4 and 9 mm). In accordance with the first experimental series, degradation of OM, fibre fractions and non-structural carbohydrates, production of NH3-N, as well as microbial CP flow and efficiency of microbial CP synthesis were higher in GS than in MS. A higher degradation of CP was found for MS than for GS, indicating interactions between forage source and soybean meal inclusion. An increase in MSS from 1 mm to 9 mm led to an improvement in the degradation of OM, CP and non-structural carbohydrates, particularly of starch in MS, as well as in the microbial CP flow for both silages. But the efficiency of microbial CP synthesis and microbial amino acid profile were less affected by MSS. In the second experimental series additionally the effect of available N on fermentation of MS was investigated. The supplementation of urea-N improved the degradation of non-structural carbohydrates, especially starch, but not that of fibre fractions in MS. The efficiency of microbial CP synthesis was increased from 26 to 35 mg microbial N per g degraded OM by urea-N supplementation to MS. The way of urea administration, either supplied together with the feed once daily or infused continuously by buffer solution, had only marginal effects on fermentation characteristics. It was concluded that microbial growth is improved by degradation of OM from GS compared to MS and by an increasing availability of N in the RUSITEC system. Meaningful associative effects of mixtures of MS and GS on ruminal fermentation characteristics are not likely to occur. However, transferability of results to other batches of MS and GS is limited, as high variations in chemical composition are known for both types of silage. Fermentation of MS- and GS-based diets in the RUSITEC system benefits more by coarse milling at MSS up to 9 mm than by fine milling at 1 mm-MSS. Consequently, variations in MSS and feed particle size distribution have to be taken into account when evaluating feeds by rumen simulation systems. The changes in composition and contribution of microbial fractions give indications to a shift in the microbial community as a result of variation of silage type and feed particle size, but further research on this aspect is needed. Moreover, the present results stated that the origin of the microbial samples is very important for measurements on microbial protein synthesis.
A shift towards succinate‐producing Prevotella in the ruminal microbiome challenged with monensin
(2022) Trautmann, Andrej; Schleicher, Lena; Koch, Ariane; Günther, Johannes; Steuber, Julia; Seifert, Jana
The time‐resolved impact of monensin on the active rumen microbiome was studied in a rumen‐simulating technique (Rusitec) with metaproteomic and metabolomic approaches. Monensin treatment caused a decreased fibre degradation potential that was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Decreased proteolytic activities resulted in reduced amounts of ammonium as well as branched‐chain fatty acids. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of the metabolism from acetate to succinate production. Prevotella species harbour a membrane‐bound electron transfer complex, which drives the reduction of fumarate to succinate, which is the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin.
Studies on the composition of the ruminal microbial community using grass silage and corn silage
(2016) Lengowski, Melanie; Rodehutscord, Markus
Grass silage and corn silage are the most commonly used silages for feeding dairy cows and fattening bulls. Due to their different chemical composition these silages have diverse effects on the ruminal microbial community and, therefore, on the fermentation process in the rumen. The major objective of this thesis was to evaluate the effect of grass silage and corn silage on the ruminal microbial community composition. The focus was on the incubation of silages without using concentrates. Furthermore, diurnal changes of the ruminal microbial community were investigated. In the first study (Manuscript 1), effects of incubation of grass silage and corn silage on the ruminal microbial community were investigated using an established rumen simulation technique (Rusitec). Furthermore, diurnal changes and changes during the first 48 hours of incubation (adaption phase) were observed. A significant decrease of the complete ruminal microbial community in the fermenter liquids, on species and group level, was observed. During the adaption phase, the silage source lost relevance because in the silage-containing fermenters as well as in the blank fermenters decreasing numbers were observed with the exception of Clostridium aminophilum. For this species, after 48 hours higher numbers were found as compared to the inoculum. As has already been described in the literature, in the current thesis changes in microbial abundance were lesser in feed residues than in fermenter liquids. The abundance of protozoa in feed residues decreased during the first 48 hours while for Prevotella bryantii and C. aminophilum higher numbers were found after 24 and 48 hours as compared to the inoculum. The adaption in the Rusitec had not yet been fully completed after 48 hours, since for several species a different abundance was found on day 13 of incubation (Period 2). The provision of fresh substrate at the beginning of Period 2 led to an increase of almost all species and groups within the following two to four hours, with the exception of the protozoa and methanogens. The reduction of the protozoal population and methanogens could have been the result of migration from fermenter liquids to new feed particles and also the fact that some species of methanogens are associated to protozoa. Likewise, sampling of the feed residues after 24 and 48 hours was too late since the microorganisms could have been already detached. However, sampling of the feed residues at shorter intervals is not possible in the Rusitec. No effect of silage was observed on the abundance of total bacteria, methanogens and Selenomonas ruminantium. Methanogens and S. ruminantium do not use the substrate itself but rather fermentation end products of other ruminal microorganisms. Although no effect of silage was observed on the abundance of methanogens, more methane was produced during the incubation of grass silage than corn silage. This is in accordance with results described in literature that an abundance of methanogens is not associated with the production of methane. The corn silage promoted the abundance of protozoa and Ruminobacter aminophilus whereby the latter could only be detected during the first 48 hours of incubation. Quantification was not possible because of the generation of unspecific products during real-time qPCR. Fibrobacter succinogenes was also promoted by corn silage even though this is one of the most active cellulolytic species in the rumen. It could be possible that this species had an advantage in the degradation of the cell wall structures of C4 plants (which corn accounts to) as compared to other cellulolytic species. The grass silage promotes the abundance of the cellulolytic Ruminococcus albus as well as P. bryantii and C. aminophilum. The last two species use amino acids and proteins as energy sources thereby the latter belongs to hyper ammonia producing bacteria which could be the reason for the higher ammonia concentration in fermenter liquids during the incubation of grass silage compared to corn silage. In the second study (Manuscript 2), the effects of supplementing corn silage with different nitrogen sources on ruminal microbial community composition and ruminal microbial crude protein synthesis was investigated. Higher efficiency of microbial crude protein synthesis with corn silage has been reported from in vivo studies, while in vitro contradictory results were found. It was proposed that the different nitrogen content of the silages, and especially the lack of rumino-hepatic circulation in vitro, could be the reason for the lower efficiency of microbial crude protein synthesis when using corn silage. Different microbial species prefer different nitrogen sources as already observed in pure cultures and partially also in mixed cultures. Thus, in the second study, corn silage was supplemented with urea, pea protein, pea peptone or mixed free amino acids. With the supplementation of peptone and urea to corn silage, the highest efficiency of microbial crude protein synthesis was observed followed by amino acids and protein supplementation. But none of the used nitrogen sources allowed corn silage to achieve the level of microbial crude protein synthesis observed for grass silage. The protozoal population was negatively correlated with the efficiency of microbial crude protein synthesis which could have been a result of the higher energy requirement of protozoa compared to bacteria. Furthermore, protozoa engulfs and ingests bacteria and uses it as the main energy and protein source which could also have a negative effect on the efficiency of microbial crude protein synthesis. The effects of different nitrogen sources on the abundance of microbial groups and species not always were in accordance with reports in the literature. This could be attributed to the use of different substrate and pure cultures. In the third study (Manuscript 3), the effect of grass silage and corn silage on the ruminal microbial community composition in vivo was investigated. Three lactating Jersey cows fitted with a permanent cannula were fed with rations based on grass silage or corn silage. The rations contained the same amount of concentrate. In order to adjust the nitrogen content of the rations the corn silage based ration was supplemented with urea. The corn silage based ration promoted the abundance of total bacteria in the solid fraction obtained from the rumen, as well as the protozoal population in the solid and liquid fraction. It could be possible that the higher content of readily fermentable carbohydrates, mainly starch, of the corn silage in combination with the urea could have had a positive effect on both groups. Furthermore, a higher abundance of F. succinogenes was observed in animals fed with the corn silage-based ration. As expected, the cellulolytic bacteria R. albus and R. flavefaciens were enhanced by the grass silage-based ration as well as P. bryantii and R. amylophilus. The latter are known to use starch and maltose as energy source and to use their proteolytic activity only to achieve access to protein-coated starch. It is assumed that the combination of grass silage and concentrate could have had an influence on this species. For S. ruminantium, a feeding effect on the abundance was only observed in the liquid fraction while in the solid fraction no effect was found. There was also no observable effect of ration on the abundance of C. aminophilum, RCC and the methanogens. Changes of the ruminal microbial community over time were observed. But no consistent increase of all species and groups after the phase with the highest dry matter intake of fresh feed became obvious. In conclusion, the results of the current thesis showed that grass and corn silages differently affected the composition of the ruminal microbial community in the liquid and solid fractions of the rumen content. Furthermore, significant diurnal changes of the ruminal microbial community could be observed in vitro as well as in vivo. These results highlighted the need of repeated sampling during the day and suggest the consideration of both ruminal compartments, liquid and solid.
Studies on the extent of ruminal degradation of phytate from different feedstuffs
(2017) Haese, Eva; Rodehutscord, Markus
The predominant storage form of phosphorus (P) in plant seeds and grains is phytate (InsP6). To cleave the phosphate group and, thus, make the bound P available for absorption by the animal, the enzyme phytase is required. Rumen microorganisms show substantial phytase activity, however, recent studies have suggested that the extent of InsP6 hydrolysis in ruminants is variable leading to an incomplete hydrolysis of InsP6 in specific conditions followed by the excretion of P from undegraded InsP6. As P is an essential element for the metabolism in animals it is important to ensure that the animals’ requirements are met. Diets for ruminants are often supplemented with mineral P (Pi). However, the global phosphate resources are finite and the excretion of surplus P contributes to eutrophication of surface water when applied to the farmland with manure in excessive amounts. Thus, dietary P supply is of environmental concern. Better knowledge about ruminal InsP6 hydrolysis could help to optimise the utilisation of InsP6 and, thus, reduce the use of Pi as well as unnecessary excretion of P. Hence, the objectives of the present thesis were to examine the InsP6 hydrolysis from different feedstuffs in ruminants and to identify factors that might affect the extent of InsP6 hydrolysis. In the first study, the total digestive tract disappearance of InsP6 from diets differing in amount and source of P was determined in lactating dairy cows. The results confirmed the high potential of rumen microorganisms to hydrolyse InsP6, but the composition of the diet influenced the extent of hydrolysis in vivo. In the second study, two in vitro experiments were conducted in order to determine the InsP6 hydrolysis from maize grain and RSM. In experiment 1, two diets differing in P- and InsP6-P concentration were fed to the donor animals of rumen fluid. In experiment 2, a diet similar to the high P diet of experiment 1 was fed to the donor animals of rumen fluid and the rumen fluid was mixed with artificial saliva containing Pi (PI: 120 mg Pi/l) or no Pi. Maize and RSM were incubated for 3, 6, 12, and 24 h in both experiments and the InsP6 concentration was analysed in fermenter fluids and bag residues. InsP6 disappearance from maize proceeded faster than from RSM. The disappearance of InsP6 was higher when the diet with high P concentration was fed (experiment 1) and lower when the rumen fluid was mixed with Pi containing buffer (experiment 2). In the third study, the in situ disappearance of InsP6 from five different concentrates was examined. Maize, wheat, RSM, heat treated RSM (hRSM), and soybean meal were incubated in the rumen of fistulated dairy cows fed with three diets differing in P- and InsP6-P concentration. Concentrations of InsP6 and isomers of InsP5, InsP4, and InsP3 were determined in the bag residues after 2, 4, 8, 16, and 24 h of incubation. The disappearance of InsP6 from cereals proceeded faster than from oilseed meals, however, averaged over the diets, after 24 h of incubation 95% had disappeared from all concentrates except for hRSM (57%). Feeding the diet with high InsP6 concentrations increased InsP6 disappearance from oilseed meals but not from cereals, while feeding the high Pi diet did not influence ruminal InsP6 hydrolysis from any concentrate. The results derived from analysis of lower InsPs suggested that intrinsic plant phytase activity plays only a minor role in the rumen and that active phytases in the rumen react differently to changes in the ruminal environment. The results of the present thesis suggest that the composition of the diet fed to ruminants affects the extent of ruminal InsP6 hydrolysis. While high InsP6 concentrations have the potential to increase InsP6 hydrolysis, a decrease of InsP6 hydrolysis can occur after addition of Pi to the diet. Differences in the pace of InsP6 hydrolysis between concentrates occurred which could be of importance at high ruminal passage rates when the time available for ruminal hydrolysis decreases.