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Browsing by Subject "Pansen"

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Comparison of plant cell wall degrading community in the rumen of N’Dama and N’Dama x Jersey crossbred cattle in relation to in vivo and in vitro cell wall degradation
(2004) Nouala-Fonkou, Simplice; Becker, Klaus
This thesis presents a unique combination of an in vivo feeding trial, the analysis of the microbial community structure in the rumen, and in vitro fermentation studies, in order to assess the impact of breeds and diets on animal performance in a West African production setting. Pure N?Dama and N?Dama x Jersey crossbred cattle were fed two basal diets, baby corn and groundnut hay, supplemented with graded levels of either conventional concentrate or moringa leaf meal, to compare animal responses in productivity. In this context, Moringa oleifera leaf meal constitutes a locally available, potential alternative to commercial concentrate for cattle production. The cell wall digesting community of N?Dama and its crosses was analysed using phylogenetically based hybridisation probes to account for the contribution of rumen microbes to differences in fermentation patterns and animal response. In vitro fermentation studies were carried out using the same diets and supplementation levels as fed in vivo, to test the accuracy of the in vitro gas production technique in predicting the optimum level of supplementation. The in vivo feeding experiment focussed on the comparison of breed performance with diets relevant for local production conditions. Six N?Dama and six N?Dama x Jersey (crossbred) animals were used in a cross over design. They were fed consecutively three combinations of roughage and supplement, baby corn stover and concentrate (BCS:Co), groundnut hay and concentrate (GNH:Co) and groundnut hay and moringa meal (GNH:Mo), each at 5 levels of supplementation (0, 10, 20, 30 and 40%). Results from this study showed that there was a clear difference in animal response to different feeding regimes between the two breeds. When averaged over all diets organic matter intake (OMI) was higher in crossbred compared to N?Dama (94 and 87.6 g/kg 0.75 d-1, respectively). When analyzed for the diets and averaged over the breeds OMI was higher when animals were fed the baby corn based diet compared to groundnut based diets (95 against 88 g/kg 0.75 d-1). Only when the diet consisted of BCS:Co, and at low levels of supplementation, N?Dama ingested more than crossbred, but the difference was not significant. With GNH:Co crossbred ingested significantly more at levels of supplementation less or equal to 20%. With GNH:Mo crossbred ingested more, whatever the level of supplementation. The optimum level of supplementation in vivo, estimated with the single slope broken line model, was 10% and 20% for both breeds when they were fed BCS:Co and GNH:Co respectively, but 30% for N?Dama and 10% for the crossbreds when animals were fed GNH:Mo. Organic matter digestibility (OMD) was higher in N?Dama (64.6% against 60.7% in crossbreds) when animals were fed BCS:Co and supplementation had no effect on OMD of BCS:Co whatever the breed. When animals were fed groundnut hay as basal diet, OMD was also significantly higher in N?Dama at low levels of supplementation, but the differences became insignificant beyond 10% and 20% of concentrate or moringa, respectively. With GNH:Co OMD showed a quadratic response (p<0.001) with increasing level of supplementation when it was fed to crossbreds and was not affected when it was fed to N?Dama. Increasing levels of moringa meal supplementation increased OMD in both breeds up to a peak at 20 and 30% for N?Dama and crossbred, respectively. Average daily weight gain (ADG) was not affected by the breed, however it was higher on BCS:Co compared to other diets. On BCS:Co ADG increased with the level of supplementation, reaching a peak at 30%, whereas supplementation had no effect on ADG when animals were fed groundnut hay based diets. As N?Dama could take in and digest more of the low quality BCS:Co diet, they were less efficient in feed conversion under this feeding regime (FCE: 14 vs. 9 for the crossbreds). On GNH based diets, however, N?Dama surpassed the crossbreds in feed conversion efficiency with ratios of 11 vs. 13 for GNH:Co and 9 vs. 27 on GNH:Mo. Rumen microbes play the key role for the digestibility of a given feed and thus also for feed intake and finally animal performance. Obviously, the community composition and activity is highly dependent on the diet. With the present set-up, however, with identical external conditions and three different, well defined diets fed to both, N?Dama and crossbred cattle, a comparison of the microbial community structure between breeds could be attempted. The in vivo and in vitro data taken in the other parts of the study allow a sensible interpretation of potential changes in microbial composition. Rumen fluid was collected from three fistulated N?Dama and three crossbred animals adapted to the experimental diets at medium supplementation level. The cell wall degrading community was analyzed using the phylogenetically based 16S rRNA hybridisation probes. The results showed that on BCS:Co diet the Fibrobacter and R. flavefaciens RNA concentrations were higher in rumen fluid of N?Dama compared to crossbred. These concentrations were also significantly affected by the diet, such that they were higher on baby corn stover compared to groundnut hay based diets. The results of the microbial community analysis suggested that the differences between breeds observed in digestibility could be partially explained by the composition of the cell wall degrading community. Parallel to the in vivo experiment, in vitro fermentation studies were undertaken to evaluate the predictability of the in vivo response to supplementation by the in vitro data. Rumen fluid from 3 N?Dama and 3 crossbred donor animals was used for 24 hour in vitro fermentations. The donor animals were fed consecutively the same three diets used in vivo (BCS:Co, GNH:Co and GNH:Mo) at 20% level of supplementation. Each of these inocula was incubated with in vitro substrates consisting of all the combinations tested in vivo (i.e. 3 diets, 5 levels of supplementation) plus supplement alone. This design should allow to analyze for both, the impact of donor breed as well as that of the donor diet and to conclude which factors may be varied while maintaining predictability. The breed of the donor animals did not significantly affect 24 hour gas production, but short chain fatty acid concentration was higher with rumen fluid from crossbreds when donors were fed BCS:Co and GNH:Mo. Moringa meal as supplement to donor animals changed the fermentation pattern of all the substrates, such that gas production and SCFA increased substantially in groundnut hay based substrates, whereas gas production of BCS:Co substrates decreased and SCFA did not substantially change. In vitro digestibility was higher with rumen fluid from N?Dama whatever the diet of donor animals and the substrate incubated. GNH:Mo as donor diet also increased IVTD of all the substrates. Even though there was no clear response in vivo, this indicates a general stimulation of microbial activity in the rumen and renders moringa leaf meal a promising supplement. Averaged over all data there was a positive correlation (r2=0.53 p<0.001) between IVTD and in vivo OMD. This correlation was much stronger when calculated for a specific diet (e.g. r2=0.90 p<0.001 for BCS:Co, averaged over the breeds). Analyzing the data for the individual breeds affected correlations only to a minor degree. Thus, when testing a supplementation strategy in vitro, it should be important that donor animals are fed the same components (roughage and supplement) that will be combined at different levels in vitro, whilst the breed of donor animals may be of second importance. This work provides conclusive evidence that in vitro incubations may be used to design supplementation strategies, thus reducing the need for in vivo experiments. Moringa leaf meal is a promising local resource to substitute for conventional concentrate. Differences in productivity between breeds could be correlated to (and may be partially manifested through) a divergent community structure of rumen microbes. That, in turn, indicates that animals of different breeds might have a ?genetic background? that favours the establishment of a certain community, even if the animals are kept under identical conditions. This relationship should be investigated by more advanced molecular techniques.
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.
Effects of monensin and tannin extract supplementation on methane production and other criteria of rumen fermentation in vitro and in long-term studies with sheep
(2013) Wischer, Gerald; Rodehutscord, Markus
Ruminants increasingly attract public concern due to their methane release and contribution to the greenhouse effect. One strategy to reduce the release of methane is to modify microbial fermentation in the rumen by the use of feed additives such as monensin and tannin extracts. However, other characteristics of fermentation including the synthesis of microbial protein may also be affected. The aim of the present studies was to provide a comprehensive evaluation of the effects of monensin and tannin extracts on ruminal fermentation and methane production. The ionophore monensin is known to increase feed efficiency in ruminants. Although the use of silages is common practice in cattle feeding, the effects of monensin on the fermentation of silages in the rumen and microbial protein synthesis are lacking. Monensin has often been described to have indirect effects on methane production resulting from its effects on feed intake, protozoa and Gram-positive bacteria. It has rarely been studied whether monensin can reduce methane production without adverse effects on other criteria of rumen fermentation. The first objective therefore was to investigate the effects of different dosages of monensin on methane production and microbial protein synthesis when supplemented to different silages in two in vitro systems (Study 1). In Experiment 1 of Study 1, 15 g of oven-dried grass silage alone or combined with a concentrate was incubated in a rumen simulation (Rusitec) over a period of 13 d to examine the effects of monensin supplementation (2 or 4 mg/d, n = 4) on the production of total gas, methane, volatile fatty acids (VFA), degradation of nutrients and microbial protein synthesis. In Experiment 2 of Study 1, different dosages of monensin (0.5, 1, 2, 6 and 10 µg) were supplemented to syringes containing 120 mg of grass silage alone, grass silage combined with concentrates, or maize silage alone. After 24 h of incubation the effects of monensin on total gas, methane and VFA production were determined. In Experiment 1 monensin inclusion to grass silage and grass silage combined with concentrate resulted in a decreased total gas, methane and acetate production, while propionate production was increased. Along with a decreased degradation of crude protein, ammonia concentration in the system was reduced. While microbial protein originating from solid associated microbes decreased with monensin inclusion, microbial protein from liquid associated microbes was increased, resulting in an increase in total microbial protein synthesis. In Experiment 2, different dosages of monensin reduced methane production in grass silage (17%), grass silage combined with concentrate (10%) and maize silage (13%) without adverse effects on total gas production. Based on these two in vitro experiments it was concluded that monensin is able to reduce methane production without a major decrease in total gas and VFA production and degradation of organic matter. Although microbial fractions were differently affected, the total microbial protein synthesis was increased upon monensin supplementation. Tannins are secondary plant compounds that are known to complex with feed and microbial proteins. Several products from this heterogeneous group have shown potential to affect rumen fermentation in vivo and, even more, in vitro, but are often accompanied by negative effects on digestibility, feed intake and microbial protein synthesis. In Study 2 of the present work, ten tannin extracts (chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed) and four monomers of rapeseed tannin (pelargonidin, catechin, cyanidin and sinapinic acid) were screened in grass silage based diets in successive runs using the Hohenheim Gas Test. The objective was to determine the optimal dosage of each tannin extract to cause a maximal methane reduction without negative effects on total gas production. Whereas the supplementation of pelargonidin and cyanidin to grass silage did not reduce methane production; catechin and sinapinic acid reduced methane production without affecting total gas production. Except tara extract, all tannin extracts reduced methane production by 8 to 28% without adverse effects on total gas production. Based on these results, chestnut, grape seed, myrabolan, sumach and valonea extract were investigated in a second step in a Rusitec to determine their effects on degradation of nutrients, VFA and ammonia production, and particularly on microbial protein synthesis. All tannin extracts were supplemented at similar dosages of 1.5 g to 15 g of grass silage. The supplementation of chestnut resulted in the greatest decrease in methane production (63%), followed by valonea (35%), grape seed (23%), sumach (18%), and myrabolan (7%; not significantly different from the control). While chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production; however, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry and organic matter was reduced by all tannin extracts, but there were no differences between tannin treatments. Crude protein degradation and ammonia production were also reduced by tannin extract supplementation. Microbial protein synthesis and its efficiency were not affected by tannin supplementation, which indicates that a reduction in methane production due to tannin extract supplementation is possible without negatively affecting microbial protein synthesis. Chestnut and valonea extract had the greatest potential in reducing methane production without negative effects on rumen fermentation of grass silage and microbial protein synthesis. Therefore, these tannin extracts were investigated for their long-term effects in sheep (Study 3). In Experiment 1 of Study 3, sheep receiving the control, chestnut or valonea treatment (each n = 4) were fed 842 g/d of hay (fresh weight). The animals on the control treatment also received 464 g/d of concentrate, and animals on the tannin treatments received the same amount of concentrate but were also fed 20 g of the respective tannin extract. Following initiation of tannin feeding, methane release from sheep was measured in 23.5 h intervals in respiration chambers on day 1, 8, 15, 29, 57, 85, 113, 148, and 190. In three balances periods faeces and urine were collected for 6 and 3 days, respectively. Effects on nutrient digestibility, nitrogen and energy metabolism were evaluated, with microbial protein synthesis estimated from the urinary excretion of purine derivatives. Based on the results of Experiment 1, a second experiment was conducted four month after the start of Experiment 1. Experiment 2 had the same study design and data collected, but the dosage of tannin extracts was doubled compared to Experiment 1 (0.9 vs. 1.7 g tannin extract/kg body weight) and the duration was shorter (85 days). Hay and concentrates used in both experiments were also evaluated using the Hohenheim Gas for their effects on total gas and methane production. In both experiments, methane release was not significantly reduced by tannin extract supplementation when analysed over the whole experimental period. In Experiment 1 the supplementation of chestnut extract on day 190 resulted in a reduced methane release. In both experiments, on day 1 a numeric reduction in methane release for the tannin treatments was observed, with a greater reduction recorded for the higher dosage used in Experiment 2. This trend disappeared by day 57. In the third balance period of Experiment 1, digestibility of dry and organic matter was reduced by tannin supplementation. The digestibility of crude protein was reduced in both experiments, whereas the digestibility of fibre fractions was not influenced. In both experiments a long-lasting shift in nitrogen excretion from urine to faeces was observed, which occurred to a greater extent in Experiment 2. The urinary excretion of purine derivatives was not significantly affected by tannin supplementation, indicating that the microbial protein synthesis was not altered in either experiment. The in vitro methane production was reduced for concentrates containing tannin extracts, but it was not significantly affected when concentrates were incubated with hay. It is concluded that monensin added to different silages caused a decrease in methane production without affecting total gas production but with an increased microbial protein synthesis. Nine of the ten considered tannin extracts and two tannin monomers decreased methane production without affecting total gas production. The Rusitec study confirmed the great potential of chestnut and valonea extract to reduce methane production without negative effects on microbial protein synthesis. However, neither chestnut nor valonea extract reduced the methane release in sheep when fed over a longer period of time. It is assumed, that rumen microbes adapted to the tannin dosages in terms of methane release but not nitrogen metabolism, as there were long-lasting effects on nitrogen excretion. The shift in nitrogen excretion can have a positive effect on the environment due to the reduced potential of ammonia emission from the urine. Both in vitro systems used in the present studies showed effects of tannin extracts that were considerably different from those observed in sheep. The monomers investigated in the present study are the basic units of condensed tannins, whereas the tannin extracts selected in vitro only contain hydrolysable tannins. It is possible that monomers of chestnut and valonea extract may reduce methane production, whereas higher dosages of these tannin extracts cause negative effects on feed intake, digestibility and microbial protein synthesis. Further investigations should focus systematically on the transfer of in vitro studies to estimate in vivo responses. Therefore, a parallel implementation of different in vitro and respiration studies would be of great value.
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 ruminal degradation of nutrients and feeding values of single feeds and compound feeds for cattle
(2020) Grubjesic, Goran; Rodehutscord, Markus
The environmental impact of intensive animal farming has been steadily increasing. Cattle can contribute to environmental pollution due to relatively low nitrogen (N) and phosphorus (P) utilisation, leading to their excess excretion. High-yielding dairy cows are commonly fed concentrate compound feed, in mash or pelleted form, to satisfy high protein and energy requirements. Main source of energy in concentrate compound feeds is starch (ST). For the accurate formulation of compound feeds, comprehensive insight into nutritive values of single feeds as well as their potential interactions (associative effects) when mixed is needed. Typically, the nutritive values of single feeds are considered to be additive, assuming that no associative effects exist. However, data supporting such assumption for concentrate feed are scarce. The present thesis had two aims: evaluation of additivity of ruminal degradation of nutrients and feeding values of single concentrate feeds in compound feeds, and evaluation of effects of pelleting on ruminal degradation of nutrients and feeding values of compound feeds. Twelve single feeds were used to formulate eight compound feeds in different combinations, targeting crude protein (CP) concentrations from 16 to 30% in dry matter (DM). Compound feeds were prepared both, in mash and pellet form in a commercial feed mill using standard industrial conditions. Ruminal degradation of single and compound feeds was evaluated using in situ and different in vitro techniques. The in situ incubations were conducted by incubating samples of all single and compound feeds in polyester bags for 2, 4, 6, 8, 16, 24, 48, and 72 hours in three ruminally fistulated dairy cows. Bag residues were analysed and the ruminal effective degradability (EDIN_SITU) of CP and ST, was calculated for passage rates of 5 and 8%/h. Phosphorus is located in plants as phytate (InsP6), and for some feed samples the EDIN_SITU of InsP6 was also determined. The in vitro gas production (GP), digestibility of organic matter (dOM), metabolisable energy (ME), and utilisable CP at the duodenum (uCP) were evaluated using Hohenheim Gas Test and extended HGT. Intestinal digestibility (IDRUP) of ruminally undegraded protein (RUP) was determined using a three-step enzymatic method through incubation with pepsin and pancreatin. Chemical fractionation of CP was performed according to the Cornell Net Carbohydrate and Protein System (CNCPS) The CP fractions can be also used to predict EDIN_SITU. Assessment of additivity was performed by comparing the observed values of compound feeds with values for compound feeds calculated from single feeds. It was concluded that additivity of single feeds in mash compound feeds was given for EDCPIN_SITU, EDSTIN_SITU (Manuscript 1), uCP, CP fractions, GP, and dOM (Manuscript 2). Here, associative effects among single feeds were considered to be small and should not affect formulation of concentrate compound feeds. The GP and proximate nutrients are necessary to estimate ME using appropriate equations, often specific for feed or feed type. The additivity of ME was given only when same ME equation for single and compound feeds was used. Additivity was not given for IDRUP (Manuscript 2). Pelleting had overall small effects on feeding values of compound feeds determined in situ and in vitro (Manuscripts 1 and 2). Presumably, the relatively low intensity of heating (up to 80–90°C) during the pelleting process was not sufficient to significantly affect nutritive value of compound feeds, with the exception of decreased IDRUP. Overall, it was concluded that additivity of ruminal degradation of nutrients and feeding values of single feeds in mash and pelleted compound feeds can be assumed for practical feed formulation. While some associative effects were detected, they might be related to methodological causes in most of the cases.
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.
Ruminal degradation characteristics of barley, rye, and triticale grains assayed in situ and in vitro, and by near-infrared spectroscopy
(2017) Krieg, Jochen; Rodehutscord, Markus
The milk yield of dairy cows and related energy and protein requirements have steadily increased in the last few decades. Since feed intake has not increased to the same extent as nutritional requirements, the concentration of nutrients in mixed rations had to be increased. An increase in energy concentration is often achieved by the inclusion of high levels of cereal grains. In the EU—apart from wheat—barley, rye, and triticale are widely cultivated cereal grains. Starch (ST), followed by crude protein (CP), is the main constituent of cereal grains. The rate and extent of ruminal CP and ST degradation can influence the performance and health of dairy cows, but data that can enable the comparison of ruminal degradation within and between barley, rye, and triticale grains are scarce. Commonly used techniques to explore ruminal degradation of feed are in situ and in vitro incubations. Both techniques require ruminal-fistulated animals, but alternative methods are being demanded by the community, in order to reduce the number of animal trials. An approach with the potential to estimate the nutritional value of various feeds is near-infrared spectroscopy (NIRS). The present thesis has two major parts. In the first part, ruminal degradation parameters and the effective degradability (ED) of DM, CP, and ST from barley, rye, and triticale grains are investigated using standardised in situ and in vitro incubation techniques. A total of 20 genotypes per grain species were used. In the second part, NIRS calibrations were developed with the aim of estimating the CP and ST concentrations of cereal grains and their incubation residues. Subsequently, data from in situ experiments were used to establish the calibrations for estimating the ruminal in situ degradation of cereal grains from their spectral data. In situ degradation studies have been conducted by ruminal incubation, utilising three lactating cows. Ruminal degradation parameters and ED (ruminal passage rate = 8%//h) were calculated. For in vitro incubations, the samples were incubated in a rumen fluid-buffer mixture (‘Hohenheim Gas Test’). The gas production was recorded for estimating gas production kinetics. In vitro gas production—in combination with crude nutrient concentrations—was used to estimate the metabolisable energy concentration (ME) and digestibility of organic matter (dOM). The degradation rates differed between and within the grain species for DM, CP, and ST. The variation within grain species was not reflected in the ED of CP and ST, due to the relatively fast and almost complete degradation of the grains. The ED of CP was 77% (69–80%) for barley, 85% (83–86%) for rye, and 82% (79–84%) for triticale. The corresponding ED of ST was 86% (82–88%), 95% (92–96%), and 94% (90–95%). Accordingly, the estimated ME (barley: 13.5 MJ/kg DM, rye: 13.9 MJ/kg DM, triticale: 13.5 MJ/ kg DM) showed only relatively minor variation within one grain species. The dOM was overall at a high level (barley: 91.3%, rye: 95.3%, triticale: 95.8%). The relatively small variation within one grain species could not be explained by the chemical and physical characteristics of the samples. Hence, it was concluded that it is feasible to use mean values for every species in feed formulation and ration planning. In the second part of this thesis, it was shown that it is possible to replace chemical CP and ST analyses of samples from in situ studies by NIRS without affecting the calculated ruminal degradation characteristics. NIRS could be used to estimate the ED of CP and ST from cereal grains. The sample set to establish the calibrations included barley, durum, maize, rye, triticale, and wheat grains. Calibrations for the CP and ST concentration were extended to pea samples. The calibrations with the best validation performance for CP and ST concentration were obtained by using the wavelength segment of 1250 to 2450 nm and the first derivative of the spectra (CP: R2 = 0.99; SEP = 0.46% DM. ST: R2 = 0.99; SEP = 2.10% DM). The results of in situ studies did not differ, irrespective of whether chemical or NIRS analysis was used. Like the CP and ST concentration, the ED was estimated with a high accuracy (ED8 CP: R2 = 0.95; SEP = 2.43%. ED8 ST: R2 = 0.97; SEP = 2.45%). However, calibrations need to be extended before they can be recommended for routine use. The present thesis demonstrates that the ED of CP and ST of barley, rye, and triticale grains differ between the species, but variation within one grain species is relatively small and not related to the chemical and physical characteristics of the grain. Hence, under the prevailing cultivation conditions, the mean values for each grain species in feed evaluation are deemed adequate. It was demonstrated that NIRS has the potential to facilitate the evaluation of the nutritive value of cereal grains for ruminants.
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.
The effects of rumen nitrogen balance on nutrient digestion, protein metabolism, and performance of dairy cows as influenced by diet composition
(2021) Kand, Deepashree Dilip; Dickhöfer, Uta
Feeding excess dietary crude protein (CP) beyond the requirements of dairy cattle and microbes in the rumen increases production costs for farmers, excretion of nitrogen (N) to the environment, and has negative effects on the cows’ health and reproductive performance. Researchers have been interested in exploring the effects that diets with negative rumen nitrogen balance (RNB) may have on the dairy cattle and their rumen function. Results so far have been inconsistent may be due to the performance level of the animal with high-yielding dairy cows being more sensitive than low performing ones. Moreover, it may be supposed that variable responses to negative RNB in different studies may at least partly be related to varying ingredient composition and the type of main carbohydrate or N sources in the animals’ diets. The overall objective of the thesis was to generate a comprehensive understanding on the effects of interactions between the RNB levels and carbohydrate and N sources in cattle diets on rumen fermentation, the efficiency of microbial CP synthesis, and on N use efficiency in vitro and in vivo. The results of the present thesis indicate that the effects of negative RNB levels may vary with dietary composition in dairy cows. Therefore, outlining a single minimum RNB balance threshold for dairy cattle diets may not be appropriate when optimizing N utilization in dairy cows, because several animal and dietary factors modify the requirements of rumen microbes.
The intestinal microbiome and metabolome of dairy cows under challenging conditions
(2022) Tröscher-Mußotter, Johanna; Seifert, Jana
The modern dairy cow is confronted with a multitude of stressors throughout live. Especially calving, transition, and microbial infections are strong challenges that can have long-lasting impacts on the cow’s health and performance. Yet, individuals can differ in their response towards these challenges, raising the question which characteristics in the dairy cow contribute to a more or less robust animal. Apart from genetics, the gut microbiome and the entailed metabolome is assumed to play an important role in buffering or promoting host stress. This is also due to the fact that the gut microbiome is strongly involved in the hosts energy metabolism and immune system. As dairy cows often show performance impairments during high energy demanding periods, it could be suggested that improving energy metabolism in these specific phases might reduce the negative phenotypic outcomes. This was tested using dietary L-carnitine, a metabolite inevitably necessary for energy metabolism. However, no supplement effects on the intestinal microbiome or metabolome have been found in the present work. Supplementation was continued throughout the complete trial. Calving functioned as an individual stimulus, and an intra-venous LPS injection induced a standardized inflammatory challenge, as a specific amount of LPS per kg of bodyweight was applied per cow. Supplemented animals were compared to a control group. In total, the animals were studied across 168 days and sampled extensively at several sites. The focus of this thesis was to analyze the bacterial consortia and metabolites of both, host and bacteria, in rumen, duodenum, and feces throughout the given period. This was to elucidate the metabolic reactions and bacterial shifts during the mentioned challenging periods and their response to the L-carnitine supplementation. First, the ruminal and duodenal fluid microbiome of eight double cannulated animals during the two respective challenges was analysed. Before calving and feed change, rumen and duodenal fluid bacterial consortia were significantly different, thereafter very alike. Strong microbial community shifts were observed throughout the complete trial irrespectively of the matrix. Both matrices varied in their metabolite patterns indicating functional variation among sites. Also, a strong increase of Bifidobacterium at three days after calving was observed in almost all animals pointing towards a strong biological purpose. This needs to be investigated in upcoming studies. The study could show increasing ketogenic activities in the animals after calving and proposes a possible protective host-microbial interaction, against a ruminal collapse induced by LPS challenge, here described as "microbial airbag". The second part included fecal samples of the same animals, which were analyzed for their bacterial consortia and targeted metabolites. Different dynamics and diversities of microbial communities amongst the individuals were observed, according to which animals could be grouped into three microbiome clusters. These showed in part fundamentally different metabolic, health, and performance parameters, indicating strong host-microbiome-metabolite interactions. The study demonstrated that microbiome clustering may contribute to identifying different metabo- and production types. Again, the study observed a strong increase of Bifidobacterium at three days after calving and even during the LPS challenge supporting the findings of the former study. This strengthens the hypothesis that also for the cow Bifidobacterium may have protective effects, as this genus is largely involved in health promoting activities. The power of this project lies in the massive sampling of different body sites in dairy cows across a very long period of time and finally, merging of the collected data. This, however, requires high computational efforts as numerous time points, matrices, animals, measurements, treatments, feeding regimen, and challenges resulted into a large bandwidth of parameters and metadata. Yet, it bears the potential to better elucidate and understand actions and reactions of the host, its microbiome and metabolism, as well as organ-axes in dairy cows and thereby gaining a more holistic picture of these complex animals. The aim of analyzing the host, its microbiome and metabolome throughout challenging periods resulted into the following main findings. Time, calving, and feed change remarkably change the microbial communities and to a lesser extent the metabolomes in all three matrices. Rumen and proximal duodenal fluid samples significantly differ in their metabolomes but not in their microbiome. In all matrices, an increase of Bifidobacterium is seen within three days after calving, which has to be further researched. Across the herd, three distinct microbiome clusters are found, which significantly differ in their production and health parameters.

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