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Degradation characteristics of feed components have been subject of numerous different studies during the last decades and on basis of this information feed-evaluation systems have developed (like for example the Dutch OEB/DVE system). These systems aim to match nutrient supply with the requirements of dairy cows and are based on protein and energy level of feed. It is unclear however where in the digestive tract and how much of the feed-stuff is digested resulting in useful end-products for the dairy cow. These systems can therefore not predict how nutrient uptake of the dairy cow will be influenced by changes in the diet, digestion of feed is not a static process and depends of several interrelating factors. Inclusion of rumen-passage is necessary to change from an animal-requirement to an animal-response system where responses of dairy cows to changes in the diet can be predicted . Efficiency of nutrient utilization can be optimized if the site and extent of digestion is known. Nutrients fed in excess leads to losses of nutrients to the environment, undersupplementation of nutrients leads to production losses. This indicates it is very important to gain more insight in digestion processes of the dairy cow.
A major component of roughage is the cell-wall fraction, Neutral Detergent Fibre (NDF). For maize-silage the NDF-content varies roughly between 30% and 40% on dry matter basis. NDF consists of several different components, since NDF represents the entire cell-wall fraction. The three different components which form together the NDF-fraction are: cellulose, hemicellulose and lignin. The NDF fractions can be divided into two parts: the digestible NDF fraction (dNDF) and the indigestible NDF fraction (iNDF). An important function of NDF is maintaining peristalsis by the dairy cow and next to the easily fermentable carbohydrates, like starch, the NDF fraction provides energy that can be used by the dairy cow. NDF is known to be a major factor in maximal rumen fill. NDF is of importance for rumen microbes as well, rumen microbes attach to the cell-wall fraction, enhancing growth. Digestibility of the NDF fraction of course is of great influence on availability of nutrients. NDF-digestibility for maize-silages varies roughly between 50% and 60%. Since NDF digestion only takes place by rumen microbes , fractional passage rate is very important for the extent of NDF digestion. NDF that passes out of the rumen will not be further degraded.
Aim of this study is to get more insight in ruminal passage behaviour of the cell-wall fraction of ensiled maize silage. Since most research has focussed on degradation characteristics, and assumed fractional passage rate to be equal for different feed-stuffs, not many data is available yet for passage kinetics. Goal of this research is to obtain quantitative data for NDF passage kinetics.
Hypothesis: maize silage with a higher NDF content passes out of the rumen faster compared to maize silage with lower NDF content, depending on NDF content and degradability, assuming equal amount of starch content.
During the past decades it has become clear that not only degradation plays a role in the nutrient degradation and absorption process, but also passage of feed components out of the rumen has a major impact. Passage rate is considered constant in current feed-evaluation systems (0.045/h for roughage and 0.060 for concentrate for the OEB system ), but several studies show a different passage rate depending on various factors, e.g. forage quality, feed intake and forage handling . Differences in passage rate have a major impact on the place and extent of digestion of feed-components. A low passage rate leads to a higher digestion of feed-components in the rumen by the rumen microflora compared to a high passage rate. The forestomach is the major site for Neutral Detergent Fibre (NDF) digestion, 93% of NDF digestion takes place in the forestomach, less than 5% of NDF digestion occurs in the hindgut . Therefore fractional passage rate is of great importance for NDF-digestion. Once it passes out of the rumen NDF will not be further degraded.
Most used current techniques to determine fractional passage rate of NDF use external markers, like Cr-NDF. It is not clear however which pool is actually being represented by this marker . New techniques involve internal markers for more accurately determining the 'real' pathway of the feed, instead of one feed-fraction only. 13C has shown to be an accurate marker for determining rumen passage . After administration of the marker, sampling of faeces and or digesta is necessary to recover the marker resulting in excretion curves. Different sampling methods are described in literature, mainly faecal and duodenal sampling are used for determining fractional passage rate. Many factors seem to influence fractional passage rate of feeds. The next sections will deal with the marker technique and fractional passage rate.
The history of markers used to determine passage of nutrients through the digestive tract of dairy cows is long. Ideal markers are markers that are indigestible, so the marker can be fully recovered. An ideal marker should not be absorbed or affected by digestion processes . This is an important feature of markers, since markers have to be recollected. Degradation or absorption of the marker will lead to a recovery rate differing from 100% which results in an over/underestimation of passage. The application of markers can be divided into two different systems: internal and external markers. Clear differences are shown between these markers. Internal markers are markers that are included in the feed-stuff, external markers are added to the feed in order to mimic the digestion and passage of the normal feed. Examples of external markers are Cr-NDF and Co-EDTA, which are used to represent the solid and liquid phase respectively of rumen contents. Examples of internal markers are 13C and lignin (which is an inert internal marker). The markers can be pulse dosed or continuously supplied to reach steady state . Pulse dosing is used to determine fractional passage rate in specific parts of the gut , resulting in less accuracy compared to a steady state approach but a higher precision. The kind of marker used (internal or external) seems to have considerable influence on fractional passage rates .
A large variety of substrates have been used as external marker for determining rumen-passage behaviour. External markers aim to mimic passage behaviour of the feed particles. It can be doubted however whether external markers succeed in determining passage behaviour. Different markers are generally used for determining passage behaviour of different fractions of the rumen content. The majority of passage behaviour studies uses transition metals, mainly Cr-NDF , or rare earth elements like Ytterbium as marker for the solid fraction in the rumen and Co-EDTA for the liquid fraction of the rumen contents. Passage studies started with colouring of indigestible feed particles as a marker, followed by simply counting coloured particles in the faeces .
The negative effects of external markers in predicting passage behaviour is illustrated here for the most used external marker in passage behaviour studies. The marker Cr-NDF consists of straw, which is impregnated with Cr, representing the indigestible NDF fraction. Cr is known not to be absorbed by the digestive tract of the dairy cow, and can therefore be greatly recollected in the faeces, 98% of this marker has been found to be recollected . It has become clear however that the Cr and the particle size of the straw do influence the digestion and passage behaviour of the straw . The impregnated straw doesn't undergo all fermentation processes, since it is greatly indigestible. Digestible cell-wall components have shown to have a lower rumen outflow rate compared to indigestible cell-wall components . Therefore it has been generally concluded the Cr-NDF method overestimates fractional passage rate, since it consists of indigestible particles only. The Cr-NDF particles represent indigestible fibres with the same functional specific gravity as the marker used only.
Internal markers form an integral part of the roughage consumed by the dairy cow and therefore have the advantage to follow the 'normal' way of digestion and absorption through the digestive tract of the dairy cow . First internal markers consisted of indigestible or insoluble parts of the feed. The assumption is that if the feed-component is not being digested or absorbed, it will behave like an ideal marker. Lignin is greatly indigestible and therefore has been used as internal marker for determining rumen passage in several studies. Advantage of this marker is that because of the indigestibility this fraction should be entirely retained by faecal collection. Literature however shows recovery of indigestible lignin is inconsistent . Another disadvantage of indigestible lignin as marker for determining fractional passage rate representing the entire particle fraction is that it has become clear that not every part of the cell-wall components behaves the same way in terms of fractional passage rate. The indigestible part, of which the lignin fraction is a major component, shows to have a higher fractional passage rate compared to the digestible part of the NDF-fraction . Therefore lignin is not a representative marker for the entire particle fraction of the diet and cannot predict the site of digestion of feed components. Inert internal markers like for example lignin, rumen indigestible neutral detergent fibre and insoluble ash have commonly shown to have low predictive value . It is not clear whether the marker recovered in the faeces is the same marker as in the diet .
A solution for the unclear recovery of internal markers is by intrinsically labelling of the feed with isotopes. 14C was one of the first isotopes used to label feed, (e.g. by Smith ) but since 14C is an unstable carbon isotope and therefore radioactive this is not an ideal marker; not for the health of the experimental animals nor for human health. Besides, labelling of forage with 14C is expensive. The search for new markers has shown two interesting stable isotopes: 13C, which is a stable carbon isotope and 15N which is a stable nitrogen isotope. These markers occur naturally at low values in nature, and therefore are useful in terms of recovery. 15N is used as marker for the NDF-fraction by Huhtanen and Hristov , but microbial contamination has shown to be considerable . To overcome the problem of microbial contamination 13C is used as marker for the NDF-fraction.
Natural differences in 13C abundance in plants has been used in order to determine passage characteristics. C3 plants are known to have lower 13C values compared to C4 plants. Tas et al. used this natural difference in 13C content for marker characteristics by replacing concentrate (50% of diet at DM-basis and consisting of C3 plants only) by a C4 based concentrate to obtain differences in enrichment. By pulse dosing with a large amount of feed naturally containing a significant different 13C/12C ratio, it is possible to determine passage by changes in 13C/12C ration in the faeces. Disadvantage of this method is that changing the ration (C3 versus C4 plants) influences rumen processes as well.
To overcome the problem of drastically changing the ration for changing the 13C content in the rumen, is by intrinsically labelling of feed components with 13C . Only small amounts (approximately 30 g) of labelled feed have to be pulse-dosed into the rumen for obtaining passage dynamics, enrichment levels are extremely compared to 13C background values. Advantage of this method is that the amount of 13C that will be ingested by the cow can be accurately determined and no changes in feeding regime have to be applied. Feeds can be labelled on the field using greenhouses . Labelling procedure is as follows: a greenhouse is placed in the field, the roughage is labelled by continuous 13CO2 infusion under greenhouse conditions. Continuous infusion is necessary to reach required constant levels of the 13C vs 12C ratio for all the plant components , therefore infusion with 13CO2 have to be done during the entire growth period of the roughage, until harvesting.
The NDF fraction consists of several components, the enrichment of these components differs as well . For grass silage these values where estimated at 628,4° for the hemicellulose fraction, 595,6° for the cellulose and 389,2° for the lignin fraction. The lower enrichment level for lignin is assumed to be because of discrimination against 13C during formation of lignin-precursors . A difference in enrichment might cause problems, since the different NDF components show differences in degradation characteristics as well. Calculations have shown however that the NDF/ADL ratios hardly change during degradation processes, so no effect of differences in enrichment level is assumed . In order to determine passage of feedstuff out of the rumen the ratio of 12C vs 13C is being used. Because the 13C is built into the plant cell-walls, it will just like the 'normal' 12C fraction be digested and fermented. Not all 13C which is pulse dosed into the rumen will be recovered in the faeces, since the 13C is just like the 12C fraction fermented in the rumen. To determine fractional passage rate it therefore is very important to look at the ratio of 12C/13C. It is assumed the 13C fraction will undergo the same degradation pathways as the 12C fraction, both fractions will be equally fermented by rumen microbes. The ratio of 12C/13C therefore does not change.
Fractional passage rate increases with increasing fractional degradation rate . Fractional degradation rates differ for the different NDF-fractions. Lignin is greatly indigestible, cellulose and hemicellulose fractions differ slightly in digestibility, depending on the amount of content . Logically the NDF-fractions are expected to behave different regarding fractional passage rate, resulting in different excretion curves in time. Differences in enrichment of the different NDF-fractions might result in differences in excretion curves (higher or lower enrichment peaks) for different NDF-fractions.
Different ways of recollection of the marker have been applied. Recollection of the marker is necessary for determining passage characteristics by calculating excretion curves. Recollection methods include for example:
Chyme sampling from the intestine
Faecal sampling is the most used, easiest to apply and least invasive for the animal technique. An indigestible marker is ingested by the cow, or pulse dosed into the rumen. Faeces are collected, concentration of the marker in the faeces is determined resulting in excretion curves.
Chyme sampling from the small intestine is done by cannulating the dairy cow. Advantage of this method over faecal sampling is that the influences of the large intestine on the chyme are excluded. Measurements are closer to the rumen, so rumen passage can be closer determined.
Rumen evacuation involves the use of fistulated dairy cows. A marker is thoroughly mixed with the rumen contents. At several pre-defined measurement points the rumen is emptied and a representative sample is taken. Concentration of the marker in the rumen-contents will decline, indicating the fractional passage rate.
For the slaughter technique an indigestible marker is ingested by the cow, after a certain period of time the cow is slaughtered. From all the different parts of the digestive tract the contents can be collected separately, marker concentrations can be determined.
Sampling of digesta from the omasum is a relatively new method and has shown to be a good alternative for determining fractional passage rates of feed particles directly after the rumen . Omasal sampling provides the possibility of investigating rumen processes directly. When compared to sampling from the abomasum or intestine, omasal chyme-samples are not biased by abomasal and endogenous secretions, which are significant, and other post-rumen degradation/absorption processes. The technique is less invasive compared to duodenal sampling, only a rumen cannula is required. Huhtanen et al. developed an omasal sampling protocol. Aim of this protocol is to interrupt the rumen-processes as least as possible. A sampling device was put through the rumen-omasal orifice with a hose attached to it. Sampling was done through this hose, the sampling device stayed into the rumen-omasal orifice during the entire experiment. It was concluded that the presence of the sampling device had no effect on dry matter intake and behaviour of the dairy cows. The method of sampling however did cause some problems, resulting in decreased dry matter intake, which again resulted in deviating fractional passage rates. Recent insight gained by a study of Krizsan et al. shows that sampling from the reticulum gives the same results compared to omasal sampling, but leads to even less interference with the animal.
Fractional passage rate
Rumen functioning includes two competing processes: degradation and passage. Fractional passage rate of rumen components determines site and extent of digestion of ingested feed, and has shown to be feed-specific. Even individual fractions within a feed (e.g. starch and cell wall-fractions) show differences in passage behaviour . The most important factors regulating the passage of the feed are the functional specific gravity and particle size . Since not all particles can escape from the rumen due to their physical position, specific gravity is expected to be the most limiting aspect for passage .
Although passage out of the rumen is a well-recognised system for a long time, main focus has been on digestion of feed. Fractional passage rate is defined as the fraction of total rumen pool size leaving the rumen per unit of time and is the reciprocal of Mean Retention Time. All feed ingested by the dairy cow passes the rumen. The feed is partly being digested here by the rumen-microbes, this fraction is absorbed or digested to form microbial protein and volatile fatty acids. Nutrients leave the rumen via two pathways. Useful end-products of digested nutrients are absorbed through the rumen wall, the fraction that passes out of the rumen consists of rumen indigestible/resistant components and microbial protein. Starch for example has a rumen resistant component, the fraction that passes out of the rumen is digested in the hindgut. The indigestible part of the rumen content consists mainly of the NDF fraction. NDF digestion takes place (only) in the rumen . NDF is a source of energy for the rumen microflora and the requirements of the dairy cow, next to of course the more easily fermentable carbohydrates (e.g. starch). NDF is only partly being digested (around 50-60% for maize silage), it is very clear however that an interaction of NDF and fermentation of other carbohydrates exists. Research shows that an increase in starch supply and digestion results in a lower NDF digestion . The positive effect of the increase in available nutrients derived from extra starch degradation is therefore partly compensated by less nutrients originating from NDF degradation.
Fractional passage rates in literature
shows the results of different passage rate studies. Different methods have been used for determining fractional passage rate, but the most used is clearly Cr-NDF. This table shows the influence of several factors on passage behaviour. Fractional passage rates in literature show a wide range of variation.
Table Fractional passage rates in literature
Bosch and Bruining researched the influence of NDF content of the diet on passage behaviour. Concluded was that a higher NDF content in the diet results in a higher fractional passage rate. Cr-NDF was used as marker in this experiment. The accuracy of Cr-NDF as marker for the entire particle phase in passage studies can be doubted however. Bruining and Bosch and Lirette and Milligan have researched the effect of particle length of the Cr-NDF marker and discovered significant differences, indicating the Cr-NDF marker represents fractional passage rate only for particles of the same size and the same functional specific gravity as the marker used. Hristov et al. emphasizes the importance of functional specific gravity for fractional passage rate. Fractional passage rate of two fractions of feed particles (functional specific gravity higher or lower than 1.02) were labelled with two different markers, significant differences in fractional passage rates were found. Huhtanen and Hristov used another approach for determining fractional passage rate. The indigestible fibre fraction was intrinsically labelled with 15N. This fraction is considered to undergo the same degradation pathways as normal feeds, so the negative aspects of the Cr-NDF are supposed to be overcome. Fractional passage rate is here found to be higher compared to the values found with the Cr-NDF method. Lund et al. researched fractional passage rate of different NDF-fractions. This research shows the Digestible NDF part behaves different from the Indigestible NDF part considering fractional passage rate indicating a selective passage of the NDF fraction out of the rumen. It was concluded however that the used technique (rumen evacuation technique) overestimates mean retention time (MRT) of DNDF. Pellikaan used 13C as marker for determining fractional passage rate. A significant influence of feed intake level is shown on fractional passage rate. Feed was intrinsically labelled with the 13C marker, and pulse-dosed into the rumen, followed by intestinal and faecal sampling. A clear difference in fractional passage rate between internal (lignin) and external markers (Cr-NDF) is shown in the research of Stefanon et al. . Recovery of lignin was inconsistent however, fractional passage rate might be underestimated. Tas et al. researched the effect of level of concentrate fed (50% or 25% of DM-uptake) on fractional passage rate of the NDF fraction. Natural abundance of 13C in the feed was used as marker, the concentrate contained mainly C3 plants, which are known to have low values of 13C compared to C4 plants. The concentrate was replaced once with another concentrate consisting of C4 plants, so higher in 13C content. This way a pulse dose of 13C is realised, just like the research of as mentioned before. Concluded was no effect of level of concentrate in total DMI on fractional passage rate exists. Van Bruchem et al. researched passage behaviour of hay and grass silage using Cr-NDF, fractional passage rate was determined at 0.053/h, no significant differences were found between diets.
Concluding these figures, a large variety is shown for fractional passage rates using different methods and/or different treatments. As shown in fractional passage rates in literature differ roughly between 0.011/h and 0.086/h, resulting in Mean Retention Times of rumen contents ranging between 11.6 up to 90.9 hours.
Modelling fractional passage rate
Modelling of fractional passage rate basically started with the research of Balch who performed research on marker excretion curves by simply counting coloured particles recovered in the faeces. Blaxter et al. designed a three-compartmental model representing the rumen, abomasum and faeces, based on the results found by Balch . The rumen was assumed to be the most important factor for variations in fractional passage rates. A time delay was introduced between duodenum and faeces. Another suggestion was that steady state has to be reached before starting recovery of the marker. Grovum and Williams used an external marker, Cr-EDTA, for determining fractional passage rate and sampled digesta from the abomasum next to faecal collection. Concluded was that the rumen is the slowest compartment. A two-compartmental mathematical model was used for calculating fractional passage rates. France et al. underlines the importance of the inclusion of a time lag in models predicting fractional passage rates. Currently fractional passage rate is determined using whole rumen models. The model used in this research is the multicompartmental model of Dhanoa et al. . If marker concentrations in the faeces are known, fractional passage rate of feed out of the rumen can be determined using this model. The model calculates the passage rates for the slowest and second slowest compartment. It is still not clear however which compartment represents the slowest compartment, the rumen or the intestines.
Strangely enough, in the Cornell model microbial growth yield is reduced when diets contain less than 20% NDF, to represent the depressing effect of pH on rumen microbial growth, but no effect on fibre degradation is included (Russell et al, 1992).
Differences in natural abundance of 13C to 12C in conventional feed was used by Tyrrell et al. (1984) to determine the source of metabolizable carbon and the contribution of dietary carbon to milk carbon. The ratio of 12C and 13C isotopes in the feces has been used to estimate the proportion of C3 and C4 plants consumed (Jones et al., 1979). Application of 13C technology to ruminant nutrition is usually beyond the reach of animal scientists because of high costs of equipment and the maintenance and technical expertise needed to operate it. Boutton et al. (1986) produced 13C-enhanced plant material using 13CO2 to overcome the high natural background of 13C, a problem encountered with using differences in natural abundance in feedstuffs. High background is not a problem encountered with use of 14C.
Importance of NDF for microbial growth: microbes attach to the NDF and grow, together with the NDF the microbes pass out the rumen.
Materials and methods
This research is part of a larger PhD-project in which an experiment will be performed at experimental accommodation 'De Ossekampen' using six high-producing dairy cows in a 6x6 latin square design. The main focus of this experiment is to determine fractional passage rate of starch. Passage will be linked to digestion. My part of the research will focus on fractional passage rate of the NDF-fraction and involve only a small part of this PhD-research. The experiment started on 2 February 2009. Goal of the research is to obtain data of fractional passage rates, so feed evaluation systems can in the end be updated.
Animals, housing and diet
Two multiparous dairy cows, fitted with rumen fistula, were assigned to the dietary treatments. A crossover design was used, as shown in table 2.
Table Distribution of maize silages per dairy cow
Both cows were about 55 days in lactation at the start of the experiment. The weight of the cows was …….. kg and the milk yield was …….. kg. The cows where housed in a tie-stall at experimental farm 'De Ossekampen' and milked twice daily at ……hrs and …… hrs. Cows had free access to water during the entire experiment. Feed intake of the cows was
The diet consists of 60% roughage and 40% concentrate at DM-basis. Roughage contains maize and grass in a 50/50 ratio. Grass silage was harvested in 2009 at the experimental farm 'De Ossekampen'. A TMR (total mixed ration) was fed twice daily after milking. The concentrate was designed to contain no C4 plants, which naturally have a higher abundance of 13C. The maize-silages (Aastar 2 and Baleric 2) were harvested on 17 September 2009. The chemical composition of the maize silages is shown in , as determined by NIRS. The calculated values for especially NDF degradability are extremes, the accuracy of the NIRS is expected to be low. To overcome this problem wet-chemical analysis of the NDF-content has been performed as well, resulting in the figures……
Two different breeds of maize silage have been selected for their genetic differences in cell-wall content and degradability and starch content. The two different maize cultivars are harvested on the same date to obtain ideally no differences in starch content. As is shown in minor differences in starch content do exist, but are not expected to bias the research results. As shown in differences in NDF content and degradability are substantial. A small amount of this maize has been labelled with the stable isotope 13C in a greenhouse (ISO-life), by continuous 13CO2 infusion. Continuous infusion of 13CO2 is a necessity for obtaining homogeneous labelled material. This highly enriched 13C labelled maize is used as a marker to determine the exact passage of this feed-component through the digestive tract of the dairy cows. Enrichment level of the labelled maize equal ……….. This high enrichment is needed, because only a small pulse-dose is put in the rumen for representing the entire rumen contents. In order to measure the passage of this maize, rumen fistulated dairy cows are used.
Table Chemical composition of maize silages used determined by NIRS
The dairy cows were selected according to their milk yield, parturition number and lactation stage before the experiment started.
Preceding every sampling period the cows where fed the ration with maize silage of interest ad-lib for a week, in order for the rumen-microbes to adapt to the different ration. During the sampling period the supply of feed was restricted at 95% to assure the cows would ingest the entire diet and steady state is maintained.
Omasal sampling technique according to the protocol developed by . A cannula was brought into the omasum 24 hrs before starting the actual sampling, passage out of the rumen is determined by determining the ratio of 13C/12C in the chyme and faecal samples collected. One sampling-period consisted of 12 days adaptation, followed by 5 days of sampling. During the experiment faecal samples will be taken to determine passage through the digestive tract. My research will focus on 2 of these cows which both have received the same maize silages () in two different periods ().
At the start of every sampling period a 30 gram 13C-labelled maize silage pulse dose is brought into the rumen through the rumen fistula. This 30 gram consists of 15 gram cob (starch fraction) and 15 gram of leaves/stems (on dry-matter basis). The total amount of enrichment necessary to determine passage behaviour has been determined by modelling. The first omasal chyme sample is taken just before the pulse-dose, to determine background level of 13C enrichment. In total 22 chyme samples were taken, every 4 hrs the first 24 hrs and every 6 hrs the four days after. Chyme samples were taken from the omasum using the omasal sampling technique designed by . A probe with a plastic tube attached was fixed through the reticulo-omasal orifice to stay there the entire sampling period. Samples of about 750 ml where taken through the tube.
Faeces were collected during periods of 3 hours every sampling period and homogenized. Subsamples of 300 gram were taken. As for omasal samples, after the first day the frequency of sampling decreased and the collection periods of 3 hours are followed by 3 hours of rest. Chyme and faecal samples were stored in a freezer immediately after sampling.
13C analysis DM and NDR
Samples are freeze-dried and ground to pass a 1 mm sieve. NDR analysis will be performed using the nylon bag-system developed by Ankom Technology. 13C of the NDR and DM fractions will be determined using an elemental analyser. Because ashing of the NDF-fraction is not possible since the 13C content has to be determined not the NDF but actually the NDR fraction is determined.
Samples are freeze-dried to get rid of most moisture. Samples are ground and homogenised for further processing. Samples are ground to pass a 1 mm sieve (Wiley Mill……….) and stored in plastic bottles of 250 mL.
Total amount of samples used in this experiment is 165. These samples include:
Chyme samples: 22*2 cows*2 periods-11 (one cow was unfortunately taken out of the experiment halfway during the first sampling-period resulting in only 11 instead of 22 chyme samples, faecal sampling did continue.) Total chyme samples: 77.
Faecal samples: 22*2 cows*2 periods: 88.
This results in a total of 165 samples to be analysed for 13CDM and 13CNDR.
The analysis of NDR is done using a protocol developed by Ankom, using filter bags (Ankom Technology, 2006). It was not possible to ash the sample after NDR determination, so actually NDR is determined. Ashing was not possible since the samples have to be researched for delta 13C. Add type and amount of amylase. Heat-stable α-Amylase. The addition of amylase is important if filterbags are used, to avoid blockage of the bag pores with starch, and starch leftovers which will be considered fibre .
Both NDR-extracted and normal ground samples are bullet milled (Retsch………..) for 5 minutes at 80 Hz, before 13C will be analysed using the elemental analyser. 0,500-1,500 mg of sample is weighed into tin cups and tightly closed before analysis. Gas chromatographycombustion isotope ratio mass spectrometry (GC-C-IRMS) with a Delta S/GC instrument (Finnifan MAT, Bremen, Germany) is used to determine the 13C : 12C ratio.
Calculation and statistical analysis
Data is analysed using the ……………. Procedure of SAS
Yt= Ae-(kp1 x t) x exp [-(N-2)e-(kp2 - kp1 x t]
With: Yt: Marker concentration on moment t
A: calculated marker concentration on t=0
Kp1: Fractional passage rate constant of the slowest compartment (/hour)
Kp2: Fractional passage rate constant of the second slowest compartment (/hour)
N: Number of compartments
t: Time of sample taken (hour)
DM and ash procedure according to the standards.
Results reporting and interpretation gained from the research.
DM and NDF samples, Faeces and chyme, cow 14 and 511.
Table shows the time peeks are clearly visible in the excretion graphs. Especially the graphs for cow 511, Aastar show nice results, number of peeks is limited and clearly visible. The amount of peeks in most cases however probably make curve-fitting difficult, if not impossible. It is clear that in most cases the points cannot be modelled into one single peak to fit the results most. Several peaks in 13C excretion are clearly visible. The occurrence of several (so more than one) peaks can probably be explained by the difference in degradation rate, and extent of the different feed components.
Lot of peeks, large variation.
Peaks in DM mainly caused by NDF
NDF: different fractions different kd, so different kp? (as researched by Lund)
Enriched particles trapped in rumen mat
Although aim of the study is to look at differences of NDF content only, other interfering factors cannot be excluded. Despite the same moment of harvest the starch content and VEM did show a difference between the two maize-cultivars, although this was only a slight difference.
Research of……….. shows no difference in sampling from the omasum or the reticulo rumen.
(Mais door houtversnipperaar. Korrels niet gekneusd, wel kapot geslagen!)
Lot of peeks, curvefitting possible?
Conclusions a thesis will generally not have more than five substantial conclusions.