FibreCap: an improved method for the rapid analysis of bre in feeding stuffs

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1 Animal Feed Science and Technology 86 (2000) 125±132 Short communication FibreCap: an improved method for the rapid analysis of bre in feeding stuffs M.A. Kitcherside, E.F. Glen, A.J.F. Webster * Department of Clinical Veterinary Science, School of Veterinary Science, University of Bristol, Langford House, Langford, Bristol BS40 5DU, UK Received 26 October 1999; received in revised form 7 April 2000; accepted 18 April 2000 Abstract An improved procedure is described for the analysis of bre in samples of feeding stuffs. The procedure uses a novel cylindrical capsule (FibreCap, patent pending) with porous walls having the same ltration characteristics as Whatman 541 lter paper. The walls are made of hydrophilic material to facilitate movement of solvents; the lid is hydrophobic to facilitate gas exchange. The procedure has been designed to speed up analysis, reduce use of solvents and reduce variability and systematic error associated with extraction and ltration. The FibreCap procedure was compared with conventional analyses for measurement of crude bre, neutral and modi ed acid-detergent bre. Mean values were identical throughout the range of bre concentration found in animal feeding stuffs. However, residual variation was consistently lower for FibreCap than for conventional analyses. Two ring tests were carried out in which results from seven and six collaborating laboratories were compared with those at Bristol. Differences between laboratories were very small although statistically signi cant. The FibreCap system is consistent with Statutory Requirements for bre analysis, more repeatable than conventional methods and quicker in operation. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Fibre; Analysis; Feeding stuffs 1. Introduction The analysis of bre in feeding stuffs for animals is one of the most time-consuming routines in any animal nutrition laboratory, whether scienti c or commercial. Measurement of crude bre (Association of Of cial Agricultural Chemists, 1984) is * Corresponding author. Tel.: ; fax: address: john.webster@bristol.ac.uk (A.J.F. Webster) /00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S (00)00153-X

2 126 M.A. Kitcherside et al. / Animal Feed Science and Technology 86 (2000) 125±132 required by the United Kingdom (UK) and European Union (EU) Feeding stuffs regulations (feeding stuffs, sampling and analysis) UK, 1982, Feed regulations, EU 1995, see Agriculture Statutory Instruments (1986). Determinations based on detergent extractions continue to be used widely as a basis for predicting diet characteristics affecting digestibility and food intake, especially in forages for ruminant animals (Van Soest, 1994). The most widely reported methods are those for neutral-detergent bre (NDF, Van Soest et al., 1991) and modi ed acid-detergent bre (MADF, Clancy and Wilson, 1966). The conventional procedure (Association of Of cial Agricultural Chemists, 1984) may involve three extraction steps followed by the collection of the residue onto Whatman 541 lter paper or a glass Sinta crucible (porosity 1). This is laborious and involves several procedures which are subject to error (e.g. transfer of samples between extraction vessels) or individual variation (e.g. the instruction to `boil gently for exactly 30 min'). There is commercial apparatus (Fibretec, Foss Tecator AB, Sweden) in which all solvent extractions are carried out in the same vessel. An alternative procedure is to enclose the sample, and thus retain the residue, within a synthetic bre bag (ANKOM, Komarek et al., 1993, 1996). This has the merit of removing both the sample transfers and the ltration stages but presents inherent problems of mixing sample and reagents. This is particularly relevant when analysing neutral-detergent bre in high-starch feeds (Van Soest et al., 1991). The current paper describes a novel approach (FibreCap, patent pending) designed both to speed up the procedures and reduce variability and systematic error associated with extractions and ltration. 2. Materials and methods 2.1. The FibreCap apparatus The system has been developed using standard laboratory hot plates and condensers. Samples of the material under test are weighed into a FibreCap cylindrical capsule, 58 mm in length, 23 mm in diameter. The `wall' of the capsule consists of a polypropylene frame which supports a polyester mesh treated so as to be hydrophilic. The ltration characteristics of the mesh have been set to match those of Whatman 541 lter paper as used in conventional Weende analysis (Association of Of cial Agricultural Chemists, 1984). The lid is also made of polyester, but treated so as to be hydrophobic. The design ensures liquid exchange across the walls of the cylinder and gaseous exchange through the lid. Six capsules are suspended in a carousel and placed in a beaker containing 350 ml solvent so that the lid is just above the liquid level. During solvent extraction, the capsules rise and fall within the carousel as gas is produced and released. This facilitates the mixing of sample and solvent. Moreover, the bottom of the capsule is not in contact with the bottom of the beaker. This avoids any localised heating. Initial trials were carried out using hand-made capsules (trials1-2 and Ring test 1). The commercial capsules (Sericol Industrial Fabrics, Broadstairs, Kent, UK) used for later trials (trials 3±4 and Ring test 2) differed only from the hand-made versions in that the shape and composition of the lid were modi ed to improve gas exchange.

3 M.A. Kitcherside et al. / Animal Feed Science and Technology 86 (2000) 125± Analysis for crude bre Conventional Weende analysis for crude bre was carried out exactly according to the directions of Association of Of cial Agricultural Chemists (1984). Analysis using the FibreCap apparatus is described in detail below Preparation Label and pre-dry capsules with lids in an oven at 1008C for 30 min. Dessicate, cool and weigh. Tare each capsule on an analytical balance, introduce subsample (0.7±1.0 g) of the milled material and weigh again Fat extraction Set up three beakers containing petroleum ether (BP 40±608C). Immerse carousel (6 capsules) and agitate vigorously (ca 30 s) in each beaker in turn. Remove carousel, allow capsules to drain and air-dry in a fume hood Acid re ux Lower carousel unit into 350 ml of boiling sulphuric acid (0.225 N) and agitate vigorously (ca 1 min) to disperse the sample. Simmer gently for 30 min. After 5 min, remove each beaker from the hot plate, agitate the carousel (10 s) and return to the heat Removal of acid and soluble fractions Remove the carousel unit and drain the capsules into a waste receptacle. Using the same extraction beaker wash once with hot (80±1008C) deionised water. Agitate carousel vigorously during washing Alkaline re ux Simmer for 30 min in 350-ml sodium hydroxide solution (0.313 M), agitating the samples as described in Stage Final rinse Rinse twice in hot deionised water, then HCl solution (1%v/v), then once again in hot water. A nal rinse with ethanol and diethyl ether is optional Drying residues Remove capsules from carousel, dry to constant weight in an oven at 1008C, dessicate and weigh for bre plus ash Ashing Prepare (dry and de-ash) tall form beakers in a furnace at 6008C for 1 h. Cool, dessicate and weigh. Slit the side of each capsule using a scalpel to avoid build-up of air pressure within the capsule and insert one into each beaker. Place beakers containing capsules in a furnace at 6008C for 4 h (or until no organic matter remains), cool, dessicate and reweigh for ash content.

4 128 M.A. Kitcherside et al. / Animal Feed Science and Technology 86 (2000) 125± Blank determinations During the development of this procedure a series of blank determinations was run to identify any losses of capsular material during solvent extraction and any contribution from the capsule to the nal measure of ash. These were small but measurable (2±3 mg loss during solvent extraction, 2±15 mg contribution to weight of ash). The contribution to ash weight has varied between batches, and in current commercial capsules it is <3 mg. Nevertheless, it is recommended that two empty capsules be incorporated into each day's analysis to establish blank correction factors for `capsule solubility' and capsule ash (c and d in the equation below) Calculation of crude bre Crude bre (CF, g/kg) is given from the following formula: CF g=kg ˆ W 3 W 1 c W 4 d W where W 1 is the initial capsule weight, W 2 the sample weight, W 3 the capsule plus residue weight, W 4 the total ash, c the blank correction factor for `capsule solubility', and d the capsule ash. The correction factor c is normally >0.9990, whereas correction factor d is (now) normally <3 mg Analysis for neutral-detergent bre (NDF) The conventional procedure for analysis of neutral-detergent bre (NDF) was carried out exactly as described by Van Soest et al. (1991). This procedure uses a-amylase at two stages in the extraction process to improve the solubleisation of starch. The Fibrecap method differs in only one step from the van Soest procedure. Each capsule is soaked in 1% w/v sodium sulphate solution for 5 min prior to the rst detergent extraction. This is, once again, intended to prevent `clumping' and so facilitate starch extraction Analysis for Modi ed Acid-Detergent bre (MADF) Analysis for MADF whether using the FibreCap or conventional apparatus exactly followed the procedure described by Clancy and Wilson (1966) Experiments Comparison of FibreCap with Weende analysis Four trials were carried out Trial 1. Measurements of CF were made in triplicate according to the Weende method and FibreCap procedure on 20 feed samples including seven commercial compound feeds, three dried forages, two silages and eight other common ingredients of animal feeds. These ingredients were selected to cover a range of crude fibre concentrations from 20 to 350 g/kg. Hand-made FibreCaps were used in this trial and

5 the fat extraction procedure was carried out before the samples were weighed into the capsules Trial 2. Measurements of MADF were made in triplicate according to Clancy and Wilson (1966), on 10 samples of grass and maize silage using the FibreCap and conventional apparatus. The hand-made capsules were used but all steps (including fat extraction) were carried out in the capsules Trial 3. Measurements were made in triplicate of CF and NDF on 30 ingredients used by feed compounders selected to cover a range of 20±380 g CF/kg, (70±700 g NDF/ kg) using conventional and FibreCap procedures. The commercial capsules were used throughout and all extraction steps were carried out in the capsules Trial 4. Two samples were selected, micronised peas and wheat straw (approximately 50 and 380 g CF/kg respectively). Three subsamples of each material were analysed by FibreCap and Weende procedures on five successive days to look for within and between-day variation Ring tests M.A. Kitcherside et al. / Animal Feed Science and Technology 86 (2000) 125± Before any new method can be accepted as a standard procedure, it is necessary to establish that its accuracy and precision are consistent between different laboratories. Two `ring tests' were, therefore, conducted to establish the consistency of the FibreCap procedure or, if not, identify and account for any differences between laboratories. Ring test 1 compared determinations of CF on eight feed samples made in seven collaborating analytical laboratories with those obtained at the University of Bristol. The eight materials including four compound feeds, two high bre forages and two high protein raw materials, were selected to cover a CF concentration range from 30 to 400 g/ kg. Each collaborating laboratory was given a demonstration of the FibreCap procedure in advance of the ring test. Hand-made capsules were used for this trial. After analysis of the results and discussion of procedures a second ring test was carried out using commercial capsules. In this case results from six collaborating laboratories (three `repeat' labs and three new ones) were compared with those from Bristol. Three feed samples (rice, oats and a compound feed) were provided by Scienti c Services (Norwich UK). These samples had previously been used (in seven different laboratories from ours) for a ring test for CF using conventional Weende analysis. 3. Results Table 1 summarises the results of the rst three trials which compared measurements of CF, MADF and NDF made using the FibreCap method (F) with the conventional procedures (C) as described in Association of Of cial Agricultural Chemists (1984). Correlation, (r 2 ) values exceeded 0.99 for NDF and for both sets of CF determinations. The r 2 value was slightly lower (0.959) for the ten comparisons of MADF in forages. All

6 130 M.A. Kitcherside et al. / Animal Feed Science and Technology 86 (2000) 125±132 Table 1 A comparison of the FibreCap procedure (F) with conventional methods (C) for measurement of crude bre (CF), neutral-detergent bre (NDF) and modi ed acid-detergent bre (MADF) Trial 1 CF 2. MADF 3. CF 3.NDF Sample no Concentration range (g/kg) 20± ±402 21±375 34±720 Regression, F/C r intercept coef cient Signi cance, method * *** n.s. n.s. methodfeed n.s. *** n.s. n.s. * Signi cance of effects of method F v. C and methodfeed interaction: * p<0.05; and *** p< regression coef cients were not signi cantly different from 1.0 and intercept terms not signi cantly different from zero. Analysis of variance (ANOVA) for the effects of feed and method (F v. C) revealed no statistically signi cant differences between the methods for CF and NDF in trial 3. There was a small but signi cant effect of method for CF in trial. Effects of method and methodfeed interaction were highly signi cant for MADF, which re ects the small departures of the coef cient and intercept from 1.0 and 0.0 respectively. Table 2 summarises the ANOVA of Trial 4 which was designed to test FibreCap and Weende procedures for CF for within and between-day variation. Between-day and residual variance were respectively 6.9 and 5.1 times greater for the Weende method than for the FibreCap method. However, the sensitivity of the test was such that the betweenday differences, though small, were statistically signi cant for both methods (p<0.001). These results demonstrated that in our laboratory at the University of Bristol the FibreCap method gave effectively identical mean values for CF, MADF and NDF compared to conventional methods through the full range of concentrations likely to be encountered in commercial laboratories, but with a reduced between-sample variability. The Ring tests were designed to discover whether this consistency could be maintained between laboratories. Table 3 examines the results from Ring test 1 by comparing the determinations made in the seven collaborating laboratories with those at The University of Bristol. In all cases r 2 values exceeded Regression coef cients did not differ signi cantly from At Table 2 Analysis of variance associated with repeated measures (3 replicates per day for 5 days) of crude bre in micronised peas and pelleted wheat straw using the FibreCap and Weende procedures DF FibreCap Weende Feed *** *** Between day *** 178.7*** Feedday *** 39.4 n.s. Residual

7 M.A. Kitcherside et al. / Animal Feed Science and Technology 86 (2000) 125± Table 3 Regression analysis of Ring Test 1 comparing determinations (y) of crude bre made using the FibreCap technique at seven laboratories with those made at The University of Bristol (x) Laboratory r 2 Coefficient y; xˆ40 y; xˆ400 Test values of 40 and 400 g CF/kg at Bristol (the independent variable) predicted values for CF in the seven collaborating laboratories ranged from 36.2 to 41.7and 370 to 402 g/kg. In general the agreement was satisfactory but laboratory 5 produced values slightly lower than the others. Table 4 summarises the analysis of variance for both Ring tests. Because of the different numbers of feed samples the two tests are not strictly comparable. Nevertheless, this table shows that while between laboratory variation had been greatly reduced in Ring test 2, it was still statistically highly signi cant. 4. Discussion The results of these trials demonstrate clearly that analysis of bre in feeding stuffs (CF, MADF and NDF) according to the FibreCap method produces mean values that are, in essence, identical to those obtained by more conventional, laborious methods. Moreover the residual variation attached to The FibreCap method is lower than for conventional methods. The method is therefore highly satisfactory both for routine analysis to determine CF in feeding stuffs for statutory purposes and for research purposes when greater precision is desirable. The lower residual variation attached to the FibreCap method may be attributed in part to the fact that all operations (including fat extraction) are carried out in the same vessel. Table 4 Analysis of variance associated with the two ring tests of measurements made of crude bre using the FibreCap method Ring test 1 Ring test 2 DF Mean square DF Mean square Feed *** *** Laboratory 7 224*** *** FeedxLab *** Residual

8 132 M.A. Kitcherside et al. / Animal Feed Science and Technology 86 (2000) 125±132 In this regard however, it is similar to the semi-automated Fibretech procedure. However it is likely that this improved consistency between replicates can also be attributed to the design of the capsules and carousel, which ensures good mixing of sample and solvent and prevents localised heating. The Association of Of cial Agricultural Chemists (1984) instruction to ``boil gently for exactly 30 min'' recognises that the amount of residue at the end of the extraction procedure is sensitive to temperature. The small but signi cant discrepancies between laboratories in the two ring tests suggest that use of the Fibrecap does not eliminate variability due to between-laboratory differences in boiling procedures. However when the three materials used in the second ring test were tested in six other laboratories, mean values obtained in different laboratories ranged from 101± 153 g/kg for oats, 46.1±50.0 g/kg for the compound feed and 1.8 to 3.6 g/kg for rice. (Kevin Self, personal communication). In the current ring test the ranges between laboratories were, respectively, 143.8±153.8, 47.8±53.4 and 1.3±2.4 g/kg. The method of Van Soest et al. (1991) for NDF analysis can generate an undesirable degree of variation especially with high starch feeds which tend to `clump' when in neutral detergent. The use of a-amylase is designed to reduce this variation. The current procedure, which involves a preliminary soak in sodium sulphate is also intended to prevent clumping of starchy materials. The natural rising and falling motion of the FibreCap capsules appears to be a further deterrent to clumping especially when compared with the ANKOM procedure using synthetic bre bags (Komarek et al., 1993, 1996). The main advantages of the FibreCap procedure are, however, operational. A single operator using the conventional manual system for CF analysis can perform approximately 15 extractions per hour. Using the ANKOM system this rises to 24 per hour, using FibreCap 90 extractions per hour can be performed as a routine. Solvent use is reduced by a factor of three. Finally, the procedure eliminates the need for vacuum ltration through Sinta crucibles or Whatman lter paper and so avoids blockages which may occur at this stage. References Agriculture Statutory Instruments 177, The Feeding Stuffs Regulations, HMSO, London Association of Of cial Agricultural Chemists, In: Williams, S. (Ed.), Of cial Methods of Analysis. 14th Ed. AOAC Arlington, VA. Clancy, M.J., Wilson, R.K., Development and application of a new chemical method for predicting the digestibility and intake of herbage samples. Proceedings 10th International Grasslands Congress, Helsinki, pp. 445±452. Komarek, A.R., Robertson, J.B., van Soest, P.J., A comparison of methods for determining ADF using the Filter Bag technique versus conventional ltration, J. Dairy Sci. 77 (Suppl. 1), 24±26. Komarek, A.R., Manson, H., Theix, N., Crude ber determinations using the ANKOM ber system. ANKOM technology publication 102. Van Soest, P.J., The nutritional ecology of the ruminant. 2nd edition. Cornell Univesity Press, Ithaca, NY. Van Soest, P.J., Robertson, J.B., Lewis, B.A., Methods for dietary ber, neutral-detergent ber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583±3597.

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