Measuring detergent fibre and insoluble protein in corn silage using crucibles or filter bags

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1 Animal Feed Science and Technology 133 (2007) Short communication Measuring detergent fibre and insoluble protein in corn silage using crucibles or filter bags Gonzalo Ferreira a,, David R. Mertens b a Dairy Science Department, University of Wisconsin, Madison, WI 53706, United States b USDA-ARS, US Dairy Forage Research Center, Madison, WI 53706, United States Received 2 January 2006; received in revised form 12 April 2006; accepted 25 April 2006 Abstract Different methods exist for the determination of fibre concentration in feeds. To determine, whether fibre recovery and the contamination of fibre by nitrogenous compounds are altered, we measured fibre concentrations in a diverse set of corn silages using three method modifications and two extraction/filtration systems. Thirty-three corn silages, obtained from a commercial feed analysis laboratory, were dried (55 C for 24 h) and ground to pass through a 1-mm screen of a cutter mill before analysis. All samples were extracted in neutral detergent with the inclusion of sodium sulphite (neutral detergent fibre or NDF), -amylase (neutral detergent residue or NDR) or both (amylase-treated neutral detergent fibre or andf), and using either Gooch crucible (CRUC) or filter bag (FBAG) systems. The andf method obtained the lowest and similar average fibre concentrations for both CRUC and FBAG (433 and 433 g/kg, respectively). Fibre concentration of NDR was greater (456 and 449 g/kg for CRUC and FBAG, respectively) than andf. Fibre concentration was greater for NDF (473 g/kg) than for NDR and andf (449 and 433 g/kg, respectively) when using FBAG. Poor extraction occurred for FBAG when -amylase was not used. For CRUC, NDF and NDR concentrations were similar (456 g/kg), although filtration of fibre residue after extraction without -amylase was difficult. Neutral detergent insoluble crude protein (ICP) was similar for NDF and andf, and slightly greater Abbreviations: ADF, acid detergent fibre; andf, amylase-treated neutral detergent fibre; CRUC, Gooch crucibles system; FBAG, filter bag system; ICP, insoluble crude protein; NDF, neutral detergent fibre; NDICP, neutral detergent insoluble crude protein; NDR, neutral detergent residue Corresponding author. Present address: Casilla de Correo 115, (6070) Lincoln, Buenos Aires, Argentina. Tel.: address: gferreira@nutrep.com (G. Ferreira) /$ see front matter 2006 Elsevier B.V. All rights reserved. doi: /j.anifeedsci

2 336 G. Ferreira, D.R. Mertens / Animal Feed Science and Technology 133 (2007) FBAG than for CRUC systems (8.4 and 9.6 g/kg, respectively). With both FBAG and CRUC, ICP was greater for NDR determined without sulphite (12.5 and 14.2 g/kg, respectively) than for andf (8.6 and 9.7 g/kg, respectively). The lower fibre concentration for andf method was attributed to less starch contamination when compared to NDF and to less protein contamination and possibly to extraction of phenolic compounds when compared to NDR. Concentration of acid detergent fibre (ADF) was greater for CRUC than for FBAG, although this difference was minimal (266 and 261 g/kg, respectively). Acid detergent ICP was similar for CRUC and FBAG systems and averaged 4 g/kg. We observed that amylase and sulphite affect the measurement of NDF concentration in corn silage. It is crucial that authors and laboratories accurately describe how they measure NDF and clearly indicate by acronym the method they used. Although the extraction/filtration system did not affect the determination of NDR and andf, the FBAG system generated higher NDF concentrations for corn silage when amylase is not used Elsevier B.V. All rights reserved. Keywords: Neutral detergent fibre; Insoluble crude protein; Fibre analysis 1. Introduction The neutral detergent fibre (NDF) method (Van Soest and Wine, 1967) was developed to separate feed s dry matter into a soluble fraction, that is readily digested and a fibrous fraction, that is slowly and incompletely digested. In plant materials, NDF consists primarily of hemicellulose, cellulose and lignin, but also contains small amounts of protein and ash. Sodium sulphite was included in the original NDF method to reduce the protein contamination of fibre (Hintz et al., 1996). Because the original NDF method did not adequately remove starch, the neutral detergent residue (NDR) method (Robertson and Van Soest, 1981) was developed to solubilize starch during neutral detergent extraction using a heat-stable -amylase. However, sodium sulphite was removed in the NDR method to minimize the loss of phenolic compounds in feeds. Sodium sulphite is necessary to remove protein contamination of cooked or heated feeds (Hintz et al., 1996). Because removal of sodium sulphite results in incomplete removal of protein from NDR, the amylase-treated NDF (andf) method (Mertens, 2002) that uses both heat-stable amylase and sodium sulphite was developed to measure insoluble dietary fibre in all feeds. The NDF, NDR and andf methods are based on the isolation of fibrous residues using coarse porosity Gooch crucibles (CRUC). The Ankom Technology Corp. (Ankom Technology Corp., Fairport, NY) developed an alternative method for isolating fibrous residues based on the extraction of samples in filter bags (FBAG) using a pressurized chamber. To our knowledge no direct comparisons among methods have been made for corn silage. To determine whether each of the modifications of the NDF method has potential for altering the concentration of fibre recovered, we measured fibre concentrations in a diverse set of corn silages using each of the three methods with each of the two extraction/filtration systems. In addition, to determine whether protein contamination affects fibre concentration of corn silages, we measured neutral detergent insoluble crude protein (NDICP) and acid detergent insoluble crude protein.

3 G. Ferreira, D.R. Mertens / Animal Feed Science and Technology 133 (2007) Materials and methods 2.1. Corn silage samples Thirty-three diverse corn silages were obtained from a commercial feed analysis laboratory. No information regarding genotype and growing or harvesting (e.g., chop-length and kernel-processing) conditions were obtained Fibre analysis All corn silage samples were dried in a forced-air oven (55 C for 24 h) and ground to pass through a 1-mm screen of a cutter mill (Wiley mill, Arthur H. Thomas, Philadelphia, PA) before analysis. For the CRUC and the FBAG systems, all corn silages were analyzed using three neutral detergent fibre modifications (i.e., NDF, NDR and andf). Acid detergent fibre (ADF) was also determined using both CRUC and FBAG systems. Extraction of detergent fibre using crucibles followed AOAC methods (Mertens, 2002). Briefly, 0.5 g (NDF) or 1.0 g (ADF) of test samples was heated in neutral detergent or acid detergent to boiling within 5 min and subsequently, refluxed for 60 min. Fibre residues were transferred into coarse porosity (50 m) 50-mL Gooch crucibles, filtered and soaked in hot ( C) water three times for 5 min each. Residues were soaked two times in acetone for 5 min each. To measure NDF, sodium sulphite (0.5 g) was added to the test sample and ND prior to refluxing. Sand was added to Gooch crucibles as a filtering aid to isolate NDF. For NDR, sodium sulphite was omitted and -amylase (Ankom Technology Corp.) previously diluted (10%, v/v) was added in two 2-mL doses (the first after 5 min of heating and the second during the first water soaking). For andf, both sodium sulphite and -amylase were used. Extraction of detergent fibre using filter bags was based on an in-house procedure using a fibre analyzer (Ankom 220, Ankom Technology Corp.). Briefly, 2 L of neutral detergent or acid detergent was poured into the extraction chamber. Filter bags (F57, 25 m, Ankom Technology Corp.) containing 0.5 g of test sample were placed in plastic trays. After inserting the plastic trays, the chamber lid was sealed, the heat turned on and the solution was heated to 100 C within 15 min. After 60 min of extraction (75 min total time) the detergent was expelled. Filter bags were washed four times within the chamber with 2 L of water at C. All washes were performed for 5 min each time with the chamber lid sealed and heat and stirrer turned on. After the last water wash, extracted filter bags were removed from the chamber and placed between two absorbent pads, and gently pressed to remove water. Filter bags were placed in a 500 ml jar, and approximately 250 ml of acetone was added. The jar was sealed with a lid and shaken three times during a 5-min extraction. Acetone was removed and replaced with 250 ml of acetone for a second 5-min extraction. Acetone residues were allowed to evaporate (air-drying) and bags were then dried in forced-air oven at 105 C for at least 8 h before being weighed. For NDF, 20 g of sodium sulphite was mixed with ND solution before it was added to the chamber for extraction. For NDR, 5 ml of heat-stable -amylase was mixed with ND solution before it was added to the chamber for extraction. In addition, 5 ml of heat-stable -amylase was added to each of the first two water washes. For the andf method, 20 g of sodium sulphite and 5 ml of

4 338 G. Ferreira, D.R. Mertens / Animal Feed Science and Technology 133 (2007) heat-stable -amylase were mixed with ND solution before this was added to the chamber for extraction. Also, 5 ml of heat-stable -amylase was added to each of the first two water washes. For all methods and systems single samples were analyzed (i.e., no replication) Insoluble crude protein analysis For the CRUC system, fibre residues in crucibles were scraped into ceramic boats and the recovered residue was weighed. Concentration of CP was measured as N 6.25 after analysis with a N analyzer (Leco FP-2000; Leco Corp., St. Joseph, MI). Insoluble N was corrected for recovery of residues from crucibles by assuming that residue remaining in the fritted disk of the crucible had the same N content as recovered residues. For the FBAG system, the N content of the filter bag plus residue was determined as described above and corrected for the N content in blank bags. For both CRUC and FBAG, detergent insoluble CP (ICP) was expressed as g of CP per kg of the original dry sample weight Statistical analysis Data were analyzed as a randomized complete block design using the MIXED procedure of SAS (release 8.2.; SAS Institute Inc., Cary, NC). The model included the effect of treatments (fixed; degrees of freedom, d.f. = 5), the effect of block or silage (random; d.f. = 32) and the residual error (d.f. = 160). Protected multiple comparisons were performed according to the method of Tukey. Statistical difference was declared at P< Results and discussion In this study, the main variable of interest was the concentration of residue after extraction in neutral detergent using three modifications with each of two extraction/filtrations systems (i.e., NDF, NDR and andf with CRUC or FBAG). Because all treatments are based on extraction with neutral detergent, the concentration of residue after extraction in neutral detergent will hereafter be referred as fibre concentration. Fibre concentration (average for all methods) was 449 ± 79 g/kg dry matter suggesting diversity among corn silages. Fibre concentration was greater for NDF than for NDR (P<0.05) and andf (P<0.05) when using the FBAG system (Table 1). Poor extraction was observed for the FBAG system when -amylase was not used (as evidenced by the gummy texture of the residue), suggesting starch gelatinization within the filter bag may have hampered the filtration of starch residues from the bags. For the CRUC system, fibre concentration using NDF and NDR methods was similar (P>0.75). However, filtration of fibre residue after extraction without -amylase (i.e., NDF) was difficult. This problem was overcome by placing the crucibles in boiling water-bath. These observations suggest that starch is not adequately removed with the NDF method, consequently overestimating fibre concentration of corn silage, especially using the FBAG system. Fibre concentration was lower for andf than for NDR in both CRUC (P<0.05) and FBAG (P<0.05) systems (i.e.,

5 G. Ferreira, D.R. Mertens / Animal Feed Science and Technology 133 (2007) Table 1 Concentration of fibre and neutral detergent insoluble crude protein (NDICP) in corn silage (n = 33) determined using six method modifications FBAG CRUC NDF NDR andf NDF NDR andf S.E.M. Fibre (g/kg dry matter) 473a 449b 433c 455b 456b 433c 14 NDICP (g/kg dry matter) 9.5c 14.2a 9.7c 8.1d 12.5b 8.6d 0.7 CRUC, residue filtration after extraction in neutral detergent using Gooch crucibles; FBAG, extraction in neutral detergent within filter bags; NDF, neutral detergent fibre (i.e., residue after extraction with neutral detergent with the use of sodium sulphite); NDR, neutral detergent residue (i.e., residue after extraction with neutral detergent with addition of -amylase); andf, amylase-treated NDF (i.e., residue after extraction with neutral detergent with addition of sodium sulphite and -amylase). Different letters (a d) in the same row differ (P<0.05). S.E.M., standard error of the mean. within systems; Table 1). Fibre concentrations of NDR and andf were similar when using CRUC (P>0.09) or FBAG (P>0.95) systems (i.e., across systems). Insoluble CP was similar for andf and NDF (Table 1), and slightly greater for FBAG than for CRUC systems. Insoluble CP was greater for the NDR than for the andf method. Insoluble CP was greater for the FBAG than for the CRUC system, although these differences were small (less than 2 g/kg) and inconsequential. The lower fibre concentration for andf method can be attributed to less protein contamination of the fibre residue (Hintz et al., 1996), although the magnitudes of these differences suggest that protein contamination explains these observations only partially. The differences in fibre concentration for NDR and andf when using the CRUC and the FBAG systems were 23 and 16 g/kg, respectively. The differences in NDICP concentration for NDR and andf when using the CRUC and the FBAG systems were 3.9 and 4.5 g/kg, respectively. This means that ICP accounted for 17 28% of the total difference between NDR and andf. Mertens (2003) reported that 76% of the difference between NDR and andf of grasses was due to differences in lignin recoveries. Sulphite attack to lignin may account for some of difference between NDR and andf methods (Van Soest, 1994; Mertens, 2003) after they are adjusted for differences in protein contamination. It is also possible that sulphite might attack proteins and weaken the protein matrix in starch granules; thereby allowing greater extraction of starch. Concentration of ADF was greater for the CRUC than for the FBAG system (P<0.01), although this difference was minimal (266 and 261 g/kg, respectively) and within the replicate variation of the procedure. Acid detergent ICP was similar (P>0.45) for CRUC and FBAG systems and averaged 4 g/kg. 4. Conclusions Poor filtration of starchy materials can result in overestimates of fibre concentration. This overestimation can be increased when using filter bags with fibre analyzer. Consequently, the NDF method should be avoided when measuring fibre concentration of corn silage. For the NDR and andf methods, fibre concentrations measured using filter bags with fibre analyzer were similar to those measured using coarse porosity Gooch crucibles. The andf

6 340 G. Ferreira, D.R. Mertens / Animal Feed Science and Technology 133 (2007) method reduces protein contamination of the fibre residue. However, this reduction does not account for all the reduction in fibre concentration due to the use of sodium sulphite. We demonstrated that the use of amylase and sulphite affects the measurement of fibre concentration in corn silage. Implications of this study are that it is crucial that authors and laboratories accurately describe how they measure fibre concentration and clearly indicate by acronym the method they used. We also conclude that the extraction/filtration system did not affect the determination of NDR and andf, but that the FBAG system generated higher NDF concentrations for corn silage when amylase was not used. References Hintz, R.W., Mertens, D.R., Albrecht, K.A., Effects of sodium sulfite on recovery and composition of detergent fiber and lignin. J. AOAC Int. 79, Mertens, D.R., Gravimetric determination of amylase-treated neutral detergent fiber in feeds using refluxing in beakers or crucibles: collaborative study. J. AOAC 85, Mertens, D.R., Challenges in measuring insoluble dietary fiber. J. Anim. Sci. 81, Robertson, J.B., Van Soest, P.J., The detergent system of analysis and its application to human food. In: James, W.P.T., Theander, O. (Eds.), The Analysis of Dietary Fiber in Food. Marcel Dekker, Inc., NY. Van Soest, P.J., Nutritional Ecology of the Ruminant, second ed. O&BBooks, Corvallis, OR. Van Soest, P.J., Wine, R.H., The use of detergents in analysis of fibrous feeds: IV. Determination of plant cell wall constituents. J. AOAC 50,

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