A comparison of filter bag methods with

Animal Feed Science and Technology 84 (2000) 33±47

A comparison of ®lter bag methods with conventional tube methods of determining the in vitro digestibility of forages Dr. D. Wilman*, A. Adesogan Welsh Institute of Rural Studies, University of Wales, Llanbadarn Campus, Aberystwyth, SY23 3AL, UK Received 13 April 1999; received in revised form 10 August 1999; accepted 4 January 2000

Abstract In vitro digestibility of forages is commonly estimated by two-stage methods in which the various samples are kept completely separate from one another, using tubes. A possible alternative approach, which may save labour, is to use larger vessels, within which up to as many as 25 samples are incubated, each contained in its own ®lter bag. The two approaches were compared for estimating apparent dry matter (DM) digestibility, apparent digestible organic matter in DM, true DM digestibility, true digestible organic matter in DM and digestibility of neutral detergent ®bre. The forage samples analysed comprised all 72 combinations of two forage species (Lolium multi¯orum and Medicago sativa), three plant parts (whole crop, leaf and stem), three degrees of particle breakdown (0.5, 1.0 and 1.5 mm sieve size when milling) and four ®eld replicates. Rumen ¯uid from sheep was used for two ®eld replicates and rumen ¯uid from cattle for the other two. There was no discernible effect on digestibility of the sieve size used when milling, e.g. true digestible organic matter in dry matter using ®lter bags was 674, 677 and 663 g kgÿ1, respectively, (SE 6.4) with the 0.5, 1.0 and 1.5 mm sieves. There were smaller differences between the two forage species (in respect of whole crop, stem and leaf) with the ®lter bag than with the tube method. The standard errors and coef®cients of variation were higher with the ®lter bag than with the tube method; of 16 coef®cients of variation calculated for each method, the mean with ®lter bags was 4.0% and the mean with tubes was 2.7%. Linear regression indicated that true digestibility using tubes could be predicted more precisely than apparent digestibility using tubes from the results using ®lter bags. The difference between apparent and true digestibility, when estimated using ®lter bags, appeared unrealistically low. The estimates of forage digestibility when using rumen ¯uid from sheep were very similar to those when using rumen ¯uid from cattle. It is concluded that the traditional methods, using tubes, are likely to give more precise results than *

Corresponding author. Tel.: 44-1970-622264; fax: 44-1970-611264. E-mail address: [email protected] (D. Wilman) 0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 1 0 - 3

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D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47

using ®lter bags, although at the cost of requiring more labour. # 2000 Elsevier Science B.V. All rights reserved. Keywords: In vitro digestibility; Forages; Filter bags

1. Introduction The two-stage technique for in vitro digestion of forage crops developed by Tilley and Terry (1963) has been widely used in many countries, often with minor modi®cations, and is widely accepted as providing a satisfactory estimate of in vivo apparent digestibility (Van Soest, 1994). The method of estimating true digestibility, based on incubation of samples in rumen ¯uid followed by boiling in neutral detergent to leave a residue of undegraded cell wall, proposed by Van Soest et al. (1966), has also been widely used. Both the Tilley and Terry (1963) and the Van Soest et al. (1966) methods involve incubating each sample in a separate container, followed by centrifugation and/or ®ltration to remove unwanted liquid. An alternative approach is to seal each sample within a small ®lter bag and incubate a large number of these samples within a single vessel containing digestion media. After incubation, the media can be drained off and the bags rinsed with water. Traxler et al. (1995) determined true dry matter (DM) digestibility of four forages, comparing a ®lter bag method with that of Van Soest et al. (1966) and concluded that the two methods gave comparable estimates. They described the ®lter bag system as considerably more ef®cient due to its batch processing at all levels of the analysis. Garman et al. (1997) determined in vitro DM digestibility of nine feedstuffs, comparing a ®lter bag method with a traditional method, and concluded that the ®lter bag approach is a feasible, labour ef®cient alternative to the traditional method. Vogel et al. (1999) determined in vitro apparent DM digestibility of 23 forages, comparing a ®lter bag with a tube method, and reported that the ®lter bag method was easier to use and required less laboratory space. On the basis of these and other ®ndings, it seems worthwhile to extend the testing of the ®lter bag approach, to determine its precision and accuracy and to identify limitations. There seem to be at least two potential disadvantages of the ®lter bag approach to estimating digestibility: (1) movement of ®ne particulate matter into and out of the bags, through the pores or through faults in the material or the seal, during incubation (as noted for bags in the rumen by Ellis et al. (1994)), which could affect residue weights and estimates of digestibility; and (2) the associative in¯uence of samples on each other when incubated in the same vessel, in which soluble, and possibly particulate, matter released into the rumen ¯uid could affect microbial activity and hence cell wall degradation in all the samples in the vessel. This paper reports a comparison of the ®lter bag approach to estimating digestibility, both apparent and true, with the traditional approaches. Three degrees of particle breakdown of the dried forage were compared to determine if that would provide evidence of movement of ®ne particles into and out of the bags. Different plant parts of two contrasting forage species were included to determine if that would provide evidence of associative effects of samples incubated in the same vessel.


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2. Experimental 2.1. Methods In vitro digestibility was determined on 72 forage samples, in each case comparing a ®lter bag with a traditional, tube method and expressing digestibility in each of ®ve ways: apparent DM digestibility; apparent digestible organic matter in DM; true DM digestibility; true digestible organic matter in DM; and digestibility of neutral detergent ®bre (NDF). Thirty-six forage samples (from ®eld replicates 2 and 3) were digested in Week 1 (beginning 15 June 1998), using rumen ¯uid from sheep, and 36 (from ®eld replicates 4 and 5) in Week 2 (the following week), using rumen ¯uid from cattle. The sheep were on a hay diet and the cattle were on pasture. The pH of the ¯uid was 6.6 in Week 1 and 6.7 in Week 2. For the ®lter bag method, `ANKOM F57' ®lter bags and a `DAISY II' incubator (ANKOM Technology, Fairport, New York, USA) were used. The bags were each 50 mm55 mm, made from polyester/polyethylene extruded ®laments in a threedimensional matrix claimed to retain particles >25 mm. The methods were based, so far as feasible, on those recommended by ANKOM Technology, using 0.5 g of sample per bag and 21 bags per incubation jar, made up of 18 experimental samples from one ®eld replicate, a high digestibility (grass) standard, a low digestibility (grass) standard and a blank. The 18 experimental samples in each jar comprised all combinations of two forage species, three plant parts and three sieve sizes during milling (see Section 2.2). In each week, two jars were used for apparent digestibility (48 h in rumen ¯uid/buffer followed by 48 h in acid pepsin) and two for true digestibility (48 h in rumen ¯uid/buffer followed by removal from the jars for boiling in neutral detergent). The four jars were mounted on slow turning rollers inside the incubator. Each jar contained a plastic divider, with holes. Each bag was heat-sealed to retain the sample. After each 48 h incubation, the jars were drained and the bags rinsed thoroughly with water. The bags for true digestibility determination were boiled individually for 1 h in 100 ml of neutral detergent solution in tall form 600 ml beakers of 80 mm internal diameter, using condensers made from 250 ml round ¯asks; because the bags ¯oated to the top of the solution, they were pushed under every 10 min to increase contact with the solution. The tube method was based on Tilley and Terry (1963) for apparent digestibility and on Van Soest et al. (1966) for true digestibility. The tubes were each of 100 ml capacity, made of polypropylene, and were sealed during incubation with a bung ®tted with a gas release valve. Each tube held 0.5 g of sample and there were 42 tubes per water bath, of which 21 were for apparent and 21 for true digestibility. Each set of 21 was made up, as for the bag method, with 18 experimental samples from one ®eld replicate (the same replicate for apparent and true), a high standard, a low standard and a blank. For true digestibility, the contents of each tube were transferred to a 500 ml ¯ask, with Liebig condenser, and boiled for 1 h in neutral detergent solution. So far as feasible, the ®lter bag and tube methods were kept comparable, drawing from the same rumen ¯uid/buffer mixture (as used by Tilley and Terry, 1963) (2 l per jar, 50 ml per tube), the same acid pepsin solution (Tilley and Terry, 1963) (2 l per jar, 50 ml per

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tube) and the same neutral detergent solution (Van Soest and Wine, 1967), and using the same incubation temperature (398C). The buffer solution was the McDougall `synthetic saliva' used by Tilley and Terry (1963), with pH 6.9, comprising mainly Na2HPO4 and NaHCO3, with smaller amounts of NaCl, KCl, MgCl2 and CaCl3 and saturated with CO2 at 388C. 2.2. Forages The 72 forage samples were made up of all combinations of two forage species, three plant parts, three sieve sizes during milling and four ®eld replicates. The forage species were Italian ryegrass (Lolium multi¯orum Lam.) and lucerne (Medicago sativa L.). The plant parts were whole crop (harvested forage above a cutting height of 4±5 cm), leaf (leaf blades of ryegrass and lea¯ets of lucerne) and stem (true stem of ryegrass and stem of lucerne). The three sieve sizes (to achieve different degrees of particle breakdown) were 0.5, 1.0 and 1.5 mm. The ®eld replicates were drawn from an experiment with three species (Italian ryegrass, lucerne and wheat) and six replicates, arranged in a randomized block design, with each plot 4 m16 m. The Italian ryegrass was harvested on 4 September 1996, after 34-day regrowth. The yield was 2.3 t DM haÿ1 and the proportions of leaf blade, leaf sheath and stem were 31, 23 and 46%, respectively. The lucerne was harvested on 8 October 1996, after 69-day regrowth; the yield was 3.9 t DM haÿ1 and the proportions of leaf and stem were 44 and 56%, respectively. The plant part samples were freeze-dried before milling and analysis. Results were adjusted to an oven DM basis for presentation. 2.3. Analysis of data The high and low digestibility standards used were of known in vivo apparent DM digestibility. There were no in vivo values for their true digestibility, nor for apparent digestible organic matter in DM, although in the latter case there were well-established in vitro values. Accordingly, in the present study, the results from the standards were used to correct the experimental results to an in vivo basis in the case of apparent DM digestibility and to the established in vitro basis in the case of apparent digestible organic matter in DM, and the results were left uncorrected in the case of true digestibility. The results were statistically analysed separately for the two weeks (Tables 2 and 5) and together (Tables 3, 4, 6 and 7), using analysis of variance and a randomized block design, with two replicates (for 1 week) or four replicates (for the 2 weeks together). The 18 combinations of species, plant part and sieve size were the experimental treatments for the analysis. For Table 6 the plant parts were analysed separately as well as together. Treatment means were compared using the least signi®cant difference test (Sokal and Rohlf, 1995). The signi®cance of differences between the variance using ®lter bags and that using tubes was tested using a two-tailed F-test (Sokal and Rohlf, 1995; Rohlf and Sokal, 1995). In each test the larger error mean square (from the analysis of variance) was divided by the smaller.


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Linear regression equations were calculated to indicate the precision with which digestibilities using the tube method might be predicted when using the ®lter bag method. 3. Results Italian ryegrass and lucerne were similar in the concentrations of NDF in stem DM and of ash in stem and leaf DM (Table 1). The concentration of NDF in leaf DM was much higher in ryegrass than lucerne. The digestibility results in Week 1, using rumen ¯uid from sheep on plant material from ®eld replicates 2 and 3, were very similar to the results in Week 2, using rumen ¯uid from cattle on plant material from ®eld replicates 4 and 5, as illustrated in Table 2 for apparent digestible organic matter in DM and digestibility of NDF. There was no discernible effect of ®neness of milling with either the ®lter bag or the tube method, as illustrated in Table 3 for apparent and true digestible organic matter in DM and digestibility of NDF. The standard errors were higher with the ®lter bag than with the tube method (Tables 2, 3 and 4). The variance using ®lter bags was signi®cantly larger than the variance using tubes, with all ®ve measures of digestibility, when the 2 weeks results were analysed together (Table 4). When the results were analysed separately for each week, the variance using ®lter bags was signi®cantly larger than the variance using tubes in Week 2 for apparent DM digestibility, apparent digestible organic matter in DM and digestibility of NDF (Table 5). The estimates of apparent digestibility were higher using ®lter bags than using tubes, except with the stems of Italian ryegrass (Tables 3 and 4). The estimates of true digestibility and of digestibility of NDF were consistently lower using ®lter bags than using tubes. According to the ®lter bag method, the lea¯ets of lucerne were very similar in digestibility to the leaf blades of Italian ryegrass, whereas according to the tube method the lea¯ets of lucerne were less digestible than the leaf blades of Italian ryegrass (p