A comparison of lter bag methods with conventional tube methods of determining the in vitro digestibility of forages

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 kg 1, 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: ddw@aber.ac.uk (D. Wilman) 0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0377-8401(00)00110-3

34 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.

2. Experimental 2.1. Methods D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 35 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

36 D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 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 Na 2 HPO 4 and NaHCO 3, with smaller amounts of NaCl, KCl, MgCl 2 and CaCl 3 and saturated with CO 2 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 (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 and lea ets of lucerne) and stem (true stem of 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, lucerne and wheat) and six replicates, arranged in a randomized block design, with each plot 4 m16 m. The Italian was harvested on 4 September 1996, after 34-day regrowth. The yield was 2.3 t DM ha 1 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 ha 1 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.

D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 37 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 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 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 (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, whereas according to the tube method the lea ets of lucerne were less digestible than the leaf blades of Italian (p<0.01) (Table 4). According to both methods, the stems of lucerne were much less digestible than the stems of Italian (p<0.001), and the NDF of the stems of lucerne was much Table 1 Neutral detergent bre (NDF) and ash content of plant parts of Italian and lucerne used in the comparison of lter bag with tube methods of determining in vitro digestibility NDF Ash (g kg 1 DM) (g kg 1 DM) Italian Whole crop 541 103 Leaf 484 109 Stem 596 78 Lucerne Whole crop 427 76 Leaf 202 110 Stem 599 72

Table 2 In vitro apparent digestible organic matter in dry matter (g kg 1 ) and digestibility of neutral detergent bre (NDF) of plant parts of Italian and lucerne, comparing a lter bag with a tube method, using rumen uid from sheep in Week 1 and from cattle in Week 2 a Apparent digestible organic matter in DM Digestibility of NDF Week 1 (sheep fluid) Week 2 (cattle fluid) Week 1 (sheep fluid) Week 2 (cattle fluid) Italian Using filter bags Whole crop 680 a 583 b 632 738 a 594 b 666 676 a 227 b 451 669 a 263 b 466 Leaf 705 a 686 a 695 728 a 756 a 742 781 a 438 b 609 706 a 285 b 495 Stem 675 a 476 b 575 662 a 444 b 553 651 a 223 b 437 560 a 261 b 410 Mean 686 a 581 b 634 709 a 598 b 654 703 a 296 b 499 645 a 269 b 457 SE plant part means 9.9 7.0 16.1 11.4 22.4 15.8 39.8 28.1 SE crop species means 5.7 9.3 12.9 23.0 Using tubes Whole crop 665 a 523 b 594 671 a 539 b 605 776 a 271 b 524 777 a 320 b 549 Leaf 690 a 627 b 659 655 a 640 a 648 838 a 453 b 646 810 a 433 b 622 Stem 698 a 424 b 561 646 a 423 b 535 759 a 287 b 523 628 a 269 b 471 Mean 684 a 525 b 605 657 a 534 b 596 791 a 337 b 564 753 a 341 b 547 SE plant part means 6.8 4.8 8.6 6.1 14.1 9.9 21.3 15.1 SE crop species means 4.0 4.9 8.1 12.3 a In each pair of crop species, different letters signify that the means are signi cantly (p<0.05) different. Lucerne Mean 38 D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47

Table 3 In vitro apparent and true digestible organic matter in dry matter (DOMD) and digestibility of neutral detergent bre (NDF) of plant parts of Italian and lucerne comparing three sieve sizes during milling, using a lter bag and a tube method, mean of weeks 1 and 2 (g kg 1 ) a Apparent DOMD True DOMD Digestibility of NDF Italian Using filter bags Sieve size (mm) 0.5 708 a 596 a 652 a 721 a 627 a 674 a 665 a 298 a 482 a 1.0 699 a 581 a 640 a 738 a 616 a 677 a 693 a 286 a 489 a 1.5 686 a 592 a 639 a 719 a 607 a 663 a 664 a 264 a 464 a Mean 698 590 644 726 617 672 674 283 478 SE sieve size means 10.2 7.2 9.1 6.4 24.2 17.1 Using tubes Sieve size (mm) 0.5 671 a 535 a 603 a 768 a 640 a 704 a 764 a 351 a 558 a 1.0 673 a 531 a 602 a 776 a 630 a 703 a 777 a 337 a 557 a 1.5 669 a 523 a 596 a 774 a 626 a 700 a 775 a 329 a 552 a Mean 671 530 601 773 632 703 772 339 556 SE sieve size means 6.5 4.6 6.3 4.4 12.9 9.1 a In each group of sieve size means, different letters would signify that the means are signi cantly (p<0.05) different. Lucerne Mean D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 39

Table 4 In vitro apparent and true dry matter digestibility (DMD), digestible organic matter in dry matter (DOMD) and digestibility of neutral detergent bre (NDF) of plant parts of Italian and lucerne comparing a lter bag with a tube method, mean of weeks 1 and 2 (g kg 1 ) a Apparent DMD Apparent DOMD True DMD True DOMD Digestibility of NDF Italian Using filter bags Whole crop 778 a 674 b 726 709 a 588 b 649 823 a 678 b 751 720 a 607 b 664 672 a 245 b 459 Leaf 787 a 794 a 791 716 a 721 a 719 876 a 870 a 873 767 a 766 a 767 743 a 362 b 552 Stem 731 a 580 b 656 668 a 460 b 564 766 a 546 b 656 691 a 477 b 584 606 a 242 b 424 Mean 765 a 683 b 724 698 a 590 b 644 822 a 698 b 760 726 a 617 b 672 674 a 283 b 478 SE plant part means 7.3 5.1 10.2 7.2 9.0 6.4 9.1 6.4 24.2 17.1 SE species means 4.2 5.9 5.2 5.2 14.0 Using tubes Whole crop 755 a 634 b 694 668 a 531 b 600 879 a 700 b 789 770 a 624 b 697 777 a 296 b 538 Leaf 763 a 734 b 748 673 a 634 b 654 915 a 888 b 901 799 a 775 b 787 824 a 443 b 634 Stem 741 a 552 b 646 672 a 424 b 548 831 a 567 b 699 750 a 497 b 624 716 a 278 b 497 Mean 753 a 640 b 696 671 a 530 b 601 875 a 718 b 796 773 a 632 b 703 772 a 339 b 556 SE plant part means 5.0 3.6 6.5 4.6 6.2 4.4 6.3 4.4 12.9 9.1 SE species means 2.9 3.8 3.6 3.6 7.5 F-test Filter bag variance/tube variance 2.09* 2.50** 2.13** 2.09* 3.51*** Tube method minus bag method Whole crop 23 a 40 a 32 42 a 57 a 50 56 a 22 b 38 50 a 17 b 33 105 a 51 a 78 Leaf 24 a 60 b 43 43 a 87 b 65 39 a 18 a 28 32 a 9 b 20 81 a 82 a 81 Stem 10 a 28 b 10 4 a 36 b 16 65 a 21 b 43 59 a 20 b 40 111 a 36 b 73 Mean 12 a 43 b 28 27 a 60 b 44 53 a 20 b 36 47 a 15 b 31 99 a 56 b 77 SE plant part means 6.1 4.3 8.7 6.1 7.3 5.1 6.9 4.9 21.2 15.0 SE species means 3.5 5.0 4.2 4.0 12.2 Lucerne Mean 40 D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 a In each pair of crop species means, different letters signify that the means are signi cantly (p<0.05) different.

D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 41 Table 5 Coef cients of variation (%) for in vitro apparent and true dry matter digestibility (DMD), digestible organic matter in dry matter (DOMD) and digestibility of neutral detergent bre (NDF) of plant parts of Italian and lucerne, comparing a lter bag with a tube method, using rumen uid from sheep in Week 1 and from cattle in Week 2 (error df in brackets) Apparent DMD Apparent DOMD True DMD True DOMD Digestibility of NDF Week 1 Week 2 Week 1 Week 2 Week 1 Week 2 Week 1 Week 2 Week 1 Week 2 (sheep (cattle (sheep (cattle (sheep (cattle (sheep (cattle (sheep (cattle fluid) fluid) fluid) fluid) fluid) fluid) fluid) fluid) fluid) fluid) Using lter 2.4 (17) 3.8 (17) 3.8 (17) 6.0 (17) 3.3 (16) 4.2 (15) 3.5 (16) 4.6 (15) 11.0 (16) 21.3 (15) bags Using tubes 2.3 (17) 2.2 (17) 2.8 (17 3.5 (17) 2.1 (17) 3.1 (17) 2.6 (17) 3.5 (17) 6.1 (17) 9.6 (17) F-test Filter bag variance/ tube variance 1.07 ns 3.39* 2.11 ns 3.54* 2.27 ns 1.67 ns 1.78 ns 1.51 ns 2.53 ns 3.47* less digestible than the NDF of the stems of (p<0.001), but these differences between the species were less with the lter bag than with the tube method (Table 4). Similarly, the whole crop samples of lucerne were less digestible than the whole crop samples of Italian (p<0.001), and the NDF of lucerne whole crop was less digestible than the NDF of whole crop (p<0.001), but the difference between the species was less with the lter bag than with the tube method. According to both methods, the leaf blades were rather more digestible than the stems in Italian and the lea ets were much more digestible than the stems in lucerne, but the difference between leaves and stems was greater with the lter bag than with the tube method (Table 4). In most cases, the coef cients of variation were higher with the lter bag than with the tube method (Tables 5 and 6). The coef cients of variation were lower when the analysis of variance was restricted to leaf alone or to stem alone than when it was restricted to whole crop alone, except that the coef cients were high when using lter bags for true digestibility of stem and NDF digestibility of leaf and stem (Table 6). The coef cients of variation from the statistical analysis which included all plant parts were generally similar to, or a little lower than, the coef cients from the analysis of whole crop alone. The coef cients of variation for DM digestibility, both apparent and true, were lower than the coef cients for digestible organic matter in DM. The coef cients of variation for true digestibility were not consistently higher or lower than the coef cients for apparent digestibility. The coef cients of variation were generally lower in Week 1 (using sheep uid) than in Week 2 (using cattle uid) (Table 5). The differences between true and apparent digestibility were less with the lter bag than with the tube method (Table 7). The standard errors relating to the differences between true and apparent digestibility were greater with the lter bag than with the tube method. The overall difference between true and apparent digestibility was similar

42 D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 Table 6 Coef cients of variation (%) for in vitro apparent and true dry matter digestibility (DMD), digestible organic matter in dry matter (DOMD) and digestibility of neutral detergent bre (NDF) of plant parts of Italian and lucerne, comparing a lter bag with a tube method over two weeks (error df in brackets) Apparent True Digestibility of NDF DMD DOMD DMD DOMD Using filter bags Whole crop 3.6 (15) 6.2 (15) 4.1 (14) 4.5 (14) 14.6 (14) Leaf 1.7 (15) 3.3 (15) 2.9 (15) 3.1 (15) 17.7 (15) Stem 1.7 (15) 3.3 (15) 5.6 (13) 6.9 (13) 16.6 (13) Mean 3.5 (51) 5.5 (51) 4.1 (48) 4.7 (48) 17.5 (48) Using tubes Whole crop 2.7 (15) 4.0 (15) 3.8 (15) 4.1 (15) 12.2 (15) Leaf 1.3 (15) 2.8 (15) 0.6 (15) 1.4 (15) 2.2 (15) Stem 2.0 (15) 3.8 (15) 2.0 (15) 2.9 (15) 6.3 (15) Mean 2.5 (51) 3.7 (51) 2.7 (51) 3.1 (51) 8.1 (51) whether digestibility was expressed as digestible organic matter in DM or as DM digestibility. The differences between true and apparent digestibility were less with stem than with leaf. True DM digestibility and true digestible organic matter in DM with the tube method were predicted fairly well using the lter bag results (with r 2ˆ0.92±0.95) and in each Table 7 Differences between true and apparent dry matter digestibility (DMD) and between true and apparent digestible organic matter in dry matter (DOMD) (true minus apparent in g kg 1 ) in plant parts of Italian and lucerne, comparing a lter bag with a tube method, mean of weeks 1 and 2 a DMD DOMD Italian Lucerne Mean Using filter bags Whole crop 45 a 3 b 24 11 a 19 a 15 Leaf 89 a 77 a 83 51 a 45 a 48 Stem 35 a 34 b 1 23 a 17 a 20 Mean 56 a 15 b 36 28 a 27 a 28 SE plant part means 10.4 7.4 12.3 8.7 SE species means 6.0 7.1 Using tubes Whole crop 124 a 66 b 95 102 a 93 a 98 Leaf 152 a 154 a 153 126 a 142 b 134 Stem 91 a 15 b 53 77 a 73 a 75 Mean 122 a 78 b 100 102 a 103 a 102 SE plant part means 4.1 2.9 4.3 3.1 SE species means 2.4 2.5 a In each pair of crop species means, different letters signify that the means are signi cantly (p<0.05) different.

D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 43 Table 8 The slopes and constants of linear regression equations predicting in vitro digestibility using a tube method from in vitro digestibility using a lter bag method (g kg 1 ) Apparent DM digestibility Apparent digestible organic matter in DM True DM digestibility True digestible organic matter in DM Week 1 (sheep uid) Constant 108 100 70 69 SE 51.7 48.1 28.0 25.6 Slope 1.136 1.112 0.954 0.941 SE 0.0723 0.0752 0.0360 0.0372 r 2 0.88 0.87 0.95 0.95 Week 2 (cattle uid) Constant 103 106 14 13 SE 31.6 30.2 38.1 36.7 Slope 0.801 0.750 1.071 1.071 SE 0.0426 0.0455 0.0500 0.0546 r 2 0.91 0.89 0.93 0.92 Over two weeks Constant 52 42 33 36 SE 34.9 31.1 23.6 22.0 Slope 0.891 0.866 1.005 0.993 SE 0.0479 0.0477 0.0306 0.0324 r 2 0.83 0.83 0.94 0.93 case the prediction equation was similar in each of the two weeks (Table 8). The scatter of points for the two weeks combined in the case of true DM digestibility is shown in Fig. 1. Apparent DM digestibility and apparent digestible organic matter in DM with the tube method were predicted less precisely than true digestibility, using the lter bag results, (with r 2ˆ0.83±0.91) and in each case the prediction equation was different in the two weeks (Table 8). 4. Discussion The pores of the lter bags should certainly have been large enough to allow microbial colonization of all forage fragments within the sample and normal rates of bre digestion (Ellis et al., 1994). The lack of a discernible effect of neness of milling with the lter bag as with the tube method suggests that the pores of the lter bags were small enough to retain the majority of forage particles. If that had not been the case, it seems likely that milling through a 0.5 mm sieve would have resulted in smaller residues, and hence higher estimates of digestibility, than milling through a 1.5 mm sieve, with the lter bag but not the tube method. Much ner milling might yield a different conclusion, but would not be relevant to routine laboratory analysis. The smaller differences between the two forage species (in respect of whole crop, stem and leaf) with the lter bag than the tube method could suggest some in uence of some samples on others in the same jar, with the lter bag method. Such in uence might bring

44 D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 Fig. 1. In vitro true dry matter digestibility (g kg 1 ) estimated using a conventional tube method plotted against equivalent values obtained using lter bags. samples closer to the mean digestibility of the population of samples in a jar via effects on the microbial activity of the rumen uid in the jar. On the other hand, the rather greater difference in estimated digestibility between leaves and stems within a species with the lter bag than with the tube method might not be expected if some samples were in uencing others within a jar. A major consideration when deciding whether or not to adopt the lter bag approach rather than tubes is the precision achieved by each. In the present study, the tube method appeared to have a clear advantage in precision, indicated by the lower coef cients of variation in Tables 5 and 6 and the lower standard errors in Tables 2, 3, 4 and 7. On the basis of this evidence, the tube method seems preferable where the priority is to detect differences between experimental treatments without the expense of using large numbers of replicates. It should be noted, however, that Vogel et al. (1999) had rather lower errors with the lter bag approach than with their tube method. The lower errors associated with the prediction of true digestibility, as determined using tubes, from true digestibility determined using lter bags, compared with the equivalent prediction of apparent digestibility, and the greater similarity between weeks in the prediction equations for true compared with apparent digestibility, may suggest that the lter bag approach is more precise and robust for estimating true rather than apparent

D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 45 digestibility. On the other hand, it should be noted that the lter bag method underestimated true `tube' digestibility as much as it overestimated apparent `tube' digestibility. The lter bag approach might be preferred where the emphasis is on analysing large numbers of samples without using as much labour as would be needed for the tube method. The lter bag approach saves labour to the extent that adding measured amounts of rumen uid/buffer and acid pepsin to each individual sample is avoided, shaking of tubes by hand is avoided, and removing liquid at the end of each stage of the determination of apparent digestibility requires much less labour. It seems that of the order of 7 and 4 person minutes per sample can be saved when determining apparent and true digestibility, respectively. In the comparison made by Van Soest et al. (1966) of the Tilley and Terry (1963) procedure for apparent digestibility with their procedure for in vitro estimation of true digestibility (48 h in rumen uid/buffer followed by boiling in neutral detergent), using 12 grass and 8 legume hays, the regression equation relating the two procedures estimated true DM digestibility by multiplying the Tilley and Terry (1963) apparent DM digestibility by 0.92 and adding 162 g kg 1. If this equation is applied to the mean apparent DM digestibility value using tubes in Table 4, 696 g kg 1, the predicted true DM digestibility is 802 g kg 1, very close to the recorded value, 796 g kg 1. If the equation is applied to the equivalent value using the lter bags, however, 724 g kg 1, the predicted true DM digestibility is 828 g kg 1, well above the recorded value of 760 g kg 1.Asin the present study, Vogel et al. (1999) recorded a higher mean apparent DM digestibility value using lter bags than using tubes. Higher apparent digestibility using lter bags rather than tubes may be linked with the thorough rinsing of the lter bags with water after each 48 h incubation; this procedure presumably removes a proportion of the microorganisms and some ne particles, reducing the weight of residue and increasing the estimate of digestibility, compared with the tube method in which the microbial matter and ne particles are retained. The recorded values for true digestibility using lter bags in the present study might have been higher if equipment such as the `ANKOM Fiber Analyzer' had been used to ensure that the lter bags were kept submerged while being boiled in neutral detergent solution. It could be argued that the use of the `ANKOM Fiber Analyzer' for the second stage of the determination of true digestibility using lter bags in the present study might have increased the precision of that approach. A point against this, however, is that the advantage in precision of the tube method was as great when determining apparent digestibility as when determining true digestibility. It may also be noteworthy that Traxler et al. (1995), who may have used the `Fiber Analyzer', recorded lower errors with the conventional than with the lter bag method when determining true DM digestibility, in agreement with the present study. The in vitro results of Van Soest et al. (1966) were close to in vivo results for true and apparent digestibility when the same hays were fed to sheep or cattle. The eld experiment from which the samples for the present study were drawn provided material which was fed to sheep and cattle (Ahmad, 1999). In this case apparent in vivo DM digestibility was 663 g kg 1 (mean of sheep and cattle and of Italian and lucerne) and the corresponding true in vivo digestibility was 141 g kg 1 greater, i.e.

46 D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 804 g kg 1. Compared with these results and those of Van Soest et al. (1966), it appears that the in vitro results using tubes (with a difference between apparent and true of 100 g kg 1 ) were closer to the relationship between apparent and true which might be expected in vivo than the in vitro results using bags (with a difference between apparent and true of only 36 g kg 1 ). Horton et al. (1980) compared sheep with cattle as a source of rumen uid for the Tilley and Terry (1963) in vitro digestion technique. When the donor animals were on a hay diet and when the samples analysed for in vitro digestibility were hay, Horton et al. (1980) generally obtained rather lower estimates of digestibility with the sheep than with the cattle uid. The present study suggests that sheep uid and cattle uid may give very similar results for the in vitro digestibility of forage, but perhaps with lower standard errors when sheep uid is used. However, it has to be noted that in the present study the comparison between sheep and cattle uid was confounded by the difference in diet between the sheep and the cattle, the use of different eld replicates, and any other small differences between weeks 1 and 2 of the study. The true DM digestibility of Italian leaf blades in the present study, whether estimated by the bag or tube method, was similar to the corresponding values for Italian leaf blades recorded by Wilman et al. (1992). Again the true DM digestibility of lucerne lea ets in the present study, whether estimated by the bag or tube method, was similar to that recorded by Wilman and Altimimi (1984). The apparent DM digestibility of lucerne lea ets was similar to that recorded by Terry and Tilley (1964) and Wilman and Asiedu (1983). It is more dif cult to compare stem results satisfactorily because of the considerable effect of stage of maturity on stem digestibility, as illustrated by Terry and Tilley (1964). It is concluded that the use of lter bags rather than tubes can give acceptable results for the in vitro digestibility of forages, if the emphasis is on saving labour rather than on obtaining the most precise results. The escape of particulate matter from the bags seems not to be a major problem, but the escape of soluble matter from samples high in solubles could in uence the microbial population and hence increase cell wall degradation in samples low in solubles which are in the same incubation vessel. The difference between apparent and true digestibility, when estimated using lter bags, may be unrealistically low. Acknowledgements We are grateful to Dr. N. Ahmad for providing the crop samples and to Dr. N.T. Faithfull and other members of the University staff for facilities, advice and assistance. References Ahmad, N., 1999. Some plant factors affecting the digestibility of forages and roughages. Ph.D. thesis, University of Wales Aberystwyth, UK. Ellis, W.C., Matis, J.H., Hill, T.M., Murphy, M.R., 1994. Methodology for estimating digestion and passage kinetics of forages. In: Fahey, G.C., Jr. (Ed.), Forage Quality, Evaluation, and Utilization. Based on the

D. Wilman, A. Adesogan / Animal Feed Science and Technology 84 (2000) 33±47 47 National Conference on Forage Quality, Evaluation, and Utilization held at the University of Nebraska, Lincoln, on 13±15 April 1994. American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison, Wisconsin, USA, pp. 682±756. Garman, C.L., Holden, L.A., Kane, H.A., 1997. Comparison of in vitro dry matter digestibility of nine feedstuffs using three methods of analysis. J. Dairy Sci. 80 (Suppl. 1), 260 (poster No. 451). Horton, G.M.J., Christensen, D.A., Steacy, G.M., 1980. In vitro fermentation of forages with inoculum from cattle and sheep fed different diets. Agron. J. 72, 601±605. Rohlf, F.J., Sokal, R.R., 1995. Statistical Tables, 3rd Edition. W.H. Freeman and Company, New York. Sokal, R.R., Rohlf, F.J., 1995. Biometry, The Principles and Practice of Statistics in Biological Research, 3rd Edition. W.H. Freeman and Company, New York. Terry, R.A., Tilley, J.M.A., 1964. The digestibility of the leaves and stems of perennial, cocksfoot, timothy, tall fescue, lucerne and sainfoin, as measured by an in vitro procedure. J. Br. Grassl. Soc. 19, 363± 372. Tilley, J.M.A., Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Soc. 18, 104±111. Traxler, M.J., Robertson, J.B., Van Soest, P.J., Fox, D.G., Pell, A.N., 1995. A comparison of methods for determining IVDMD at three time periods using the Filter Bag Technique versus conventional methods. J. Dairy Sci. 78 (Suppl. 1), 274 (poster No. 366). Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant, 2nd Edition. Cornell University Press, Ithaca. Van Soest, P.J., Wine, R.H., 1967. Use of detergents in the analysis of brous feeds. IV. Determination of plant cell-wall constituents. J. Assoc. Off. Anal. Chem. 50, 50±55. Van Soest, P.J., Wine, R.H., Moore, L.A., 1966. Estimation of the true digestibility of forages by the in vitro digestion of cell walls. In: Proceedings of the Xth International Grassland Congress, Helsinki. Finnish Grassland Association, Helsinki, pp. 438±441. Vogel, K.P., Pedersen, J.F., Masterson, S.D., Toy, J.J., 1999. Evaluation of a lter bag system for NDF, ADF, and IVDMD forage analysis. Crop Sci. 39, 276±279. Wilman, D., Altimimi, M.A.K., 1984. The in-vitro digestibility and chemical composition of plant parts in white clover, red clover and lucerne during primary growth. J. Sci. Food Agric. 35, 133±138. Wilman, D., Asiedu, F.H.K., 1983. Growth, nutritive value and selection by sheep of sainfoin, red clover, lucerne and hybrid. J. Agric. Sci. Cambr. 100, 115±126. Wilman, D., Walters, R.J.K., Baker, D.H., Williams, S.P., 1992. Comparison of two varieties of Italian (Lolium multi orum) for milk production, when fed as silage and when grazed. J. Agri. Sci. Cambr. 118, 37±46.