Losses of particulate N during filtration and handling of feed and rumen incubation residues

Animal Feed Science and Technology 125 (2006) 123–137

Losses of particulate N during filtration and handling of feed and rumen incubation residues D. Silke a,b,∗ , P. Ud´en a a

Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, S-753 23 Uppsala, Sweden b Faculty of Agriculture, Latvia University of Agriculture, Liela Street 2, Jelgava LV-3001, Latvia Received 26 January 2005; received in revised form 13 May 2005; accepted 24 May 2005

Abstract Unintended losses of feed N occur during filtration procedures in the laboratory as well as during washing of in sacco residues and other procedures to remove microbial contamination. Our aim was to find the most reliable method for recovery of buffer and neutral detergent (ND) insoluble N (BIN and NDIN, respectively) in feed residues. In Experiment 1, paper filtration (PF; 20–25 ␮m retention) and sintered glass crucible filtration (GF; 40–90 ␮m retention) and centrifugation at 3000×g (LSC) were compared for recovery and loss of feed N after feeds were incubated with a borate–phosphate buffer or ND. The same ND removal techniques and centrifugation at 5000×g (HSC) were also applied to in vitro-fermented residues. In Experiment 2a, in vitro fermentation residues were transferred to in sacco bags (28 ␮m pore openings), washed, extracted with ND using GF or LSC techniques to remove detergents. In 2b, the in vitro residues were homogenised, stomached and/or centrifuged in different ways, then extracted with ND using HSC to remove ND. The NDIN was assumed to exclude microbial N. Samples used in Experiments 1 and 2a consisted of two grasses, birdsfoot trefoil (Lotus corniculatus), wheat bran, rapeseed cake (RSC), dried distillers grain with solubles from wheat (DDGS), de-hulled barley grain (barley) and solvent extracted soybean meal (SBM). For Experiment 2b, we used only one grass and barley. Highest recovery of BIN was by PF > GF > LSC, whereas NDIN recovery was highest for LSC with no differences between filtration techniques for Abbreviations: BIN, buffer insoluble N; ND, neutral detergent; NDIN, neutral detergent insoluble nitrogen; RSC, rapeseed cake; DDGS, dried distillers grain with solubles from wheat; SBM, solvent extracted soybean meal; PF, paper filtration; GF, filtration in sintered glass crucibles; LSC, centrifugation at 3000×g for 5 min; HSC, centrifugation at 5000×g for 5 min ∗ Corresponding author. Tel.: +46 18 672058; fax: +46 18 672946. E-mail address: [email protected] (D. Silke). 0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.05.022

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most feeds. After in vitro fermentation, recovery of NDIN was 1.2–7.1- and 1.2–2.8-fold higher by LSC versus GF and PF, respectively. Washing of in vitro residues in sacco caused NDIN reductions of up to 0.35 and 0.85 when GF or LSC was used to remove ND. Losses of NDIN from in vitro fermented feeds, including in sacco and GF losses after ND extraction, were 0.64–0.70 in forage, and 0.81 and 0.83 in SBM and DDGS, respectively. Losses of NDIN from homogenisation, pummelling and centrifugation of in vitro residue were smaller (0.02–0.42) than after sample washing in sacco bags. Oven drying of in vitro residues at 65 ◦ C prior to ND extraction inflated NDIN values and must be avoided. A higher centrifugal force, i.e., 5000×g instead of 3000×g and fewer centrifugations (three instead of five) resulted in the highest recovery and are recommended to minimise sample losses during centrifugation of in vitro residues. © 2005 Elsevier B.V. All rights reserved. Keywords: Filtration; Centrifugation; Filtration loss; Bacterial attachment; Bacterial removal; Neutral detergent insoluble nitrogen; Rumen; In vitro

1. Introduction To estimate rumen degradability of a feed component using in vitro or ruminal in sacco incubation, isolation of the desired residue is required. Normally this is done by successive use of filters. Synthetic fibre bags are used for the in sacco procedure but sintered glass filters are normally used to recover the in vitro residue, and also during isolation of fibre fractions in the original feed and fermented residue. If feed particles are lost through the filters, degradability measurement will be compromised. Standard retention sizes of sintered glass filters for fibre or in vitro residue isolation are 40–90 and 90–150 ␮m (porosities 2 and 1), respectively. Filter papers retaining 20–25 ␮m and larger particles (Whatman #541, 54) are commonly used for determining buffer soluble N. Cherney et al. (1993) reported higher neutral detergent insoluble N recoveries in crucibles (10–15 ␮m retention size, porosity C) compared to filter paper (Whatman #54, 20–25 ␮m retention size). These analyses were completed using forages after a 48 h in vitro fermentation. Legay-Carmier and Bauchart (1989) reported that 0.24–0.30 of cows rumen content consisted of particles, which sediment after centrifugation at 500×g for 30 min, but were small enough to pass a 0.1 mm screen. With such a high proportion of fine particles, it is important to ensure that they are not lost in filtration. In sacco procedures commonly recommend a porosity of polyester bags between 40 and 60 ␮m (see review by Vanzant et al., 1998), a recommendation that is a compromise between the need for solute and microorganism exchange across the membrane, and prevention of particle flux. Ud´en and Van Soest (1984) showed in sacco influx of particles, but escape of small particles has to our knowledge not been quantified in ruminal in sacco procedures. If microbial degradation procedures are used to measure feed protein degradation, then microbial contamination must be removed or corrected. Homogenisation and stomaching are often used as procedures to detach bacteria from rumen-incubated feed residues (Merry and McAllan, 1983; Robinson and Sniffen, 1985; Hsu and Fahey, 1990; Cecava et al., 1990; Calsamiglia et al., 1996). Such procedures run the risk of losing undegraded feed N, and Beckers et al. (1995) showed that pummelling in a stomacher, followed by washing in

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polyester bags with 43 ␮m pore openings, resulted in N losses from unfermented feed. As neutral detergent (ND) is assumed to remove all microbial matter (Van Soest, 1994), ND insoluble N (NDIN) in fermentation residues should be virtually of feed origin. Isolation of NDIN would therefore be a possible indicator of particulate feed N recovery or loss when different microbial detachment procedures are compared. The first objective was to compare routine laboratory filtration and centrifugation techniques to recover insoluble N fractions in common feeds before and after in vitro fermentation. The second objective was to quantify losses of NDIN that may occur during washing of residues after fermentation in polyester bags. The third objective was to evaluate the effects of homogenisation, stomaching and centrifugation procedures on recovery of NDIN.

2. Materials and methods Two experiments were completed. In the first, filtration and centrifugation techniques were compared for their effectiveness in recovering buffer insoluble N (BIN) in feeds and NDIN in feeds before and after in vitro incubation using rumen inocula. In the second experiment, procedures for detachment and removal of microbial population were also examined for recovery of NDIN. 2.1. Feeds Poor and medium quality grass hay (grass hay-1 and -2, respectively), birdsfoot trefoil (Lotus corniculatus), wheat bran, rapeseed cake (RSC), dried distillers grain with solubles from wheat (DDGS), de-hulled barley grain (barley) and solvent extracted soybean meal (SBM) were dried at 55 ◦ C and ground in a Wiley cutting grinder to pass a 1.5 mm screen. All eight feeds were used in Experiments 1 and 2a, but in Experiment 2b only grass hay-2 and barley were used. 2.2. Experiment 1 2.2.1. Unfermented samples Recovery of BIN was measured after incubating 0.5 g of feed in triplicate in 50 ml of borate–phosphate buffer (8.91 g/l disodium tetraborate decahydrate, 12.20 g/l sodium dihydrogenphosphate monohydrate) at pH 6.7–6.8 (Licitra et al., 1996). Incubations were completed in a water bath at 39 ◦ C for 1 h with manual stirring every 15 min. The NDIN recovery was measured after incubating 1 g feed and 50 ml of normal strength ND solution without sulphite in an oven at 85 ◦ C for 16 h (Chai and Ud´en, 1998). About 100 ␮l of bacterial amylase Termamyl, 300 l, type DX (Novozymes A/S, Denmark) was added to wheat bran, barley and SBM 2 h before the end of the ND incubation. 2.2.2. In vitro samples The feed samples were fermented in vitro using the medium described by Goering and Van Soest (1970) and the procedures adapted from Mbwile and Ud´en (1991). Rumen fluid with some particulate matter was collected approximately 2 h after the morning feeding

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from the ventral sac of a rumen fistulated cow which was fed grass hay (4 kg/d) and concentrates (1.5 kg/d) in two equal meals. In the laboratory, the rumen fluid was reduced with approximately 5 ml of ferrous sulphide precipitate per litre of fluid, homogenised at moderate speed in a domestic blender for 1 min, filtered through a single layer of cheesecloth and centrifuged at 1000×g for 5 min at 24 ◦ C to remove small particles. Carbon dioxide was flushed over the fluid during all processing. Incubations were in CO2 flushed Kjeldahl tubes (250 ml) capped with rubber stoppers with capillary rods. Triplicate samples of 1.5 g of feed, 120 ml buffer containing medium and 6 ml of reducing solution were pre-warmed to 39 ◦ C for approximately 1 h before 30 ml of centrifuged rumen fluid was added. Tubes were kept in a water bath at 39 ◦ C and manually stirred twice daily. After 24 h, samples were put in ice water to stop fermentation. Cooled samples were transferred to 250 ml centrifuge bottles and centrifuged at 5000×g for 5 min at 4 ◦ C. Forage samples were kept in a vacuum chamber for 10 min prior to centrifugation, to release gas associated with feed particles and supernatant was removed by suction. Initially, residues were dried in a forced draught oven at 65 ◦ C for at least 36 h and ground with a mortar and pestle. This procedure resulted in the apparent creation of NDIN with values exceeding that of the original feed by a factor as high as 14, and so the procedure was abandoned. New samples were prepared and extraction with 50 ml of ND was then completed directly on the centrifuged in vitro residues, in the same way as with feed samples except for exclusion of amylase. 2.2.3. Techniques for removal of solubles Filter paper, sintered glass crucibles and centrifugation at 3000×g were used to remove soluble N after buffer and ND extraction of the unfermented feeds. For in vitro samples, centrifugation at 5000×g was also used. 2.2.3.1. Paper filtration (PF). Samples were transferred to a Munktell OOR paper (retention size 20–25 ␮m, Munktell Filter AB, Grycksbo, Sweden) fitted in a B˝uchner funnel (70 mm diameter). Buffer incubated samples were washed repeatedly with a total of 250 ml of de-ionised water and samples incubated in ND were washed with boiling de-ionised water until no foaming was observed. 2.2.3.2. Sintered glass filtration (GF). Filtration was in sintered glass crucibles, porosity 2, retention size 40–90 ␮m, and washing was with similar volumes of water as described for paper filtration. 2.2.3.3. Centrifugation at 3000×g (LSC). Incubated samples were transferred to 250 ml centrifuge bottles and centrifuged at 3000×g for 5 min (fixed-angle rotor Suprafuge 22 centrifuge Heraeus Sepatech GmbH, Osterode, Germany). After centrifugation, supernatant was removed by suction and the pellet re-suspended in 250 ml of de-ionised water of room temperature (BIN) or hot water (NDIN). The centrifugation suction procedure was repeated twice. 2.2.3.4. Centrifugation at 5000×g (HSC). The same procedure was followed as centrifugation at 3000×g, but centrifugal force was 5000×g.

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Fig. 1. Experiment 2: recovery for neutral detergent insoluble N of in vitro residues after 24 h fermentation. C1–C3 = control treatments from Experiment 1; A1 and A2 = detachment and removal of loose microbes simulated by washing in sacco; B1–B6 = detachment and removal of loose microbes simulated by different centrifugation methods, by homogenisation (H) and stomaching (S) treatments (see Section 2.3.2 for details); GF = filtration in sintered glass crucibles.

All samples were finally dried in a forced draught oven (80 ◦ C) and analysed for Kjeldahl N, using a 2020 Digestor and a 2400 Kjeltec Analyser unit (FOSS Analytical A/S, Hillerød, Denmark) with Cu as a catalyst. 2.3. Experiment 2 Feed N in the form of fine particles can be lost during efforts to remove microbes. The extent of these losses was examined in 24 h in vitro fermentation residues obtained by the same procedure as in Experiment 1. In Experiment 2a, washing in sacco was completed, and in Experiment 2b, homogenisation, stomaching and centrifugation were completed (see also Fig. 1). After these treatments, all residues were ND extracted, as described in Experiment 1, but without amylase. The ND extraction from Experiment 2a samples was removed either by GF (A1) or LSC (A2). The ND extraction from Experiment 2b samples was removed by HSC (B1–B6) as in Experiment 1. Results from Experiment 2 were also compared to controls, using results from respective soluble removal techniques obtained for in vitro samples in Experiment 1. The controls are referred to as C1 after GF, C2 after three times LSC, and C3 after three times HSC.

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2.3.1. Experiment 2a: washing in sacco In vitro residues were transferred to polyester bags (28 ␮m openings, 125 mm×60 mm internal size), which were then mounted on loops and rinsed in a conventional washing machine for 2×12 min, according to the standard in sacco procedure of the laboratory. After rinsing, bags were dried in a forced draught oven at 45 ◦ C for a minimum of 20 h, emptied, and contents were ground with a mortar and pestle. 2.3.2. Experiment 2b: homogenisation, stomaching and centrifugation In vitro residues were transferred to bottles and the following tests were completed: (1) (2) (3) (4) (5) (6)

five times LSC, five times HSC, homogenisation and stomaching, followed by four times LSC, homogenisation and stomaching followed by four times HSC, homogenisation and stomaching followed by two times LSC, and homogenisation and stomaching and two times HSC.

Homogenisation and stomaching was completed by transferring in vitro residues to 250 ml bottles to be centrifuged at 5000×g for 5 min at 4 ◦ C. Supernatant was decanted to leave 100 ml of sample volume. Homogenisation was done three times for 10 s, with 10 s intervals, using an Ultra Turrax probe (T25, IKA Janke & Kunkel, Staufen, Germany) set at 8000 rpm. The sample was then transferred to a stomacher bag (size 177 mm×304 mm) and pummelled for 5 min (Seward stomacher 3500, Worthington, England), and then transferred back to the centrifuge bottle. Centrifugations were completed as outlined above and supernatants were removed each time by suction and the pellet re-suspended in de-ionised water. 2.4. Statistical analyses The effects of fixed factors – technique, feed and technique×feed interaction – were tested by the GLM procedure of SAS (2001). Results from Experiment 1 are reported as least square means with minimum significant differences among techniques within feeds, that were determined using the Tukey test at P=0.05 following a significant F-test. In Experiment 2, results are reported in tables as least square means with comparisonwise error probabilities for differences obtained by PDIFF of SAS (2001). Differences were declared significant if P
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logarithms when effects of techniques were analysed. Results from Experiment 2a were analysed together with in vitro controls (C1, C2, and C3), obtained in Experiments 1 and 2b results were analysed together with C3 from Experiment 1.

3. Results 3.1. Experiment 1: unfermented samples For both BIN and NDIN, effects (P<0.001) of technique, feed and technique×feed interactions were observed (Table 1). Recovery of BIN was in the order PF > GF > LSC. However, LSC resulted in the highest NDIN recovery, with no differences between filtration techniques in six feeds out of eight. Exceptions were SBM, where PF recovered less BIN than GF and in RSC, where GF recovered less BIN than LSC. For wheat bran, NDIN recovery after LSC was less than both types of filtration. Ranking of feeds within the techniques differed for BIN and NDIN. In RSC, GF recovered about 22 and 15% less BIN than by PF or LSC, whereas no differences occurred among techniques for NDIN. Recovery of NDIN in DDGS was 3.4-fold higher by LSC, compared to GF, but was the same for BIN. 3.2. Experiment 1: in vitro samples After 24 h in vitro fermentation, centrifugation resulted in the highest NDIN values, and it greatly differed from recoveries after both filtration techniques (P<0.05) in all feeds (Table 2). LSC recovered 1.22–7.10 and 1.20–2.80 of the NDIN measured by GF and PF, respectively. Paper filtration resulted in higher (P<0.05) recoveries than GF in six fermented Table 1 Buffer insoluble N and neutral detergent insoluble N (g/kg total N) in feeds, measured after paper filtration (PF, retention size 20–25 ␮m), sintered glass crucible filtration (GF, retention size 40–90 ␮m) and by centrifugation at 3000×g (LSC) were used to remove solubles (Experiment 1) Grass hay-1

Grass hay-2

Birdsfoot trefoil

Wheat bran

Rapeseed cake

DDGSa

Barley

SBMb

Buffer insoluble Nc PF 751 c GF 689 b LSC 636 a

690 b 680 b 609 a

682 b 660 b 601 a

613 b 587 a 567 a

429 c 336 a 397 b

687 b 658 a 667 ab

786 b 731 a 743 a

799 b 847 c 765 a

Neutral detergent insoluble Nd PF 331 b 305 a GF 323 a 305 a LSC 358 c 339 b

71 a 67 a 94 b

211 b 211 b 196 a

97 a 104 ab 106 b

210 b 88 a 297 c

82 a 82 a 83 a

23 a 24 a 34 b

Technique

Data with different index letters (a, b, c) on the same column within N fraction differ (P=0.05) after the Tukey test. a DDGS = distillers dried grains with solubles from wheat. b SBM = soybean meal. c Minimum significant difference = 23.2; SED = 9.64. d Minimum significant difference = 8.0; SED = 3.35; For DDGS after centrifugation at 3000×g, n = 2, minimum significant difference = 9.0; SED = 3.74.

130

Feeda

Experiment 1b PFc

Grass hay-1 153 b Grass hay-2 78 b Birdsfoot trefoil 57 a Wheat bran 58 b Rapeseed cake 85 a DDGS 133 b Barley 47 b SBM 16 b

Experiment 2a: in sacco wash followed by

Probability for differenced

Relative difference

GF (C1) LSC (C2) HSC (C3) GF (A1) LSC (A2) C1–A1 C2–A2 C3–A1 A1–A2 C1–A1

C2–A2 C3–A1 A1–A2

132 a 69 a 54 a 49 a 84 a 40 a 19 a 8a

<0.001 <0.001 <0.001 <0.001 0.424 <0.001 <0.001 <0.001

344 c 154 c 110 b 114 c 102 b 168 c 132 c 32 c

352 c 209 d 124 c 139 d 115 c 182 c 179 d 40 d

123 65 45 42 97 30 12 8

148 86 84 53 97 56 20 12

0.07 0.06 0.17 0.13 −0.16 0.25 0.35 0.06

0.57 0.44 0.24 0.54 0.05 0.66 0.85 0.63

0.65 0.69 0.64 0.70 0.16 0.83 0.93 0.81

0.17 0.24 0.47 0.20 0.01 0.47 0.41 0.36

0.177 NS 0.213 NS 0.001 0.006 0.007 <0.001 <0.001 0.215 NS

<0.001 <0.001 <0.001 <0.001 0.001 <0.001 <0.001 <0.001

0.001 <0.001 <0.001 <0.001 0.864 <0.001 <0.001 <0.001

Data with different letters (a, b, c, d) within row differ at P=0.05 for the Tukey test, ln minimum significant difference = 0.10, SED = 0.035, except for PF of DDGS and barley, for LSC of grass hay-2 and rapeseed cake, HSC of grass hay-1 with ln transformed minimum significant difference = 0.11, SED = 0.039; NS: not significant, P>Pcrit. 0.013 after sequential Bonferroni adjustment. a DDGS = distillers dried grains with solubles from wheat; SBM = soybean meal. b Comparisons were made on ln transformed data. c PF = filtration on paper filters (retention size 20–25 ␮m); GF = filtration on sintered glass crucibles (retention size 40–90 ␮m); LSC = three times centrifugation at 3000×g for 5 min; HSC = three times centrifugation at 5000×g for 5 min. d Comparisonwise error probability for difference between laboratory tests obtained by PDIFF test, ln transformed SED = 0.051, except for C1 of grass hay-2 and rapeseed cake, C3 of grass hay-1, A2 for DDGS, where ln transformed SED = 0.057.

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Table 2 Neutral detergent insoluble N (g/kg total feed N) in feed residues after 24 h in vitro fermentation measured by different techniques for removal of neutral detergent solubles (Experiments 1 and 2a)

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feeds out of eight. Increasing centrifugal force from 3000 to 5000×g, increased (P<0.05) recovered NDIN in six feeds. 3.3. Experiment 2a: washing in sacco Although in sacco washing reduced the difference between LSC and GF (Table 2, A1 versus A2 and C1 versus C2), NDIN recovery was higher after LSC (A2) than after GF (A1) for all feeds (P<0.001), except for RSC. For DDGS and birdsfoot trefoil, 87% more NDIN was recovered by LSC (Experiment 2a) compared with GF (A1). No washing losses from the bags occurred for either of the grass hays or SBM when NDIN was measured by Table 3 Neutral detergent insoluble N (g/kg total feed N) in feed residues after 24 h in vitro fermentation Treatmenta

C3 B1 B2 B3 B4 B5 B6

Least square means Grass hay-2

Barley

209 123 155 147 167 162 183

179 119 144 111 150 134 175

Probability of differenceb

C3–B1 C3–B2 C3–B3 C3–B4 C3–B5 C3–B6 B1–B3 B2–B4 B3–B5 B4–B6 B1–B2 B3–B4 B5–B6

Grass hay-2

Barley

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.007 0.001 0.001 <0.001 <0.001 <0.001

<0.001 <0.001 <0.001 <0.001 <0.001 0.417 NS 0.075 NS 0.181 NS <0.001 <0.001 <0.001 <0.001 <0.001

In vitro residues were subjected to simulated bacterial removal treatments by either centrifugation (B1, B2) or homogenisation + stomaching + centrifugation (B3–B6). Neutral detergent solubles were removed by three times centrifugation at 5000×g for 5 min (see Fig. 1) (Experiment 2b). NS: not significant, P>Pcrit. 0.017 after sequential Bonferroni adjustment. a C3 = no pre-treatment (control); B1 = five times centrifugation at 3000×g; B2 = five times centrifugation at 5000×g; B3 = homogenisation and stomaching, followed by four times centrifugation at 3000×g; B4 = homogenisation and stomaching followed by four times centrifugation at 5000×g; B5 = homogenisation and stomaching followed by two times centrifugation at 3000×g; B6 = homogenisation and stomaching followed by two times centrifugation at 5000×g. b Comparisonwise error probability for difference between laboratory tests obtained by PDIFF test, SED = 4.2.

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GF (C1–A1). In contrast, if LSC was used to remove ND extraction (C2–A2), losses due to bag washing were evident (P<0.001) in all feeds, except for RSC. In sacco bag losses were generally higher in concentrates versus forages, with RSC as an exception. Combined NDIN losses from in vitro fermented residues from filtration (GF) and from in sacco bag washing (C3–A1) were generally very high, with forages ranging from 0.64 to 0.70 and concentrates, excluding RSC (0.16), from 0.70 to 0.93. 3.4. Experiment 2b: homogenisation, stomaching and centrifugation Treatments induced NDIN losses of 0.02–0.41 (Table 3). Homogenisation + stomaching and centrifugation at the higher g-force (Test B6) applied to barley did not differ from control and caused the least reduction of NDIN in grass hay (0.12). Increasing the g-force from 3000 to 5000×g improved (P<0.001) recovery of the NDIN in both feeds (B1–B2; B3–B4; B5–B6). Extending number of centrifugations (B3–B5; B4–B6) increased NDIN losses at both g-force levels (P<0.001). Homogenisation and stomaching applied in addition to centrifugation did not reduce NDIN recovery in barley (B1–B3; B2–B4) and improved NDIN recovery in grass hay at both g-force levels (P<0.01).

4. Discussion 4.1. Principles of filtration and centrifugation Particle retention of a filter is often expressed in terms of the particle size at which a retention level of 0.98 of the total number of particles greater than this particle size is obtained. In a centrifugation process, sedimentation rate is proportional to the size of a particle, as well as to the density difference of the particle and solute, force applied, and inversely proportional to viscosity of the medium (Sprot et al., 1994). Density is obtained by a direct mass and volume measurement that is not convenient with feed particles in rumen degradation studies. Density can also be interchanged with specific gravity (SG), estimated as a ratio between a sample mass and mass of liquid displaced by the sample. Unit specific gravity of feed particles is defined as the product sum of the proportions and specific gravity of solids, gas and internal liquid (Wattiaux et al., 1992). This is likely to be the most relevant measure of feed particle specific gravity under the centrifugation conditions used in current study. Wattiaux et al. (1993) showed that solid SG, and also solution SG, changes as solutes are released from feed particles into solution. There are density differences among water, buffer or ND and differences in release of solubles into these media. Therefore, the same centrifugal force applied to a sample may recover particles differing in size and SG as a result of the media used. Using a filter, only size determines particle retention. It is therefore understandable that centrifugation and filtration can rank feeds differently in terms of residue recovery. A choice between filtration and conventional centrifugation could be made according to particle size and density difference. Particle size can be defined as “small” if it is smaller than the aperture of the filter. Density difference refers to that of the particle and the suspension media, which has to be positive for particles to sediment. Both filtration and centrifugation

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techniques can be used for “large” and “dense” feed particles. Filtration should be chosen for “large” non-positive density particles and centrifugation should be chosen for “small” particles with a positive density difference. Neither technique will suffice when particles are “small” with a non-positive density difference. In the current experiment, releasing gas associated with feed particles in a vacuum chamber increased the density difference between particle and solute. Theoretically, filters retaining very small particles could be used, but slow filtering speed and clogging of the filters usually prevents this. These problems may be overcome by increasing filter surface area and using a smaller sample size. 4.2. Unfermented samples BIN constitutes a major part of the protein in feeds such as barley grain, meal from soybean oil extraction, maize gluten and rapeseed meals (Hedqvist, 2004). NDIN contents may also be high in brewers grains and DDGS from maize (NRC, 2001). Precise and accurate measurements are required for these fractions, as they are important components of some feed evaluation systems (Sniffen et al., 1992; NRC, 2001). Filtration in sintered glass crucibles (porosity 2) is normally used after extraction with ND (Mertens et al., 2002), and paper filtration (Whatman #54 or #541) is commonly used after buffer extraction (Licitra et al., 1996). Centrifugation is sometimes used after buffer incubations (Madsen and Hvelplund, 1985). We examined all these techniques for recovery of BIN and NDIN fractions, expecting that PF and GF, and possibly centrifugation, would rank feeds similarly for both BIN and NDIN recovery. In most cases, similar or higher amounts of feed BIN and NDIN were measured by PF versus GF. Centrifugation generally resulted in the lowest BIN, and the highest NDIN, values compared to filtration techniques, especially in forages. Incomplete hydration during extraction could have been a reason for the lower BIN concentrations obtained by LSC versus filtration (Table 1). Sedimentation, rather than floating, is expected as hydration of particles proceeds and three to 5 h were needed to hydrate forage samples in the autoclaved rumen fluid buffer solution used by Wattiaux et al. (1992). Licitra et al. (1996) included tertiary butyl alcohol in the buffer incubation medium to facilitate hydration, but no wetting agent was used for the 1 h incubation used in the present experiments. 4.3. In vitro samples Recovery of NDIN was relatively consistent within filtration technique and within centrifugation level (Experiment 1, Table 2). However, markedly higher NDIN recovery was obtained after centrifugation versus filtration for all residues except RSC. In this feed, in vitro NDIN disappearance was negligible (compare Tables 1 and 2) and recovery was not influenced by technique. The NDIN recovery from DDGS was >4-fold higher than by GF. Clogging impeded filtration and sufficient rinsing was difficult. This should have increased NDIN recovery, but instead it was lowered. Particle losses through the glass filter were evident and could be seen in the filtrate. These differences in NDIN recovery for DDGS were of such magnitude that they could have considerable nutritional and economic consequences for this feed.

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During centrifugation, concentrates, but not forages, formed solid pellets. Barley was an extreme, as the pellets formed were difficult to re-suspend. It is likely that removal of the soluble fraction from barley was incomplete, since higher NDIN values were obtained after versus before fermentation (Tables 1 and 2). A similar observation was made by Figroid et al. (1972), and was assumed to have contributed to slower degradation in 0.4–0.6 mm than 0.6–0.8 mm barley particles. Centrifugation of birdsfoot trefoil also recovered more NDIN after, versus before, in vitro fermentation (Tables 1 and 2). Birdsfoot trefoil contains tannins that form complexes with proteins at neutral pH. Makkar et al. (1997) suggested that high centrifugal force (20 000×g for 30 min at 4 ◦ C) prior to ND extraction increases the affinity of tannin–protein complexes to such an extent that these complexes become insoluble in ND. The tannin concentration in birdsfoot trefoil (cv. Leo), used in our study, is relatively low and less than 10 g/kg DM (Hedqvist et al., 2000), centrifugation was milder (3000 or 5000×g for 5 min), and occurred after (not before) the ND extraction. Since centrifugation was also applied to unfermented samples, increased NDIN values for in vitro samples cannot be explained by increased tannin–protein affinity during centrifugation. The casein peptone-N from the in vitro incubation medium could have bound to the tannins and formed additional NDIN. If formed tannin–protein complexes also included polysaccharides and minerals (Jansman, 1993), their mass and density could have been enough to sediment by centrifugation, but pass the filters used in our experiment. In only two unfermented feeds, was more NDIN recovered by PF versus GF. After fermentation, however, differences occurred in six feeds (Tables 1 and 2). Higher NDIN concentrations were also obtained in fermented feeds by increasing the centrifugal force from 3000 to 5000×g (Table 2). No difference was found between these centrifugation levels in unfermented feeds (grass hay, wheat bran, barley and SBM, P=0.20, results not shown). Only small changes in NDIN recovery occurred in unfermented DDGS by centrifugation either at 3000, 6000 or 9000×g (Ud´en, unpublished data). It appears that differences in feed N recovery between filtration and centrifugation techniques increase after feeds are fermented, and could be attributed to the reduction in feed particle size that is known to occur due to digestion and detrition in the rumen (McLeod and Minson, 1988). 4.4. Washing of in vitro residues in sacco Residue washing in bags after rumen incubation may partially remove attached microbes. The problem is that feed particles are also lost. Small particle escape through the bag pores during washing was expected to eliminate differences between filtration and centrifugation techniques used after ND extraction (Experiment 2a). However, further losses of NDIN occurred by GF after the in vitro residue was washed in bags. The highest recovery of NDIN was for HSC and no bag wash (C3) and lowest for bag wash and GF (A1). Comparing these values, unacceptable NDIN losses of up to 0.93 occurred from the combined effects of losses from bags with 28 ␮m openings and through the sintered glass filter discs with 40–90 ␮m retention. In contrast to other feeds, NDIN recovery for RSC had no, or positive, effects of bag washing, and there were no differences between filtration and centrifugation techniques applied after washing the bags (Table 2). Washed bags containing RSC were greasy. Apparently fat

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clogged the pore openings of the bags during washing in cold tap water, thereby preventing particle escape. 4.5. Homogenisation, stomaching and centrifugation As washing of fermentation residues in bags resulted in NDIN losses, removal of microbial contamination and fermentation products was completed using alternative procedures. Low-speed centrifugation at 150×g for 10 min does not sediment all feed particles, and bacterial pellet collected after the resulting supernatant is subjected to high speed centrifugation are contaminated with feed particles (John, 1984). However, centrifugal force higher than 5000×g were not useful relative to the objectives of the current experiment, since 3000–5000×g for 5–10 min harvests intact bacterial cells (Sprot et al., 1994). Repeated pellet washing after centrifugations (B1 and B2) was expected to remove loose and loosely attached microbes (Legay-Carmier and Bauchart, 1989). Homogenisation and stomaching procedures were applied prior to centrifugation (B3–B6), as they are known to detach microbial mass from feeds (Merry and McAllan, 1983). All tests of homogenisation, stomaching and centrifugation treatments (Table 3) reduced NDIN, but the reduction was less than that obtained after washing of bags (Table 2). As repeated centrifugation of in vitro residues at 3000 and 5000×g reduced NDIN values, it could be inferred that either a considerable proportion of feed NDIN is in a fine particle form that will not sediment easily, or feed losses occur during supernatant removal, or not all NDIN in untreated control samples originates from feed but from attached microbes. 5. Conclusions High NDIN losses occurred in 24 h in vitro fermented feeds after filtration in sintered glass crucibles (porosity 2). Sample washing in polyester bags (28 ␮m pore openings) after in vitro fermentation, as is common in many standard in sacco procedures, also results in high NDIN losses. Additional losses occur if ND extraction is done by filtration in glass crucibles instead of by centrifugation. Oven drying of in vitro residues at 65 ◦ C prior to ND extraction inflates NDIN values and must be avoided. Losses of NDIN after homogenisation, stomaching and centrifugation are smaller than after sample washing in bags. When residue recovery constitutes a problem, higher g-force (i.e., 5000×g instead of 3000×g) and fewer (three instead of five) washing times is recommended to minimise sample losses. Acknowledgement Financial support from the Royal Swedish Academy of Agriculture and Forestry is gratefully acknowledged. References Beckers, Y., Thewis, A., Maudoux, B., Francois, E., 1995. Studies on the in situ nitrogen degradability corrected for bacterial contamination of concentrate feeds in steers. J. Anim. Sci. 73, 220–227.

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