Ruminal large and small particle kinetics in dairy cows fed red clover and grass silages harvested at two stages of growth

Animal Feed Science and Technology 155 (2010) 86–98

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Ruminal large and small particle kinetics in dairy cows fed red clover and grass silages harvested at two stages of growth A.R. Bayat a,1 , M. Rinne a,∗ , K. Kuoppala a , S. Ahvenjärvi a , A. Vanhatalo b , P. Huhtanen c a b c

MTT Agrifood Research Finland, Animal Production Research, 31600 Jokioinen, Finland University of Helsinki, Department of Animal Science, Finland Swedish University of Agricultural Sciences, Department of Agricultural Research for Northern Sweden, S-901 83 Umeå, Sweden

a r t i c l e

i n f o

Article history: Received 17 January 2009 Received in revised form 7 October 2009 Accepted 12 October 2009 Keywords: Steady-state model Indigestible NDF Passage rate Particle comminution rate Digestion rate Red clover Grass

a b s t r a c t Passage, comminution and digestion rates of large and small particles were estimated using a rumen evacuation technique and total faecal collection with five lactating dairy cows in a 5 × 5 Latin square experiment. Two grass and two red clover silages harvested at early and late primary growth stages and a 1:1 mixture of late harvest grass and early harvest red clover were the dietary treatments. Cows received 9.0 kg supplementary concentrate per day. Ruminal contents and faeces were divided into large (>1.25 mm) and small (1.25–0.038 mm) particles by wet sieving. Indigestible neutral detergent fibre (iNDF) was determined by 12 days ruminal in situ incubation followed by neutral detergent extraction. Plant species did not affect ruminal particle size distribution, whereas advancing forage maturity decreased the proportion of large particles for both grass and red clover silage diets. Ruminal pool size of iNDF was higher (P<0.001) with red clover compared to grass silage diets. Ruminal passage rates of iNDF and potentially digestible NDF (pdNDF) increased with decreasing particle size (P<0.01). Passage rate of iNDF for small particles was slower (P<0.01) when red clover compared to grass silage diets were fed. Particle comminution rate in the rumen was slower (P<0.001) with red clover compared to grass silage diets and it increased (P<0.01) with advancing forage maturity. The contribution of particle comminution to ruminal mean retention time of iNDF in the ruminal large particle pool was smaller (P<0.01) in red clover compared to grass silage diets and it increased (P<0.05) with the mixed silage compared to the separate silages. Passage rate of pdNDF for both large and small particles was not affected by dietary treatments. Digestion rate of pdNDF for large particles was faster (P<0.001) with red clover compared to grass silage diets. Differences in ruminal passage and digestion rates of the large and small particles, in addition to differences in the passage and digestion rates of red clover compared to grass silage diets, emphasize the need to consider particle size and forage type in metabolic models predicting feed intake and fibre digestibility in ruminants. © 2009 Elsevier B.V. All rights reserved.

Abbreviations: CP, crude protein; DM, dry matter; kd , digestion rate; ki , intake rate; kp , passage rate; kr , comminution rate; LP, large particles; SP, small particles; NDF, neutral detergent fibre; iNDF, indigestible NDF; pdNDF, potentially digestible NDF; MRT, ruminal mean retention time. ∗ Corresponding author at: MTT Agrifood Research Finland, Animal Production Research, Building H, FI-31600 Jokioinen, Finland. Tel.: +358 50 5700 811; fax: +358 3 4188 3661. E-mail address: marketta.rinne@mtt.fi (M. Rinne). 1 Current address: Shiraz University, Department of Animal Science, Shiraz 7144165186, Iran. 0377-8401/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2009.10.005

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1. Introduction The ruminant digestive system has developed to selectively retain undigested fibrous material in order to maximize ruminal fibre digestion (Allen and Mertens, 1988). A drawback of selective retention is reduced feed intake due to the filling effect of the retained material in the rumen, especially with low quality forages (Romney and Gill, 2000). Rates of digestion and passage are important determinants of fibre digestibility (Mertens, 1993). In situ and in vitro incubations have usually been used to determine rate of digestion (kd ) while rate of passage (kp ) is usually estimated using marker techniques. It is necessary to validate these constants with more direct measurements such as rumen evacuations (Bruining et al., 1998). For instance, kd estimations derived from in situ incubations have been lower than those derived using rumen evacuations (Aitchison et al., 1986; Tamminga et al., 1989; Bruining et al., 1998). Neutral detergent fibre (NDF) is not a homogenous entity, because indigestible NDF (iNDF) and potentially digestible NDF (pdNDF) have different digestion and passage rates (Mertens, 1993; Huhtanen et al., 2007). It is well documented that pdNDF is selectively retained in the rumen (Tamminga et al., 1989; Rinne et al., 2002; Huhtanen et al., 2007). In addition, Allen and Mertens (1988) indicated that escapable (i.e., small) and non-escapable (i.e., large) particles have different passage and digestion properties which affect digestibility. Hence, rumen evacuations and faecal collection together with wet sieving and iNDF determination have been used to study the kinetics of particle comminution and passage (Huhtanen et al., 2007; Bayat et al., 2007). Grasses and legumes have different digestion, passage and comminution kinetics due to different histological characteristics (Van Soest, 1994), and forage maturity also affects them. Accurate and precise estimates of the kinetic parameters defining ruminal digestion are needed for dynamic mechanistic models for predictions of feed intake and digestion. Therefore, the purpose of this study was to investigate effects of plant species (i.e., grass versus red clover) and forage maturity on ruminal pool sizes and digesta kinetics of small (i.e., escapable) and large (i.e., non-escapable) particles in dairy cows. A rumen evacuation technique, wet sieving and iNDF determination were used in a rumen assumed to be in steady state. One advantage of estimating ruminal mean retention time (MRT) of iNDF using rumen evacuation and wet sieving is that the distribution of MRT between the escapable and non-escapable ruminal pools is taken into account. Data on feed intake, milk production, digestion and metabolism of nutrients and digesta kinetics of total amounts of NDF and iNDF are reported elsewhere (Vanhatalo et al., 2009; Kuoppala et al., 2009). 2. Materials and methods 2.1. Animals, diets, and experimental design Conduct of the experiment, animals and experimental diets were described in detail by Vanhatalo et al. (2009). In brief, five ruminally fistulated multiparous Finnish Ayrshire dairy cows in early lactation with a body weight of 620 ± 71.3 kg were assigned randomly to a 5 × 5 Latin square design with 21 days periods. Experimental silages were prepared from primary growths of timothy (Phleum pratense) and meadow fescue (Festuca pratensis) mixture, and red clover (Trifolium pratense) swards grown at Jokioinen, Finland (60◦ 49 N, 23◦ 28 E) and harvested at two stages of maturity. The grass silages were harvested on 17 June (early harvest grass) and on 26 June (late harvest grass), and the red clover silages on 2 July (early harvest red clover) and on 16 July (late harvest red clover). The swards were cut with a mower conditioner, wilted (approximately 3 h for grass and 7 h for red clover) and harvested with a precision chop harvester. Silages were preserved with a formic acid-based additive (5 L/ton fresh weight for grass and 6 L/ton for red clover silages). The four pure silages and a mixture of late harvest grass and early harvest red clover (1:1 on DM basis) were fed ad libitum to cows during days 1–15 of each period, and restricted to 0.95 during the collection period (i.e., days 16–21). The forages were supplemented with 9.0 kg/day of a concentrate containing (g/kg as fed) barley grain (405), oats grain (400), rapeseed expeller meal (160) and a proprietary vitamin and mineral supplement (35). Cows were housed in individual tie stalls with continuous access to water and diets were offered four times daily at 0600, 0900, 1800 and 2000 h. All experimental procedures were approved by the MTT Agrifood Research Finland Care and Use of Animals Committee. 2.2. Procedures and chemical analyses Ruminal contents were evacuated before the morning feeding at 0600 h on days 13 and 6 h after morning feeding at 1200 h on day 15 of each experimental period, respectively, as described by Kuoppala et al. (2009). Ruminal contents were collected into plastic barrels kept in a warm water bath, weighed, mixed thoroughly in a feed cart, and sampled. The average weight of ruminal contents of the two evacuations was used as the estimation of the diurnal mean. Total faecal collection occurred during days 18–21 of each period, and representative samples were taken for further analyses. The particle size distribution of dietary concentrate, ruminal digesta (separately for the two sampling times) and faeces was determined by a Retsch AS200 Digit wet sieving apparatus (Retsch GmbH, Haan, Germany). The samples were divided into seven particle size fractions by wet sieving using sieves with pore sizes of 2.5, 1.25, 0.63, 0.315, 0.16, 0.08 and 0.038 mm. Poppi et al. (1980) proposed a critical particle size of 1.18 mm for cattle, and hence the size of 1.25 mm was defined as the boundary between large and small particles.

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To avoid errors in mixing of the two ruminal samples, each sample was sieved separately, but the average particle size distribution of them was used in further calculations. Ten, five and ten replicates of 5.0, 30.0 and 30.0 g fresh weight were sieved for dietary concentrate, and each ruminal and faecal sample, respectively. The samples were sieved for 10 min using a water flow of 3.5 L/min. After sieving, material from each sieve was quantitatively collected to pre-weighed nylon bags (60 mm × 120 mm, pore size 0.038 mm, Swiss Silk Bolting Cloth Mfg. Co., Ltd., Zurich, Switzerland), dried at 60 ◦ C for 48 h, weighed to determine the DM distribution of different particle size fractions and ground to pass a 1 mm screen. The neutral detergent fibre (aNDFom) concentration was determined by ANKOM220 Fiber Analyser (ANKOM Technology Corporation, Macedon, NY, USA) using sodium sulphite and heat stable ␣-amylase (Sigma Chem. Co., St. Louis, MO, USA) according to Van Soest et al. (1991). To determine the iNDF concentration (i.e., aiNDFom), 1.0 g DM of concentrate, ruminal digesta or faecal samples were weighed into nylon bags (60 mm × 120 mm, pore size 0.017 mm, Swiss Silk Bolting Cloth Mfg. Co. Ltd., Zurich, Switzerland). Duplicate bags were incubated for 12 days in the rumen of two cows fed a grass silage based diet (∼0.3 concentrate on a DM basis). After ruminal incubation, bags were rinsed in cold water for 25 min using a household washing machine, incubated for 1 h in boiling neutral detergent solution, rinsed, and dried to a constant weight at 60 ◦ C. All aNDFom and iNDF concentrations are reported ash-free. Crude protein (CP) was analysed by Dumas method using Leco FP 428 N analyser (Leco Corp., St. Joseph, MO, USA) and ash was determined after incineration at 600 ◦ C for 2 h.

2.3. Calculations and statistical methods Particle size distribution of chewed silages is derived from a study carried out in the same laboratory with the same cows (Table 2; Rinne, M., Animal Production Research, MTT Agrifood Research Finland, Jokioinen, Finland; personal communication). Particle size distribution was determined by feeding grass and red clover silages to the cows and catching the bolus from the emptied rumen through the rumen cannulae. It was assumed that the particle size distribution of concentrate does not change during eating and that there was no NDF in the material that was <0.038 mm. The aNDFom and iNDF distribution between the different particle size fractions of the chewed silages were adopted from the above-mentioned study, and aNDFom and iNDF concentrations in small particles were 0.937 and 0.827 of those in large particles. Calculations of ruminal pool sizes, kinetics of iNDF and pdNDF, MRT and median retaining aperture are described by Huhtanen et al. (2007) and Bayat et al. (2007). It was assumed that there was no NDF in ruminal and faecal material <0.038 mm, and therefore NDF recovered from the sieves was corrected to correspond to faecal NDF output and the rumen NDF pool. Due to incomplete recovery of iNDF in faeces (0.86 ± 0.012), iNDF intake rather than faecal output was used in kinetic models. The following model (Latin square split-plot) was used to analyse the data using the random statement of mixed model procedure of SAS (2003): Yijkl =  + Ci + Pj + Tk + eijk + Sl + Tk × Sl + εijkl where  is the overall mean, Pi , Tj , Sk and Tj × Sk are the fixed effects of period (i = 1–5), treatment (j = 1–5), particle size (k = 1–2 or 7, depending on the model used) and the interaction between treatment and particle size, respectively and the eijk is the main-plot error term. Effects of cow, Ci (i = 1–5), and eijk were considered as random effects, and εijkl is the subplot error term. The estimation method was REML and the degrees of freedom method was Kenward–Rogers. Because of significant interactions between forage factors and particle size, data on chemical composition and digesta kinetics were analysed separately for large and small particles and eijk was used as the error term to compare effects of treatments. Contrasts were used to compare: (1) differences between the plant species (grass versus red clover) and forage maturities (early harvest versus late harvest) and their interaction, (2) differences between late harvest grass and early harvest red clover fed separately versus mixed, (3) effects of particle sizes and its interaction with other factors. One observation was missing from the mixed silage diet because of digestive disorders and therefore the standard error of means (SEM) of the mixture silage diet should be multiplied by 1.19 when making comparisons with the other mean values.

3. Results The chemical composition of the experimental silages and dietary concentrate is in Table 1. The DM intake of the silages was 13.2, 12.0, 14.0, 11.3, and 12.1 kg/day, and that of concentrates was 7.6, 7.8, 7.4, 7.4 and 8.0 kg/day for the cows consuming early and late harvest grass, mixed silage, and early and late harvest red clover, respectively. The detailed chemical composition and fermentation quality of the silages as well as nutrient intake and milk production of the cows are in Vanhatalo et al. (2009) and Kuoppala et al. (2009). Ruminal pool sizes of total DM, aNDFom and iNDF before and 6 h after the morning feeding are in Fig. 1. There was no treatment by time interaction. Particle size distribution of chewed silages as well as dietary concentrate and aNDFom and iNDF concentrations of dietary concentrate, used to calculate amounts of DM, aNDFom and iNDF entering the ruminal pools, are in Table 2. Rumen evacuations did not affect daily DM intake (20.5 and 20.6 kg/day, respectively; SEM = 0.14 kg/day) and milk production (28.6 and 28.9 kg/day, respectively; SEM = 0.21 kg/day) when comparing data from 3 days prior to 3 days during rumen evacuations.

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Table 1 Chemical composition (g/kg DM) of the silages and concentrate consumed by the cows (n = 5 for each feed). Feed

Grass silage a

DM (g/kg as fed) Ash Crude protein Neutral detergent fibre (aNDFom) iNDFb a b

Red clover silage a

a

Concentrate a

Early

Late

Early

Late

249 86 134 500 57

257 75 111 570 84

214 102 212 375 70

212 93 181 463 138

890 54 167 205 70

Growth stage. Indigestible neutral detergent fibre determined by 12 days in situ incubation.

Fig. 1. Total ruminal DM (a), aNDFom (b) and iNDF (c) contents of dairy cows fed early harvest grass (GE ), late harvest grass (GL ), early harvest red clover (RE ), late harvest red clover (RL ) silage diets and the mixture of GL and RE silage diets (GL RE ). Standard errors of the means were 0.91 and 0.97 kg for DM, 0.59 and 0.52 kg for aNDFom and 0.26 and 0.26 kg for iNDF for prior to and 6 h after morning feeding, respectively.

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Table 2 Particle size distribution of the feed DM, and neutral detergent fibre (aNDFom) and indigestible neutral detergent fibre (iNDF) concentrations in different particle size fractions of the dietary concentrate. Particle size (mm)a >2.5

SEMb

1.25

0.63

0.315

0.16

0.08

0.038

50 39

31 29

15 24

7 10

4 5

164 311

15.0 28.2

109 417 157

93 421 188

65 373 185

49 326 125

34 238 87

420 – –

7.1 – 20.3

Silage after ingestive mastication (g/kg DM)c Grass 699 31 Red clover 543 37 Intact concentrate feed (g/kg DM) DM 70 aNDFom 270 iNDF 61

160 234 37

<0.038

a >2.5: particles larger than 2.5 mm; 1.25: particles between 2.5 and 1.25 mm; 0.63: particles between 1.25 and 0.63 mm; 0.315: particles between 0.63 and 0.315 mm; 0.16: particles between 0.315 and 0.16 mm; 0.08: particles between 0.16 and 0.08 mm; 0.038: particles between 0.08 and 0.038 mm; <0.038: particles smaller than 0.038 mm. b Standard error of means, n = 2 for silages and iNDF concentration, n = 5 for concentrate DM distribution. c Rinne, M., Animal Production Research, MTT Agrifood Research Finland, Jokioinen, Finland; personal communication.

3.1. Distribution and chemical composition of particles Data of ruminal and faecal particle size distribution, and aNDFom, iNDF and CP concentrations in large (LP, >1.25 mm) and small (SP, 1.25–0.038 mm) particles are in Table 3. Forage maturity decreased the proportion of large particles in both Table 3 Dry matter (DM) distribution, and neutral detergent fibre (aNDFom), indigestible neutral detergent fibre (iNDF) and crude protein (CP) concentrations in large (>1.25 mm) and small (1.25–0.038 mm) particles of ruminal digesta and faeces of dairy cows fed different forages. Forage

Mixturea

Grass silage d

Early

d

489 741 246 332 134

Faeces (g/kg DM unless otherwise stated) Large particles 109 DM distributione aNDFom 838 iNDF 245 iNDF (g/kg aNDFom) 293 CP 50 Small particles DM distributione aNDFom iNDF iNDF (g/kg aNDFom) CP

891 761 330 433 82

d

Early

Late

521 802 249 310 84

529 784 363 464 113

480 819 479 584 92

7.3 7.9 8.2 9.9 3.4

511 733 258 351 125

479 716 310 434 159

471 700 348 497 185

520 721 449 622 165

7.3 7.0 10.4 13.7 4.5

116 843 271 322 43

106 815 296 363 56

147 823 410 498 63

124 838 530 632 58

884 771 352 456 78

894 745 389 523 96

853 754 445 591 95

876 772 534 692 91

Ruminal digesta (g/kg DM unless otherwise stated) Large particles 511 489 DM distributione aNDFom 823 823 iNDF 179 185 iNDF (g/kg aNDFom) 217 225 CP 68 67 Small particles DM distributione aNDFom iNDF iNDF (g/kg aNDFom) CP

d

Late

SEMb

Red clover silage

Contrastsc S

M

S×M

X

†

ns ns

ns

***

*

*

*

***

***

***

***

***

***

**

**

*

**

*

*

ns

*

ns

***

†

**

ns

*

***

***

**

***

***

**

***

***

†

ns ns ns ns ns

9.9 0.5 10.6 12.8 0.1

*

ns

ns

†

***

***

***

***

***

***

***

**

***

***

**

*

***

***

***

***

9.9 3.7 13.5 18.4 2.5

*

ns **

ns ns

†

ns ***

**

*

***

**

*

ns ns

***

†

ns

**

*

ns, non-significant. a Mixture of late harvest grass silage and early harvest red clover silage (1:1 DM basis). b Standard error of means, n = 5 per treatment. c S, grass versus red clover; M, early versus late harvest time (maturity); S × M, interaction of plant species and forage maturity; X, mixture of the late harvest grass silage and the early harvest red clover silage versus feeding these silages separately. d Growth stage. e In particulate DM. † P<0.10. * P<0.05. ** P<0.01. *** P<0.001.

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Fig. 2. Particle size distribution of ruminal digesta () and faeces () of dairy cows (g/kg particulate DM). The data are pooled over the treatments. Standard errors of the means for the particulate DM of ruminal digesta and faeces were 3.4 and 4.4 g/kg, respectively.

grass and red clover silage diets (P<0.001). With increasing maturity, aNDFom concentration in both ruminal LP and SP did not change with the grass silage diet, but it increased with the red clover silage diet (P<0.05 for plant species × forage maturity interaction). The aNDFom concentration was higher (P<0.001) in LP compared to SP irrespective of plant species and maturity. Ruminal iNDF concentration (g/kg DM or aNDFom), being higher for both LP and SP of red clover compared to the grass silage diets, increased with advancing maturity for red clover but did not change markedly with advancing maturity for grass silage diets for both LP and SP (P<0.01 for plant species × forage maturity interaction). Ruminal iNDF concentration (g/kg DM) was higher in SP compared to LP of grass silage diets, while the opposite occurred with red clover silage diets (P<0.001 for plant species × particle size interaction). Feeding the mixed silage diet decreased ruminal iNDF concentration in LP (P<0.01) but not in SP compared to the values of the separate silages. The proportion of faecal LP in particulate DM was lower than SP (Table 3). The composition data are expressed on a particulate DM basis (i.e., particles retained on all sieves). Red clover silage diets had higher faecal LP fractions compared to grass silage diets (136 and 113 g/kg particulate DM, P<0.05). Faecal aNDFom concentration was lower in SP compared to LP in all diets (P<0.001). Faecal aNDFom concentration in both LP and SP increased with advancing maturity (P<0.01) but in LP the difference was greater with red clover silage diets (P<0.001 for plant species × forage maturity interaction). Feeding the mixed silage diet reduced the faecal aNDFom concentration in both LP and SP (P<0.01). The iNDF concentration in faecal particles had almost the same trend as in ruminal particles but values were higher. Ruminal and faecal CP concentrations were higher with red clover compared to grass silage diets (P<0.001) and they decreased with advancing maturity of the harvested forages, except for faecal SP (P<0.01). Based on the model with seven particle size pools, half of the ruminal particles were larger than 2.5 mm and the majority of faecal particles were smaller than 0.63 mm (Fig. 2). The aNDFom concentration in ruminal particles had a curvilinear trend; the decrease taking place in particles smaller than 0.315 mm (P<0.001; Fig. 3a). The concentration of iNDF (g/kg aNDFom) in ruminal digesta had a quadratic trend as the medium particles had the highest concentration of iNDF (P<0.001; Fig. 3b). The iNDF concentration in all ruminal particles was higher in cows fed red clover compared to those fed grass silage diets and forage maturity increased it only when red clover silage diets were fed (P<0.001 for plant species × forage maturity interaction). The aNDFom concentration of faecal particles had a similar trend as ruminal digesta (Fig. 3c). The faecal iNDF concentration increased quadratically with decreasing particle size (P<0.001) and it was higher with red clover compared to grass silage diets (P<0.001). Forage maturity increased faecal iNDF concentration only with red clover silage diets in all particle size fractions (P<0.001 for plant species × forage maturity interaction; Fig. 3d). 3.2. Ruminal pool sizes and faecal output Data on ruminal pools and daily faecal excretion of DM, iNDF, pdNDF and CP in addition to MRT of iNDF and pdNDF in LP and SP are in Table 4. Advancing maturity decreased intake of LP with the grass silage diets, but increased it with the red clover silage diets (P<0.05 for plant species × forage maturity interaction). Feeding the mixed silage diet increased intake of LP (P<0.01). Both LP and SP ruminal pools were greater with grass compared to red clover silage diets (P<0.05) and it increased with advancing maturity in both LP and SP (P<0.05 and P<0.001, respectively). Feeding the mixed silage diet increased ruminal LP (P<0.05). The ruminal iNDF content in both LP and SP increased with advancing maturity, but the increase was higher with red clover silage (P<0.01 for plant species × forage maturity interaction). Ruminal pdNDF content was higher in both LP and SP of grass compared to red clover silage diets (P<0.001). Faecal DM excretion of SP with grass silage diets was higher than that with red clover silage diets (P<0.01) and it increased with advancing maturity in both LP and SP (P<0.05 and P<0.01, respectively). Faecal excretion of iNDF increased with advancing maturity, and the difference was greater with the red clover silage diet for LP and SP (P<0.10 and P<0.001 for plant

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Fig. 3. The aNDFom (g/kg DM) and indigestible neutral detergent fibre (iNDF; g/kg NDF) concentrations in the particle size fractions of ruminal digesta and faeces of dairy cows fed early harvest grass (), late harvest grass (), early harvest red clover (), late harvest red clover () silage diets and the mixture of late harvest grass and early harvest red clover silage diets (×). Standard errors of the means for the aNDFom and iNDF concentrations of ruminal digesta were 6.8 and 9.7 g/kg, respectively, and those of faeces were 7.8 and 22.0 g/kg, respectively.

species × forage maturity interaction, respectively). Faecal iNDF excretion was considerably higher in the SP fraction than in the LP fraction for all treatments (P<0.001). The median retaining aperture of chewed material was higher for grass compared to red clover silage diets (P<0.001; Table 5) and the mixed silage diet had a higher median retaining aperture compared to the separate silages (P<0.05). In ruminal digesta, the median retaining aperture was not affected by plant species or maturity. In faecal particles, it was higher with red clover compared to grass silage diets (P<0.01) and it increased with advancing maturity of forages (P<0.05). Ruminal and faecal DM passing through the smallest sieve (<0.038 mm) was higher with red clover compared to grass silage diets (P<0.001) and it decreased with advancing maturity (P<0.01). However the maturity effect was more for faecal red clover silage diets (P<0.05 for interaction of plant species × forage maturity). 3.3. Ruminal digesta kinetics The kp of iNDF was faster for both LP (P<0.05) and SP (P<0.01) of grass compared to red clover silage diets (Table 6), and it was faster for SP compared to LP irrespective of diet fed (P<0.001). The kr was faster for grass compared to red clover silage diets (P<0.001) and it increased with advancing maturity (P<0.01). The contribution of particle comminution in clearance of iNDF from ruminal LP, calculated as kr /(kr + kp ) of iNDF, was higher with grass compared to red clover silage diets (P<0.01). Feeding the mixed silage diet increased the contribution of particle comminution in clearance of iNDF compared to the average of the separate silages (P<0.05). The kp of pdNDF in both LP and SP was not affected by plant species, forage maturity or mixing the silages, and it was faster for SP compared to LP irrespective of the diet fed (P<0.001). The kd of pdNDF for LP being faster for red clover compared to grass silage diets, decreased with advancing maturity for grass silage diets but increased for red clover silage diets (P<0.01 for plant species × forage maturity interaction). The kd of pdNDF for SP tended (P<0.10) to be slower for red clover compared to grass silage diets. The contribution of digestion in clearance of pdNDF from ruminal pools for LP, calculated as kd /(kr + kp + kd ), was higher for red clover compared to grass silage diets, and decreased with advancing maturity although the decrease was more with grass silage diets (P<0.01 for plant species × forage maturity interaction). The contribution of digestion in the clearance of pdNDF for SP was not affected by plant species, forage maturity or mixing the silages. The MRT of iNDF was longer with red clover compared to grass silage diets in LP (P<0.001) and SP (P<0.05; Table 4). The MRT of iNDF decreased with advancing maturity for LP (P<0.01), and it increased for pdNDF of LP with advancing maturity for grass silage diets, but decreased with advancing maturity for red clover silage diets (P<0.001). Selective retention, calculated as kp of iNDF/kp of pdNDF, was greater for LP of grass compared to red clover silage diets (P<0.01), while it was not affected by plant species, forage maturity or mixing the silages for SP.

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Table 4 Intake, ruminal pools and faecal excretion of dry matter (DM), indigestible neutral detergent fibre (iNDF), potentially digestible neutral detergent fibre (pdNDF) and crude protein (CP), and ruminal retention time of iNDF and pdNDF of large (LP, >1.25 mm) and small (SP, 1.25–0.038 mm) particles in dairy cows fed different forages. Forage

Mixturea

Grass silage d

d

SEMb

Red clover silage d

S

M

S×M

X

***

ns

*

**

***

***

***

*

***

ns

ns

**

**

***

*

***

ns ns

Early

Late

11.41 0.75 5.52 1.59

10.53 0.97 5.50 1.26

10.74 1.01 5.10 1.74

8.27 0.77 3.31 1.68

8.85 1.54 3.71 1.57

0.549 0.068 0.288 0.083

Small particles DM iNDF pdNDF CP

4.06 0.53 1.36 0.63

3.99 0.57 1.37 0.60

4.06 0.57 1.36 0.67

3.80 0.54 1.15 0.69

4.12 0.69 1.29 0.71

0.155 0.026 0.048 0.026

4.97 0.88 3.20 0.33

5.28 0.97 3.37 0.34

5.54 1.39 3.06 0.44

4.36 1.59 1.83 0.45

4.94 2.36 1.68 0.43

0.301 0.098 0.185 0.033

* *** ***

Small particles DM iNDF pdNDF CP

4.72 1.16 2.34 0.60

5.48 1.41 2.61 0.66

5.13 1.59 2.06 0.76

3.88 1.36 1.36 0.65

5.36 2.41 1.46 0.83

0.307 0.129 0.131 0.041

Faeces (kg/d) Large particles DM iNDF pdNDF CP

0.44 0.11 0.26 0.02

0.53 0.14 0.30 0.02

0.45 0.13 0.23 0.02

0.44 0.18 0.18 0.02

0.52 0.28 0.16 0.02

0.046 0.020 0.022 0.002

Small particles DM iNDF pdNDF CP

3.60 1.17 1.56 0.24

3.99 1.40 1.68 0.28

3.73 1.44 1.33 0.28

2.56 1.13 0.80 0.18

3.66 1.96 0.87 0.26

Ruminal mean retention time (h) Large particles 28.7 iNDFe 13.9 pdNDFf Small particles iNDFe pdNDFf

23.8 15.2

Late

Contrastsc

Ingested (kg/d) Large particles DM iNDF pdNDF CP

Ruminal digesta (kg) Large particles DM iNDF pdNDF CP

Early

d

ns

ns

ns

*

**

*

**

†

ns

ns ns

*

**

*

ns

*

***

***

ns

ns ns

ns ns

*

*

***

ns

***

***

**

***

ns

ns

ns ns ns

**

**

†

*

**

ns

ns

ns

*

ns

***

***

†

***

ns

†

†

*

ns

ns ns ns ns

0.189 0.069 0.107 0.021

**

**

†

†

**

***

***

†

***

ns

†

**

ns ns

†

24.3 14.8

32.8 14.3

49.8 13.4

37.6 11.0

2.56 0.67

***

**

†

**

ns

*

24.6 14.9

26.3 14.8

29.0 17.9

29.8 16.2

2.28 0.95

*

ns ns

ns ns

*

ns

ns ns ns ns

ns, non-significant. a Mixture of late harvest grass silage and early harvest red clover silage (1:1 DM basis). b Standard error of means, n = 5 per treatment. c S, Grass versus red clover; M, early versus late harvest time (maturity); S × M, interaction of plant species and forage maturity; X, mixture of the late harvest grass silage and the early harvest red clover silage versus feeding these silages separately. d Growth stage. e Calculated as 1/(kp + kr ) for each particle size fraction; kp and kr are from iNDF kinetics (Table 6). f Calculated as 1/(kp + kr + kd ) for each particle size fraction; kr is the same as kr of iNDF and kp and kd are from pdNDF kinetics (Table 6). † P<0.10. * P<0.05. ** P<0.01. *** P<0.001.

4. Discussion Use of rumen evacuations relies on assumptions that normal rumen function must not be disturbed, and a steady-state rumen pool size must be estimated. In meal-fed animals, rumen pool size is not in a steady state, and therefore rumen evacuation times should be chosen to estimate the average pool size. The study of Huhtanen et al. (2007) suggested that

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Table 5 Median retaining aperture of ingested, ruminal and faecal materials and the proportion of ruminal and faecal DM passing through the smallest sieve (0.038 mm). Forage

Mixturea

Grass silage d

d

Early

d

Late

Median retaining aperture (mm) Ingested DM 3.16 Ruminal DM 1.35 Faecal DM 0.29

DM not retained on the sieves (g/kg DM) Rumen 214 177 Faeces 277 233

3.00 1.49 0.31 225 288

M

S×M

X

0.111 0.054 0.011

***

ns ns

ns

*

ns

†

**

*

ns

ns ns

6.7 8.3

***

***

ns

***

***

*

Late

2.43 1.55 0.34

2.39 1.35 0.36

260 368

223 278

Contrastsc S

d

Early

2.96 1.38 0.32

SEMb

Red clover silage

ns ns

a

Mixture of late harvest grass silage and early harvest red clover silage (1:1 DM basis). Standard error of means, n = 5 per treatment. c S, grass versus red clover; M, early versus late harvest time (maturity); S × M, interaction of plant species and forage maturity; X, mixture of the late harvest grass silage and the early harvest red clover silage versus feeding these silages separately. d Growth stage. † P<0.10. * P<0.05. ** P<0.01. *** P<0.001. b

Table 6 Rates (1/h) of intake (ki ), passage (kp ), particle comminution (kr ) and digestion (kd ), and efficiency of selective retention of large (LP, >1.25 mm) and small (SP, 1.25–0.038 mm) particles of ruminal digesta of dairy cows fed the different forages. Forage

Mixturea

Grass silage d

Early

d

Small particles ki kp

0.0194 0.0428

0.0175 0.0424

Potentially digestible neutral detergent fibre (pdNDF) Large particles 0.0736 0.0689 ki 0.0034 0.0038 kp 0.0391 0.0288 kd kd /(kr + kp + kd ) 0.53 0.42

kp

d

Late

Indigestible neutral detergent fibre (iNDF) Large particles 0.0330 0.0386 ki kp 0.0050 0.0062 kr 0.0280 0.0325 kr /(kr + kp ) 0.85 0.84

SEMb

Red clover silage d

Early

Late

0.0293 0.0041 0.0251 0.86

0.0187 0.0046 0.0141 0.75

0.0260 0.0049 0.0212 0.81

0.00211 0.00048 0.00182 0.016

0.0155 0.0393

0.0171 0.0356

0.0121 0.0343

0.00164 0.00330

0.0709 0.0032 0.0404 0.57

0.0761 0.0041 0.0563 0.74

0.0925 0.0039 0.0661 0.71

Contrastsc M

S×M

X

***

**

*

ns

ns ns ns

*

S

ns

***

**

**

ns

†

*

**

*

**

ns

ns ns

ns ns

0.00362 0.00037 0.00234 0.015

**

ns ns ns

*

***

***

**

ns ns ns ns

ns ns ns ns

ns ns ns ns

ns ns ns ns

ns ns

†

ns ns

ns ***

Small particles ki kp kd kd /(kp + kd )

0.0245 0.0280 0.0384 0.58

0.0222 0.0271 0.0416 0.60

0.0283 0.0274 0.0417 0.60

0.0354 0.0242 0.0322 0.57

0.0371 0.0252 0.0378 0.60

0.00151 0.00230 0.00289 0.029

***

iNDF /(kp pdNDF ) Large particles Small particles

1.50 1.54

1.66 1.57

1.29 1.45

1.17 1.51

1.25 1.43

0.098 0.103

**

ns †

ns

ns

ns **

ns

ns

ns, non-significant. a Mixture of late harvest grass silage and early harvest red clover silage (1:1 DM basis). b Standard error of means, n = 5 per treatment. c S, Grass versus red clover; M, Early versus late harvest time (maturity); S × M, Interaction of plant species and forage maturity; X, Mixing of the late harvest grass silage and the early harvest red clover silage versus feeding these silages separately. d Growth stage. † P<0.10. * P<0.05. ** P<0.01. *** P<0.001.

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the steady state pool size was approximated from the mean minimum and maximum values. In the present study, rumen evacuation prior to feeding was chosen to represent the minimum, and 6 h after feeding, to represent maximum pool size. The choice of rumen evacuation time may not have a strong influence on the estimates of comminution and passage kinetics, since they are based on iNDF pool size that showed small diurnal variation even in cattle fed twice daily (Huhtanen et al., 2007). Rumen evacuation may further disturb normal rumen functions by destroying (at least temporarily) the normal stratification of particles within the rumen. Towne et al. (1986) reported that ruminal evacuation did not seem to disrupt rumen anaerobiosis, microbial population, volatile fatty acid concentrations or liquid kinetics. Huhtanen et al. (2007) found no effect on in situ degradability of forage samples. The interval between the two rumen evacuations in the current study was 54 h, which can be considered adequate for the ruminal contents to reach normal stratification. In addition, because rumen evacuations did not affect feed intake or milk yield, we conclude that the procedure had minimal, if any, effect on rumen functions. 4.1. Distribution and chemical composition of particles The proportion of LP (>1.25 mm) of ruminal particulate DM was higher (about 500 g/kg) with all diets than in the study of Bayat et al. (2007) who, using a red clover-grass silage diet alone, observed that only one-third of particulate DM was composed of ruminal LP. However, our results agree with those of Bruining et al. (1998), Ahvenjärvi et al. (2001) and Rinne et al. (2002) that approximately half of the particulate DM was >1.25 mm. Poppi et al. (1980) defined the critical particle size of 1.18 mm in terms of ruminal escape as the size that retains 50 g/kg of faecal particulate matter. The proportion of particles >1.25 mm in faecal DM (68 g/kg on average) indicates that the critical particle size in this study was near the 1.18 mm value proposed by Poppi et al. (1980), which is in accordance with Shaver et al. (1988) and Bruining et al. (1998), who both used diets containing a 600:400 forage to concentrate ratio. However, Ahvenjärvi et al. (2001), Rinne et al. (2002) and Bayat et al. (2007) observed that less than 50 g/kg of faecal DM was retained on the 1.25 mm sieve, suggesting that critical particle size was smaller than 1.18 mm in those studies. The lower aNDFom concentration in ruminal SP compared to LP was associated with higher CP concentration in SP. The higher CP concentration in the SP fraction may be attributed to a more microbial attachment to them. Higher iNDF concentration in ruminal particulate DM with red clover compared to grass silage diets is consistent with the concentration of the truly undegradable fraction reported by Bruining et al. (1998). Grasses have a higher NDF concentration than legumes, but a lower concentration of lignin and iNDF (Van Soest, 1994; Huhtanen et al., 2006b), which together with a faster ruminal digestion rate, and slower passage rate, explains the higher iNDF:NDF ratio with diets based on red clover compared to those based on grass silage. Lignin in grasses is distributed at low concentrations in most cell types whereas in legumes it is not distributed equally among the different tissues (i.e., the lignified xylem tissue is highly indigestible while unlignified pith and cortex tissues are digested rapidly and completely (Wilson and Hatfield, 1997)). Furthermore, grasses have phenolic ester links with hemicelluloses that are susceptible to comminution with fungal esterases while legumes do not (Van Soest, 1994), which may contribute to lower iNDF:NDF ratio in grasses compared to legumes, as was observed in our forages (Kuoppala et al., 2009). The lack of a maturity effect on aNDFom and iNDF concentrations in ruminal particles with grass silage diets is contrary to the results of Gasa et al. (1991) and Rinne et al. (2002). Mertens (1993) pointed out that iNDF:NDF ratio increases with advancing maturity of forage plants. In this experiment, the iNDF and lignin (see Kuoppala et al., 2009) concentrations (g/kg NDF) increased with advancing maturity of the forages, but the increase was proportionally higher in red clover silage compared to grass silage (0.60 and 0.42 for red clover compared to 0.29 and 0.16 for grass, respectively). This probably explains the greater increase in iNDF concentration in ruminal LP and SP with red clover compared to the grass silage diet with advancing maturity. Changes in aNDFom and iNDF concentrations in ruminal digesta particles based on the seven pool model are in agreement with Ahvenjärvi et al. (2001) and Bayat et al. (2007). The lower aNDFom concentration in smaller particles may be attributed to greater attachment of bacteria to these particles compared to larger particles (Legay-Carmier and Bauchart, 1989), which is consistent with higher particle-associated enzyme activities (Huhtanen et al., 1993) and higher CP concentration of SP (Fig. 4a). Small particles may also originate from different morphological parts of the forage plant as leaves have a higher CP concentration than stems in both species (Rinne and Nykänen, 2000), or from the concentrate feed (including e.g., rapeseed expeller meal). 4.2. Ruminal pool sizes and faecal excretion The higher ruminal particulate DM pool with grass compared to red clover silage diets was mainly due to higher NDF intake. Smaller rumen pool size of NDF with red clover compared to grass silage diets suggests that rumen fill was not the major factor limiting intake of red clover silages (Kuoppala et al., 2009). The higher ruminal iNDF pool with red clover compared to grass silage diets originated partly from the higher iNDF intake and partly from the longer MRT of iNDF with red clover in relation to the grasses.

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Fig. 4. Crude protein (CP) concentration (g/kg DM) in the particle size fractions of ruminal digesta and faeces of dairy cows fed early harvest grass (), late harvest grass (), early harvest red clover (), late harvest red clover () and the mixture of late harvest grass and early harvest red clover silage diets (×). Standard errors of the means for the CP concentration of ruminal digesta and faeces were 2.8 and 4.2 g/kg, respectively.

Increasing total ruminal particulate DM pool with advancing maturity was a result of the increase in ruminal iNDF pool rather than ruminal pdNDF pool, especially with red clover silage diets. Bowman et al. (1991) observed that the ruminal DM pool size increased with advancing maturity in both LP and SP fractions, but that the difference was more in the LP fraction while, in the current experiment, the increase was more considerable in SP. 4.3. Ruminal digesta kinetics The faster kp of iNDF in large and small particles of grass compared to red clover silage diets was probably due to higher intake and rumen pool size of NDF. Meta-analysis of Nordic data (Huhtanen et al., 2006a) indicated a stronger positive relationship with iNDF passage rate and NDF intake than DM intake. The faster kp of iNDF and pdNDF in SP, compared to the LP fraction, has been observed in other studies using rumen evacuation technique (Bruining et al., 1998; Huhtanen et al., 2007; Bayat et al., 2007). Poppi et al. (1980) proposed that large particles must be reduced to a specific size to be eligible for passage through the reticulo-omasal orifice. Digestion of the digestible materials increases gas production, thereby decreasing the functional specific gravity of the particle (Wattiaux et al., 1992) and preventing small but digestible particles from being able to escape the rumen. As digestion proceeds, the amount of digestible NDF in the particle decreases, while that of iNDF is not reduced, meaning that the concentration of iNDF, and simultaneously, the likelihood for ruminal escape increase. Particle comminution depends on the histological structure of the forages (Grenet, 1989) and, as the anatomical structures of legumes and grasses differ (Wilson, 1990), the presence of supportive tissues, lignified in grasses and non-lignified in legumes, the volume of xylem tissues (always lignified) and their arrangement in the plant, have been reported to be the factors affecting resistance of plants to physical comminution (Grenet, 1989). Greater kr with grass compared to red clover silage diets in our study is contrary to Grenet (1989), Wilson (1993) and Bruining et al. (1998), who concluded that grasses are more resistant to particle size reduction by mastication compared to legumes. This discrepancy may be related to plant species (e.g., in the studies of Grenet (1989) and Bruining et al. (1998) lucerne and perennial ryegrass were compared, whereas in our study red clover was compared to mixed grass from timothy and meadow fescue). However, ruminants spend more time chewing grasses than legumes and more time chewing mature than immature forage (Buxton and Redfearn, 1997), which promote kr . It should be noted that the kr calculated in our study is a combined effect of mastication during eating and ruminating, microbial digestion by increasing the fragility of particles and physical effects caused by rumen contractions. Increasing kr with advancing maturity is comparable to observations of Rinne et al. (2002) who reported that kr of LP increased linearly with advancing maturity. Reasons for the slower comminution rate of early harvest grass silages are unclear. The kr values for all treatments were slower than the values reported by Huhtanen et al. (2007) and Bayat et al. (2007). Digestion facilitates comminution of LP (Wilson and Kennedy, 1996) and feeding concentrates reduces fibre digestion. Therefore, it is possible that concentrate supplementation reduces kr of LP. Further research is needed to assess possible effects of concentrate supplementation on kr . Lack of effects of plant species, forage maturity and mixing of silages on kp of pdNDF in both LP and SP suggest that digestibility of pdNDF is mainly regulated by rate of digestion. In agreement, Rinne et al. (1997) reported that kp of total pdNDF was not affected by maturity of grass silages. Rinne et al. (2002) reported that kp of pdNDF was not affected by forage maturity for large and small particles while it decreased with advancing maturity for medium particles. The faster kd of pdNDF of LP for red clover compared to grass silage diets is consistent with Bruining et al. (1998) who compared grass and lucerne silage with grass and maize silage using rumen evacuations. Legumes usually have a faster kd of pdNDF than grasses (Buxton and Redfearn, 1997), although large differences exist within legumes and grasses, and management factors within species, such as growth stage at harvest, also affect kd . Bayat et al. (2007), using a mixture of red clover-grass (3:1), reported that kd of LP was much faster than that of SP. Bruining et al.

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(1998) using rumen evacuations, observed that, with a grass silage diet, kd of potentially degradable DM was faster in the SP fraction compared to that in the LP fraction but, with a lucerne silage based diet, it did not differ between particle fractions. However, Huhtanen et al. (2007) reported that kd of medium particles was faster than that of large or small particles. Greater selective retention values for LP of grass compared to red clover silage diets could be related to faster kr and slower kd of them. Our observed selective retention values for SP are consistent with the values of Rinne et al. (2002). Overall, differences in kp of iNDF and pdNDF of the SP fraction were relatively small, and could partly be attributed to digestion in the omasum and hind-gut. This suggests, in agreement with Ahvenjärvi et al. (2001), that in dairy cows at high feed intake particle passage is regulated by size rather than other characteristics such as specific gravity or potential digestibility. In earlier studies, differences in kp of iNDF and pdNDF were probably overestimated due to incomplete recovery of pdNDF in faecal samples. Longer MRT of iNDF with red clover silage diets compared to grass silage diets was due to both slower kr of LP and kp of SP. Longer MRT of iNDF compared to that of pdNDF for both particle size fractions in all the treatments is in agreement with the results of a previous study conducted in the same laboratory (Bayat et al., 2007). 5. Conclusions Larger ruminal iNDF and smaller aNDFom pool sizes in both large and small particle size fractions with red clover compared to grass silage based diets indicate differences in digestion and passage characteristic of aNDFom in red clover and grasses. The high proportion of particles smaller than 1.25 mm in ruminal contents indicates that mechanisms besides particle size control outflow of feed particles from the rumen (e.g., entrapment of small particles to large particles). The faster kp of iNDF compared to pdNDF for the small particle size fraction indicates that pdNDF is selectively retained in the rumen. Increased forage maturity increased ruminal aNDFom and iNDF pool sizes probably due to increases in intake and cell wall lignification. Differences in ruminal passage and digestion rates of large and small particles, in addition to the differences in ruminal passage and digestion rates of red clover compared to grass silage diets emphasize the need to consider particle size and forage type in metabolic models that predict DM intake and fibre digestibility in ruminants. Acknowledgements The project was partly funded by the Finnish Ministry of Agriculture and Forestry. The grant of Dr. Bayat from the Academy of Finland is greatly acknowledged. References Ahvenjärvi, S., Skiba, B., Huhtanen, P., 2001. Effect of heterogeneous digesta chemical composition on the accuracy of measurements of fiber flow in dairy cows. J. Anim. Sci. 79, 1611–1620. Aitchison, E., Gill, M., France, J., Dhanoa, M.S., 1986. Comparison of methods to describe the kinetics of digestion and passage of fibre in sheep. J. Sci. Food Agric. 37, 1065–1072. Allen, M.S., Mertens, D.R., 1988. Evaluating constraints on fiber digestion by rumen microbes. J. Nutr. 118, 261–270. Bayat, A.R., Rinne, M., Khalili, H., Valizadeh, R., Huhtanen, P., 2007. Estimation of digesta kinetics of different particle size fractions using rumen evacuation technique in dairy cows fed red clover-grass silage. J. Anim. Feed Sci. 16, 538–554. Bowman, J.G.P., Hunt, C.W., Kerley, M.S., Paterson, J.A., 1991. Effects of grass maturity and legume substitution on large particle size reduction and small particle flow from the rumen of cattle. J. Anim. Sci. 69, 369–378. Bruining, M., Bakker, R., Bruchem, J.V., Tamminga, S., 1998. Rumen digesta kinetics in dairy cows fed grass, maize and alfalfa silage. 1. Comparison of conventional, steady-state and dynamic methods to estimate microbial degradation, comminution and passage of particles. Anim. Feed Sci. Technol. 73, 37–58. Buxton, D.R., Redfearn, D.D., 1997. Plant limitations to fiber digestion and utilization. J. Nutr. 127, 814S–818S. Gasa, J., Holtenius, K., Sutton, J.D., Dhanoa, M.S., Napper, D.J., 1991. Rumen fill and digesta kinetics in lactating Friesian cows given two levels of concentrates with two types of grass silage ad lib. Br. J. Nutr. 66, 381–398. Grenet, E., 1989. A comparison of the digestion and reduction in particle size of lucerne hay (Medicago sativa) and Italian ryegrass hay (Lolium italicum) in the ovine digestive tract. Br. J. Nutr. 62, 493–507. Huhtanen, P., Ahvenjärvi, S., Weisbjerg, M.R., Nørgaard, P., 2006a. Digestion and passage of carbohydrates. In: Sejrsen, K., Hvelplund, T., Nielsen, M.O. (Eds.), Ruminant Physiology: Digestion, Metabolism and Impact of Nutrition in Gene Impression, Immunology and Stress. ‘Proceedings of the X International Symposium on Ruminant Physiology’. Copenhagen, Denmark. Wageningen Academic Publishers, Wageningen, The Netherlands, pp. 87–135. Huhtanen, P., Asikainen, U., Arkkila, M., Jaakkola, S., 2007. Cell wall digestion and passage kinetics estimated by marker and in situ methods or by rumen evacuations in cattle fed hay 2 or 18 times daily. Anim. Feed Sci. Technol. 133, 206–227. Huhtanen, P., Dakowski, P., Vanhatalo, A., 1993. Composition, digestibility and particle-associated enzyme activities in rumen particles as influenced by particle size and time after feeding. J. Anim. Feed Sci. 1, 223–235. Huhtanen, P., Nousiainen, J., Rinne, M., 2006b. Recent developments in forage evaluation with special reference to practical applications. Agric. Food Sci. 15, 293–323. Kuoppala, K., Ahvenjärvi, S., Rinne, M., Vanhatalo, A., 2009. Effects of feeding grass or red clover silage cut at two maturity stages in dairy cows. 2. Dry matter intake and cell wall digestion kinetics. J. Dairy Sci. 92, 5634–5644. Legay-Carmier, F., Bauchart, D., 1989. Distribution of bacteria in the rumen contents of dairy cows given a diet supplemented with soya-bean oil. Br. J. Nutr. 61, 725–740. Mertens, D.R., 1993. Kinetics of cell wall digestion and passage in ruminants. In: Jung, H.G., Buxton, D.R., Hatfield, R.D., Ralph, J. (Eds.), Forage Cell Wall Structure and Digestibility. Am. Soc. Agron.-Crop Sci. Soc. Am. Soil Sci. Soc. Am., Madison, WI, USA, pp. 535–570. Poppi, D.P., Norton, B.W., Minson, D.J., Hendricksen, R.E., 1980. The validity of the critical size theory for particle leaving the rumen. J. Agric. Sci. Camb. 94, 275–280. Rinne, M., Huhtanen, P., Jaakkola, S., 1997. Grass maturity effects on cattle fed silage-based diets. 2. Cell wall digestibility, digestion and passage kinetics. Anim. Feed Sci. Technol. 67, 19–35. 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Rinne, M., Nykänen, A., 2000. Timing of primary growth harvest affects the yield and nutritive value of timothy-red clover mixtures. Agric. Food Sci. Finl. 9, 121–134. Romney, D.L., Gill, M., 2000. Intake of forages. In: Givens, D.I., Owen, E., Axford, R.F.E., Omed, H.M. (Eds.), Forage Evaluation in Ruminant Nutrition. CABI Publishing, pp. 43–63. SAS User’s Guide, 2003. Statistics, Version 9.1 Edition. SAS Inst., Inc., Cary, NC, USA. Shaver, R.D., Nytes, A.J., Satter, L.D., Jorgensen, N.A., 1988. Influence of feed intake, forage physical form, and forage fiber content on particle size of masticated forage, ruminal digesta, and feces of dairy cows. J. Dairy Sci. 71, 1566–1572. Tamminga, S., Robinson, P.H., Vogt, H., Boer, H., 1989. Rumen ingesta kinetics of cell wall components in dairy cows. Anim. Feed Sci. Technol. 25, 89–98. Towne, G., Nagaraja, T.G., Owensby, C., Harmon, D., 1986. Ruminal evacuation’s effect on microbial activity and ruminal function. J. Anim. Sci. 62, 783–788. Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant, 2nd ed. Cornell University Press, Ithaca, NY, USA. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Vanhatalo, A., Kuoppala, K., Ahvenjärvi, S., Rinne, M., 2009. Effects of feeding grass or red clover silage cut at two maturity stages in dairy cows.1. Nitrogen metabolism and supply of amino acids. J. Dairy Sci. 92, 5620–5633. Wattiaux, M.A., Satter, L.D., Mertens, D.R., 1992. Effect of microbial fermentation on functional specific gravity of small forage particles. J. Anim. Sci. 70, 1262–1270. Wilson, J.R., 1990. Influence of plant anatomy on digestion and fibre breakdown. In: Akin, D.E., Ljungdahl, L.G., Wilson, J.R., Harris, P.J. (Eds.), Microbial and Plant Opportunities to Improve the Lignocellulose Utilization by Ruminants. Elsevier Sci. Publ. Co., NY, USA, pp. 99–117. Wilson, J.R., 1993. Organization of forage plant tissues. In: Jung, H.G., Buxton, D.R., Hatfield, R.D., Ralph, J. (Eds.), Forage Cell Wall Structure and Digestibility. Am. Soc. Agron.-Crop Sci. Soc. Am. Soil Sci. Soc. Am., Madison, WI, USA, pp. 1–32. Wilson, J.R., Hatfield, R.D., 1997. Structural and chemical changes of cell wall types during stem development: consequences for fibre degradation by rumen microflora. Aust. J. Agric. Res. 48, 165–180. Wilson, J.R., Kennedy, P.M., 1996. Plant and animal constraints to voluntary feed intake associated with fibre characteristics and particle breakdown and passage in ruminants. Aust. J. Agric. Sci. 47, 199–225.