Species and variety of herbage affects release of cell contents during ingestive mastication in dairy cows fed indoors

Animal Feed Science and Technology 132 (2007) 28–48

Species and variety of herbage affects release of cell contents during ingestive mastication in dairy cows fed indoors A. Acosta a , A. Boudon b,∗ , J.-L. Peyraud b a

b

Universidad de Buenos Aires, Facultad de Agronom´ıa, Av. San Martin 4453, Ciudad de Buenos Aires, Argentina Unit´e Mixte de Recherches INRA/Agrocampus Production du Lait, Institut National de la Recherche Agronomique, 35590 St.-Gilles, France Received 11 May 2005; received in revised form 8 March 2006; accepted 9 March 2006

Abstract To achieve a better understanding of the nutritional value of grazed forages to ruminants, it is necessary to improve our knowledge of the accessibility and nature of nutrients available to microorganisms in the rumen. The objectives of this experiment were to compare release of cell contents and comminution of particles during ingestive mastication in dairy cows fed four temperate species or varieties of fresh forages, and to analyse the respective effects of chewing behaviour and mechanical properties of plant tissue on release of cell contents. Six Holstein dairy cows were fed indoors with four species/varieties of fresh forages being white clover (WC) (Trifolium repens L., cv Alice), tetraploid perennial ryegrass (tPR) (Lolium perenne L., cv Ch´eops), diploid perennial ryegrass (dPR) (Lolium perenne L., cv Ohio) and tall fescue (TF) (Festuca arundinacea Schreb., cv Barcel). The design was two 4 × 4 Latin squares with 4 periods of 1 day each and two replications of two treatments on two cows each period. One Latin square was completed in May and the other in June. Ingestive boli were collected directly at the cardia of the cows during the morning meal after manually emptying the rumen. The intake rate averaged 62.5 g DM/min, and chewing behaviour parameters were unaffected

Abbreviations: ADFom, acid detergent fiber; DM, dry matter; dPR, diploid perennial ryegrass; ICs, intracellular constituents; Lignin (sa), Lignin determined by solubilization of cellulose with sulphuric acid; NDFom, neutral detergent fiber; NDS, neutral detergent soluble; TF, tall fescue; tPR, tetraploid perennial ryegrass; WC, white clover; WSC, water-soluble carbohydrates ∗ Corresponding author. Tel.: +33 2 23 48 50 86; fax: +33 2 23 48 51 01. E-mail address: [email protected] (A. Boudon). 0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2006.03.006

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by herbage. The average proportions of intracellular constituents (ICs) released during ingestive mastication were 265, 203 and 364 g/kg of ICs ingested for neutral detergent soluble (NDS), intracellular N and water-soluble carbohydrates (WSC), respectively. For most ICs considered, the proportions released during mastication were higher for white clover and fescue compared with ryegrasses. The proportions of intracellular N released were higher for tetraploid compared with diploid ryegrass. The median size of the bolus particles was unaffected by the herbage species or variety. In contrast, the median size of the particles in the boli was strongly affected by individual cow (P<0.01), whereas the proportions of ICs released showed no such effect. It appeared that those cows with the lowest reduction in particle sizes by ingestive chewing were those with a smaller molar surface area. None of the parameters measured (i.e., release of ICs, median size of particles in the boli and chewing behaviour) could be related to the shear properties of the herbage. Results illustrate the complexity of the interaction between the structure of the plant tissue and the physical damage of the particles during chewing. © 2006 Elsevier B.V. All rights reserved. Keywords: Mastication; Cell contents; Particle size; Ruminal digestion; Shear strength; Forage

1. Introduction To improve our understanding of the nutritional value of grazed forages for ruminants, a better description of the accessibility and nature of nutrients available to the ruminal microorganisms is required. In grazed forages, most nitrogenous compounds (and all soluble carbohydrates) are intracellular constituents (ICs) (Butler and Bailey, 1973; Sanderson and Wedin, 1989) that are still metabolically active during ingestion. The ICs are enclosed within a plasmalemma and cell wall that have to be ruptured before cell contents can be released and made available to the micro-organisms in the rumen. Therefore, to clarify the nature of the nutrients, and their availability to ruminal micro-organisms, a first step is to improve the description of the cell-content release during ingestion of herbage. The release kinetics of ICs among plant species, varieties and maturities has not been well characterized. It has been shown that release kinetics of ICs during ingestive mastication and digestion depends primarily on the individual ICs (Reid et al., 1962; Mangan et al., 1976; Boudon et al., 2002b). Water-soluble carbohydrates (WSC), medium-sized molecules contained in vacuoles, are released more rapidly than chlorophyll and protein contained in chloroplasts. It has also been shown that chewing during eating determines release of ICs, since more than 90% of WSC and 40% of chlorophyll, are released after ingestive mastication and a mean rumen residence time of 1.5 h (Boudon et al., 2002b). However, little or no variation in the proportion of ICs released during ingestive mastication was observed with increasing stage of maturity, at least within a plant species (Bryant, 1964; Boudon and Peyraud, 2001; Boudon et al., 2002a). The central focus in this experiment was to characterize variability of the proportions of ICs released during ingestive mastication among four herbages chosen because of their wide range in the mechanical properties of their tissues (Wilman et al., 1996; Henry et al., 1997). This experiment also aimed to determine the extent that chewing behaviour, and the nature of the plant tissue, can modulate release of ICs during ingestive mastication

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and how these parameters interact. The ICs examined were WSC, total intracellular N and chlorophyll. WSC are vacuolar compounds, whereas intracellular N is both cytoplasmic and chloroplastic. Chlorophyll is not of major nutritional interest, but it has been used as a tracer for chloroplastic compounds (Mangan and West, 1977). Neutral detergent solubles (NDS) was also studied to give a gross estimate of the total cell contents.

2. Materials and methods 2.1. Experimental treatments and animals The experiment compared release of ICs during ingestion, chewing behaviour and comminution of particles in four temperate herbages commonly used for grazing in the oceanic climates of Europe (i.e., white clover (WC, Trifolium repens L. cv Alice), tetraploid or diploid perennial ryegrass (tPR, Lolium perenne L. cv Ch´eops or dPR, Lolium perenne L. cv Ohio) and tall fescue (TF, Festuca arundinacea Schreb. cv Barcel)). Ingestive boli were collected from six lactating dairy cows fed indoors with fresh herbage. The experimental design consisted of two 4 × 4 Latin squares with four herbages, 4 periods of 1 day each and 6 cows. Two pairs of cow were defined among the six cows and, in each period, two treatments were replicated on both cows of each pair. Both pairs of cows were always the same for all periods. The first Latin square was completed from May 27 to 30, 2002 and the second from June 24 to 27, 2002 at the INRA experimental farm at Mejussaume near Rennes (France). Each Latin square was preceded by a pre-experimental period of 7 days for adaptation to indoor feeding. During the pre-experimental period, cows were fed indoors with fresh perennial ryegrass. Cows were fitted with large ruminal cannulae (i.d., 123 mm) and housed in tie stalls on rubber mats in an artificially ventilated barn with free access to water and a mineral block. At the beginning of the experiment, the mean stage of lactation of the cows was 215 ± 24.1 days, the body weight was 592 ± 40.6 kg and the daily milk production 18.1 ± 4.22 kg/day. The cows were milked twice daily at 07:00 and 17:00 h. Procedures relating to the care and use of animals were approved by the French Ministry of Agriculture in accordance with French regulations (Decree-law 2001-464, May 29, 2001). 2.2. Sward management and animal feeding The herbage fed during the 4 days of each Latin square was cut on four 0.1 ha pastures. The pastures were sown in 1999 with either WC, tPR, dPR or TF. The grass pastures received 260 kg N/ha/year and the white clover pasture received no N fertilisation. The four pastures were cut on May 2 and the regrowth was fed during the first Latin square in May. The swards were entirely cut on May 31, after the first Latin square, and the regrowth was fed during the second Latin square in June. The stage of maturity of the pastures at the beginning of each Latin square was 25 days of regrowth. The herbage fed during the pre-experimental periods was fresh Lolium perenne L. cv Belfort, cut on a 2 ha pasture that received 160 kg N/ha/year. The stage of regrowth during the pre-experimental periods ranged between 20 and 27 days.

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During the two Latin squares, the cows were fed a new herbage, among the four tested, each day at 10:00 h and the ingestive boli were collected the next morning, before 10:00 h. Each day, the cows were fed ad libitum during two main meals at 10:00 and 18:30 h, but their access to the feed was limited to 6 h after 10:00 and 3 h after 18:30 h. Throughout the study, the herbage was cut each day at 16:00 h with a mower. A portion of this herbage was fed directly, while the remainder was stored at 4 ◦ C in the dark and in open plastic bins, until the morning meal. 2.3. Sampling and measurements on the ingested boli Fifteen consecutive ingestive boli were collected from each of the six cows, during a test meal of a few minutes between 8:30 and 10:00 h (i.e., after a full day of adaptation to the herbage). The rumen contents of each cow were previously removed by hand and 16 consecutive ingestive boli were collected directly at the cardia while the cow was eating the test meal. The quantity of herbage fed for the test meal was 7 kg on a fresh matter basis. People were trained such that the molded part of the boli was, as much as possible, entirely collected. The first bolus of a series was discarded because it was assumed to be incomplete. After collection of the boli, remaining herbage was removed from the feed trough and weighed, and the rumen of the cow was refilled. The 15 boli thus collected were individually weighed and all the boli of each cow were pooled to a composite sample, which was gently mixed by hand to avoid rupturing the plant cells that remained intact after ingestive mastication. Three subsamples of 350 g were collected for determination of the proportions of IC contents, a subsample of 300 g was kept at −20 ◦ C for measurement of the particle-size distribution and two subsamples of 300 g were collected for determination of the dry matter (DM) content at 80 ◦ C for 48 h. The proportion of ICs released during ingestive mastication was measured by the method described by Boudon and Peyraud (2001). Subsamples of ingestive boli were rinsed with 3 l of distilled water per 100 g of boli to remove ICs released from the plant cells during ingestive mastication, and their IC contents were compared with the IC contents of herbage. To achieve this, a Buchner funnel was covered with a 100 ␮m mesh nylon cloth and connected to a vacuum pump. The subsamples were gently mixed with a spatula while rinsed. The rinsing liquid was poured 0.5 l at a time, the subsamples being sucked dry between washings. The rinsed subsamples were pooled before freeze-drying and stored at −20 ◦ C in the dark. For the second Latin square in June, the particle-size distribution of the boli was measured by wet sieving on a Fritsch electromagnetic siever (Analysette 3, Fritsch, Idar-Oberstein, Germany) containing seven sieves. Sieve sizes (i.e., length of the side of square hole) were 16, 10, 4, 2, 1, 0.5 and 0.063 mm. A subsample of 20 g (fresh weight) of boli was thawed and stirred into 200 ml of water with 5 ml of neutral detergent (Valordetergent, ValleyYvay, France) to achieve a preliminary separation and prevent matting. The water and bolus particles were poured onto the top sieve and sieving was completed in two cycles of 3 min each, with an amplitude of 80 and a water flow of 1.3 l/min. Material retained on each sieve was washed onto tared filter papers and oven-dried at 80 ◦ C for 48 h. The soluble fraction was defined as the DM passing the 0.063 mm sieve, and its proportion was calculated as the difference between the total DM sieved and the sum of DM retained on all the sieves.

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The length and width of the particles retained on the top sieve were analysed by image analysis. Before being filtered on filter paper, the contents of the top sieve was washed and spread onto three large white enamelled trays. A small quantity of water was kept in each tray to cover the particles and prevent dehydration, while particles were manually separated with pliers. To avoid producing shade, the light source was placed perpendicularly to each tray and centred. A standard particle measuring 5 cm × 1 cm was added to each tray to indicate the scale. Pictures of each tray were taken with a digital camera (Coolpix 775, Nikon, Tokyo, Japan; resolution 1024 × 768) and the picture was analysed with the SigmaScan Pro® 5 software (SPSS Science Inc., Chicago, IL, USA). The camera was placed 40 cm above the centre of the tray, in a plane parallel to the tray. The contrast of the picture was maximised by the software Sigma Scan Pro 5, and an overlay was adjusted to discriminate particles according to an intensity threshold. From the binary image so obtained, direct measurements were made of the number of particles, as well as the length and width of each particle. The length was defined as the length of the major axis of the particle, being taken as the separation between the two most distant points on the particle. The width was defined as the length of the minor axis of the particle, taken as the separation between the two most distant points on the particle forming a line perpendicular to the major axis (SSPS, 1999). To eliminate artefacts, such as, dust or soil residue, the few particles with length and width smaller than 0.2 mm were discarded. 2.4. Characterisation of chewing behaviour and jaw movements of the animals Eating time, number of jaw movements and number of boli were manually recorded during collection of boli. The week following the experiment, the dental formula of the six cows was recorded. Measurements were taken of the length of the incisor breadth, the protrusion of the incisor arcade (Perez-Barberia and Gordon, 2001), the external lateral length of the premolars and molars, as well as the space between the canines and premolars, using a measuring tape on the lower jaw. The six cows were successively anaesthetised with 1.5 ml of Rompun® (Bayer AG, Leverkusen, Germany) administered intravenously, while their mouth was maintained open with a retractor. 2.5. Sampling and measurements on herbage To determine the DM content of the offered herbage, two representative 700 g samples of each herbage were oven dried at 80 ◦ C for 48 h, each day before feeding the test meal. A further 300 g sample was frozen at −20 ◦ C in the dark and freeze dried for analysis of organic matter, aNDFom, ADFom, Lignin (sa) and IC contents. To characterize the morphological composition and particle size of the herbage, a further 300 g sample was collected before feeding the test meal each day and kept at −20 ◦ C. The herbage DM mass of the sward above 5 cm was measured weekly using a motor-scythe. For morphological separation and measurement of the tiller or foliole length, 40 g (fresh weight) of herbage were subsampled from the 300 g samples mentioned above, thawed and manually separated into four organs (i.e., stems/pseudostems, pseudostems alone, laminae, heads) and dead tissue for grasses and six organs (i.e., folioles, petioles of folioles, petioles of flower, flowers, stolons) and dead tissue for WC. For each sample, the contents in each

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category were oven dried at 80 ◦ C for 48 h. For morphological separation, the organs of the tillers or petioles were separated with scissors. For measurement of length distribution, the total length and the length of the longer sheath for the grasses were measured on one of five tillers or petioles before morphological separation, while the length of each organ was measured after morphological separation. The over all mechanical properties of the herbage cell walls was characterized as the energy required to break up each organ by shearing, which was assumed to be similar to the energy expended during mastication. The shear energies of the herbage samples were measured in June only. Thirty tillers for each grass (tPR, DPR and TF) and 30 petioles for WC were randomly chosen and cut at their base, directly in the pasture, the day before and after each Latin square. To avoid dehydration of plant organs before measurement of shear properties (Henry et al., 2000), the tillers/petioles were stored at 100% relative humidity, (i.e., they were placed between water saturated sheets of paper towelling), in an aluminium tray and sealed in an air-tight plastic bag. All measurements of shear properties were performed during the day of sampling. The shear energy of the main organs of each herbage was measured by shearing the organ perpendicularly to its axis, with a guillotine, using an Instron® universal texture analyser (Model 4500, Instron Corporation, Canton, MA, USA). A few particles of each category of organ were randomly selected, measured for length and width at their midpoint, rolled into bundles of five particles and held together with adhesive tape. Particles were aligned within the bundles so that they could be sheared at their midpoint. Particles were held on the device platform with clamps positioned 5 mm from the midpoint on each side of the bundle. The texture analyser was fitted with a modified Warner–Bratzler test cell consisting of a flat single blade designed to pass through a slot at a speed of 81 mm/min. The blade was attached to a load cell of 1 N. The rake angle and closing angle of the blade were 60◦ and 0◦ , respectively (Henry et al., 1996). The wear of the blade was regularly checked by means of 5 mm wide paper strips (Brochure and flyer paper, two-sided gloss, 162 g/m2 , Hewlett-Packard, Houston, TX, USA). After measurement, fragments were oven-dried and weighed for calculation of linear density (DM weight/length), which was assumed to be a gross estimate of the crosssectional area. Measurements were repeated in triplicate for each sample and each category of organ. 2.6. Chemical analysis Chemical analyses were on freeze-dried samples of offered herbage and rinsed ingestive boli, after grinding through a 0.8 mm screen. Organic matter was determined after ashing at 550 ◦ C for 5 h. Neutral detergent fiber (aNDFom), acid detergent fiber (ADFom) and lignin (sa), were analysed on a Fibersac extraction unit (Ankom, Fairport, NY, USA) using the method described by Van Soest and Wine (1967) with addition of a heat stable alphaamylase and without sodium sulphite for determination of aNDFom (Giger et al., 1987). The aNDFom and ADFom contents were expressed exclusive of residual ash. Total N was determined by the Dumas method (Association Franc¸aise de Normalisation, 1997) on a Leco apparatus (Leco, St. Joseph, MI, USA). Intracellular N was calculated as the difference between total N and N in the NDF residue. Chlorophyll was analysed by spectrophotometry after extraction with N, N-dimethylformamide (24 h at 4 ◦ C in the dark) by the method of

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Inskeep and Bloom (1985), and WSC were measured using the anthrone method of Yemm and Willis (1954). 2.7. Calculations The proportion of IC released from ingested boli was defined as the difference between the quantity of IC ingested in the bolus and the quantity of IC remaining in the bolus after rinsing, divided by the quantity of IC ingested for the bolus. The quantity of IC ingested for the bolus was calculated assuming that the quantity of aNDFom in the bolus was the same as the quantity of aNDFom in the ingested herbage before mastication. The final formula is (Boudon and Peyraud, 2001):   IC boli × aNDFom Herbage IC released = 1 − 100 IC herbage × aNDFom Boli where “IC released” is the proportion of IC released during eating (g/100 g of IC intake), “IC boli” and “IC herbage” are the IC contents of rinsed boli and offered herbage (g/100 g of DM), respectively, and “aNDFom Boli” and “aNDFom Herbage” are the aNDFom contents of rinsed boli and offered herbage (g/100 g of DM), respectively. The median size of the particles in the boli and their size dispersion were calculated using a logarithmic normal distribution (Waldo et al., 1971). The distribution of particle sizes between the sieves was first expressed as cumulative fractions of the weight retained on each sieve. A logarithmic normal distribution was then fitted to each particlesize distribution using the PROBIT procedure of SAS (1999). The median size of the particles was the common logarithm of the fractile corresponding to 50% of the distribution (d50). The difference between the fractiles corresponding to 15% and 85% of the distribution (d15–d85) was considered as an index of the particle-size dispersion in the ingestive boli. The shear energy of each organ was calculated by integrating the force displacement curve observed while shearing the bundles on the texture analyser. Before the calculation, the mechanical friction due to the device was eliminated by subtracting the force displacement curve observed on the empty device from the force displacement curve observed while shearing the bundles. The friction of the plant material along the blade after the bundles were cut was discarded by integrating only the part of the curve observed at the time of cutting the bundles. The index of shear tenacity (mJ cm/g DM) was defined as the ratio between the shear energy (mJ) and the linear density (g DM/cm). This index represents a gross estimate of shear tenacity, which is the ratio between shear energy and the crosssectional area (Wright and Illius, 1995). The linear shear energy was defined as the ratio between the shear energy and the cut width. 2.8. Statistical analyses Data were analysed by analysis of variance using the MIXED option of SAS (1999). Since the four cows refused to eat the white clover in May, only the data for the three grasses were analysed in May and the data for the four herbages were analysed in June. Because of this, two different statistical models were used.

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In the first model, the data obtained for the grasses in May and June were analysed together, according to the following model. For the proportions of ICs released, the parameters related to the particle-size distribution and the chewing behaviour parameters, the model sums of squares were separated into effects due to month, herbage and cow, as well as the interaction between month and herbage. The interaction between month and period was also included as a random effect. For parameters related to the offered herbage, the effect due to cow was removed from the model. The effects of fescue versus ryegrasses (TF versus tPR + dPR) and tetraploid versus diploid ryegrass (tPR versus dPR) were analysed by orthogonal contrasts. In the second model, the second Latin square (i.e., June), was analysed independently with the four herbages including WC. For the proportions of ICs released, the parameters related to the particle-size distribution and the chewing behaviour parameters, the model sums of squares were separated into effects due to herbage and cow. The period was also included as a random effect. For parameters related to the offered herbage, the effect due to cow was removed from the model. The effects of WC versus grasses (WC versus tPR + dPR + TF), fescue versus ryegrasses (TF versus tPR + dPR) and tetraploid versus diploid ryegrass (tPR versus dPR) were analysed by orthogonal contrasts.

3. Results The interaction between month and herbage was barely significant (i.e., P<0.05) for the data obtained on grasses in May and June. When it tended to be significant (P<0.10), the data of May and June are presented in the tables with the standard error of the interaction and the contrasts were tested among each month. 3.1. Characteristics of the offered herbages In grasses in May and June, the average herbage DM mass of the sward above 5 cm was 2618 kg DM/ha and the average total length of the offered herbage was 180 mm (Table 1). The interaction between the herbage and the month for the three grasses was significant (P<0.001) for the total length and the length of the longest sheath (P<0.05). In May, the offered tPR was longer than dPR and TF (227 mm versus 173 mm), but the length of the longest sheath was not affected by herbage. In June, both ryegrasses were shorter than TF (147 mm versus 201 mm) and the length of the longest sheath was higher in both ryegrasses than in TF (48 mm versus 31 mm). The aNDFom and ADFom and lignin (sa) contents were slightly higher in ryegrasses compared to TF whereas they were unaffected by grasses in May (interaction month × herbage P<0.05). The lignin (sa) content was higher with ryegrasses compared to TF and was higher with tPR compared with dPR. The intracellular N content was lower with ryegrasses compared to TF and was higher with tPR compared with dPR. None of the other chemical composition parameters differed between the three grasses. The interaction between month and grasses was significant for all the parameters of the morphological composition (Table 2, P<0.05). In May, both ryegrasses were cut at a vegetative stage with a negligible content of stems or heads, and TF was in an early

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Table 1 Biomass of the sward, length and chemical composition of offered herbages (only grasses) in May and June (common statistical model for both months) Treatmentsa

Available herbage DM

massc

(kg DM/ha)

Contrast analysesb

tPR

dPR

TF

PR vs. TF

tPR vs. dPR

S.E.

2547

2476

2832

Length of offered herbage (mm) May June

227 148

173 145

186 201

ns ***

*** ns

14.1

Length of longest sheath (mm) May June

56 54

47 42

55 31

ns **

ns 0.08

6.2

Dry matter (DM) (g/kg) Organic matter (g/kg DM) Total N (g/kg DM)

188 909 28.1

203 907 26.3

205 909 28.2

ns ns ns

0.08 ns 0.09

10.3 1.7 0.83

aNDFom (g/kg DM) May June

440 497

446 500

456 464

ns **

ns ns

9.3

ADFom (g/kg DM) May June

222 244

215 241

223 229

ns *

ns ns

4.6

Lignin (sa) (g/kg DM) Intracellular N (g/kg DM) Chlorophyll (g/kg DM) Water-soluble carbohydrates (g/kg DM)

23 20.7 7.78 129.9

20 18.5 7.54 134.6

18 21.1 7.61 128.4

** * ns ns

* * ns ns

1.0 0.77 0.272 5.56

a

tPR: tetraploid perennial ryegrass, dPR: diploid perennial ryegrass, TF: tall fescue. *P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, S.E.: standard error of the means. When the interaction month × herbage tended to be significant (P<0.10), contrasts tested in each month and S.E. of the interaction are presented. c The available herbage DM mass (kg DM/ha) was measured above 5 cm rising plate metre. b

reproductive stage with 17 g/kg DM of stems and 10 g/kg DM heads. In June, both ryegrasses were in a reproductive stage while TF was in a vegetative stage. The lamina content was lower (P<0.001) with ryegrasses compared to TF (734 g/kg versus 922 g/kg) and the stem and head contents were higher. Similarly, in the same way in June, tPR contained less lamina, and more stems and heads, compared to dPR. In June, the herbage DM mass of the sward above 5 cm was, on average, 2832 kg DM/ha and the total length of the offered herbage was 158 mm (Table 3). The offered WC was shorter than the grasses (139 mm versus 165 mm). The DM, OM, aNDFom and watersoluble carbohydrate contents were lower in white clover compared to the three grasses, whereas the total N, intracellular N and lignin (sa) contents were higher. Total N content was, on average, 35.8 g/kg DM for WC and 25.5 g/kg DM for the three grasses, while the aNDFom was, on average, 344 g/kg DM for WC and 487 g/kg DM for the three grasses. The chlorophyll content was not affected by herbage.

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Table 2 Morphological composition of offered herbages (only grasses) in May and June (common statistical model for both months) Treatmentsa

Contrast analysesb

tPR

dPR

TF

PR vs. TF

tPR vs. dPR

S.E.

Proportions (g/kg DM) Lamina May 771 June 681

815 786

853 922

ns ***

ns *

28.2

Pseudostem May June

223 84

162 90

103 44

*** 0.06

* ns

19.7

Stem May June

0 130

4 50

17 14

ns ***

ns **

14.4

Head May June

0 76

3 16

10 0

ns **

ns **

10.6

Dead tissue May June

6 28

16 59

17 20

ns **

ns ***

4.6

a

tPR: tetraploid perennial ryegrass, dPR: diploid perennial ryegrass, TF: tall fescue. *P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, S.E.: standard error of the means. When the interaction month × herbage tended to be significant (P<0.10), contrasts tested in each month and S.E. of the interaction are presented. b

In June, both ryegrasses and WC were in a reproductive stage while TF was in a vegetative stage with no heads (Table 4). The shear energy of the WC folioles was higher than that of TF laminae, which was itself higher than that of perennial ryegrasses. However, the shear tenacity indexes of the laminae were unaffected by the herbages among the three grasses and the shear tenacity index of the WC folioles was lower (P<0.01) than that of laminae of the three grasses (9.2 mJ mm/mg DM versus16.1 mJ mm/mg DM). The shear energy of the petioles and stems of WB were lower (P<0.05) than the grasses. However, the shear tenacity index of the WC petioles and stems was in the same range as that of the stems and pseudostems of the grasses. The shear tenacity indexes weighted by the proportions of the organs, with dead tissue and flowers excepted, led to average values of 14.0, 19.4, 17.7 and 15.6 mJ mm/mg DM for WC, tPR, dPR and TF, respectively and tended (P=0.07) to be lower for WC compared to the grasses. 3.2. Effect of herbage on chewing behaviour of the animals The average time taken to collect the 16 boli was 254.9 s, and the average fresh and dry intakes per bolus were 73.9 and 14.0 g, respectively. For grasses in May and June, the time to swallow a bolus tended (P=0.06) to be higher with ryegrasses than TF, while the fresh intake per bolus, the fresh intake rate and the frequency of jaw movements

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Table 3 Biomass of the sward, length and chemical composition of the four offered herbages in June Treatmentsa

Available herbage DM massc (kg DM/ha) Length of offered herbage (mm) Dry matter (DM) (g/kg) Organic matter (g/kg DM) Total N (g/kg DM) aNDFom (g/kg DM) ADFom (g/kg DM) Lignin (sa) (g/kg DM) Intracellular N (g/kg DM) Chlorophyll (g/kg DM) Water-soluble carbohydrates (g/kg DM) a b c

Contrast analysesb

WC

tPR

dPR

TF

2507

2614

2828

3379

139

148

145

159 893 35.8 344 230 57

222 911 26.3 497 244 27 18.7 7.41 116.1

28.9 6.72 50.1

WC vs. Grass

PR vs. TF

tPR vs. dPR

S.E.

201

*

***

ns

9.1

230 906 24.0 500 241 23

235 911 26.3 464 229 20

*** *** *** *** ns ***

ns ns ns * 0.07 0.09

ns ns ns ns ns ns

16.5 2.7 1.30 12.0 5.6 2.3

16.5 6.87 121.1

20.0 7.45 127.2

*** ns ***

ns ns ns

ns ns ns

1.25 0.476 7.695

WC: white clover, tPR: tetraploid perennial ryegrass, dPR: diploid perennial ryegrass, TF: tall fescue. *P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, S.E.: standard error of the means. The available herbage DM mass (kg DM/ha) was measured above 5 cm rising plate metre.

tended (P=0.06, 0.07, 0.06, respectively) to be lower with ryegrasses compared to TF (Table 5). In June, the DM intake per bolus, the DM intake rate and the DM content and dry weight of boli were lower (P<0.05) in WC compared to grasses (Table 6). The fresh intake per bolus, the fresh intake rate and the fresh weight of the boli were unaffected by herbages, and no chewing behaviour parameter was affected by the grasses in June. 3.3. Effect of herbage on IC release during mastication and size distribution of chewed particles Average proportions of NDS, intracellular N, chlorophyll and WSC released during ingestive mastication were 265, 222, 173 and 377 g/kg individual IC intake, respectively. Among grasses, in May and June (Table 7), the proportions of NDS, intracellular N and WSC released were lower (P<0.05) for the perennial ryegrasses compared with TF (respectively 203 g/kg versus 338 g/kg for NDS, 169 g/kg versus 246 g/kg for intracellular N and 331 g/kg versus 449 g/kg for WSC). The proportions of chlorophyll released also tended (P=0.08) to be lower for the perennial ryegrasses compared with TF. Among ryegrasses in May and June (Table 7), higher (P<0.05) proportions of intracellular N were released from tPR compared with dPR (210 g/kg versus 127 g/kg). Higher proportions of NDS also tended (P=0.08) to be released from tPR compared with dPR. In June, WC was characterized by a higher (P<0.05) proportion of NDS and intracellular N released, compared with grasses (Table 7, respectively 367 g/kg versus 264 g/kg for NDS and 385 versus 215 for intracellular N). This effect was not reflected in the proportions of chlorophyll and WSC released.

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Table 4 Morphological composition of the four offered herbages and shear energy of each organ in June Treatmentsa

Proportions (g/kg DM) Foliole/lamina Petiole/pseudostem Stem Flower/head Dead tissue Shear energyc (mJ) Foliole/lamina Petiole/pseudostem Stem Head

Contrast analysesb

WC

tPR

dPR

TF

WC vs. Grass

PR vs. TF

tPR vs. dPR

S.E.

419 243 87 68 34

681 84 130 76 28

786 90 50 16 59

922 44 14 0 20

*** *** ns 0.06 ns

*** * * * *

* ns * * *

31.5 18.0 19.2 15.1 8.3

7.4 4.0 10.0

2.1 13.2 19.2 16.5

2.2 10.2 18.4 22.3

4.2 12.0

** * *

* ns

ns ns ns ns

0.46 1.76 1.52 2.81

Shear tenacity indexc (mJ mm/mg DM) Foliole/lamina 9.2 15.5 Petiole/pseudostem 18.4 21.6 Stem 29.3 27.6 Head 27.0 Weighted meand 14.0 19.4

16.8 20.5 27.5 29.2 17.7

15.9 26.4

** ns 0.09

ns ns

15.6

0.07

ns

ns ns ns ns ns

1.00 3.22 0.90 1.59 1.00

a

WC: white clover, tPR: tetraploid perennial ryegrass, dPR: diploid perennial ryegrass, TF: tall fescue. *P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, S.E.: standard error of the means. c The shear energy was defined as the quantity of energy required to cut the organ perpendicularly to its axis. The shear tenacity index was defined as the ratio between the shear energy and the linear density around the width cut of the organ. For both these parameters, the statistical analyses were with two data per cow and per period (instead of four for other parameters). d The mean of the shear energy index weighted by the proportions of the organs, with dead tissue and flowers excepted. b

Table 5 Chewing behaviour of the dairy cows fed grasses during the bolus collection in May and June (common statistical model for both months) Treatmentsa

Duration to swallow a bolus (s) Fresh intake/Bolus (g) Dry matter intake/Bolus (g) Intake rate (g DM/min) Intake rate (g fresh weight/min) Frequency of jaw movements (min) Number of jaw movements per bolus Fresh weight of boli (g) DM content of boli (g/100 g) Dry weight of boli (g) a b

Contrast analysesb

tPR

dPR

TF

PR vs. TF

tPR vs. dPR

S.E.

14.7 73.9 14.0 65.8 354 73.6 18.0 127.8 9.6 12.3

15.0 75.7 15.0 68.6 350 71.1 17.7 130.8 10.4 13.6

17.8 67.2 13.7 60.2 295 67.5 19.1 120.6 10.3 12.4

0.06 0.06 ns ns 0.07 0.06 ns ns ns ns

ns ns ns ns ns ns ns ns * ns

1.55 3.43 0.62 4.35 30.5 3.21 1.15 4.19 0.34 0.63

tPR: tetraploid perennial ryegrass, dPR: diploid perennial ryegrass, TF: tall fescue. *P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, S.E.: standard error of the means.

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Table 6 Chewing behaviour of the dairy cows fed with the four herbages, during the bolus collection in June Treatmentsa

Duration to swallow a bolus (s) Fresh intake/Bolus (g) Dry matter intake/Bolus (g) Intake rate (g fresh weight/min) Intake rate (g DM/min) Frequency of jaw movements (/min) Number of jaw movements per bolus Fresh weight of boli (g) DM content of boli (g/100 g) Dry weight of boli (g) a b

Contrast analysesb

WCa

tPRa

dPRa

TFa

WC vs. Grass

PR vs. TF

tPR vs. dPR

S.E.

16.5 83.9 12.5 380 57.5 71.0 18.9 121.3 8.6 10.5

14.9 77.5 17.0 367 78.8 78.1 19.4 137.3 10.5 14.6

15.1 78.9 17.3 358 78.4 78.9 19.8 139.4 11.2 15.5

17.2 68.7 15.9 295 66.4 72.5 20.9 130.2 10.9 14.2

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

1.57 9.09 1.08 59.7 6.59 2.85 1.34 7.43 0.51 1.10

WC: white clover, tPR: tetraploid perennial ryegrass, dPR: diploid perennial ryegrass, TF: tall fescue. *P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, S.E.: standard error of the means.

In June, the average median size of chewed particles was 9.1 mm while the average proportions of particles smaller than 2 mm, and the soluble fractions were 135 and 316 g/kg bolus DM, respectively (Table 8). None of the particle-size parameters were affected by herbage, except the median size that tended (P=0.06) to be higher for both ryegrasses compared with TF and the proportion of particles retained on the 2 mm sieve that tended (P=0.09) to be lower. The median length of the major axis of the long particles (i.e., their length) was 1.05 cm, and the median length of the minor axis (i.e., the width) was 0.16 cm. The length of the major axis (length) of the long particles was lower (P<0.05) for WC compared with the three grasses (0.87 mm versus 1.11 mm) and higher (P<0.05) for ryegrasses compared with TF (1.19 mm versus 0.96 mm). The length of the minor axis (width) tended (P=0.05) to be higher for WC compared with the grasses. The ratio between the lengths of the major and the minor axes was clearly lower (P<0.01) for WC compared with the three grasses (5.12 versus 7.46, P<0.001) and higher (P<0.05) for ryegrasses compared with TF (8.16 versus 6.08). Table 7 Proportions of intracellular constituents (IC) released during ingestive mastication (in g/kg of individual IC intake) of the dairy cows fed grasses in May and June (common statistical model for both months) Treatmentsa

Neutral detergent soluble (g/kg IC intake)c Intracellular nitrogen (g/kg IC intake)c Chlorophyll (g/kg IC intake)c Water-soluble carbohydrates (g/kg IC intake)c a b c

Contrast analysesb

tPR

dPR

TF

PR vs. TF

TPR vs. dPR

S.E.

235 210 172 377

171 127 141 285

338 246 220 449

*** * 0.08 *

0.08 * ns ns

24.8 27.3 29.2 41.3

tPR: tetraploid perennial ryegrass, dPR: diploid perennial ryegrass, TF: tall fescue. *P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, S.E.: standard error of the means. Proportions of IC released were expressed as proportions of individual IC consumed.

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Table 8 Proportions of intracellular constituents (IC) released during during ingestive mastication (in g/kg of individual IC intake) of the dairy cows, size distribution and image analyses of the chewed particles for the four herbages in June Contrast analysesb

Treatmentsa WC

tPR

WC vs. Grass

PR vs. TF

tPR vs. dPR

S.E.

Proportion of IC released during ingestive mastication (g/kg IC intake)c Neutral detergent soluble 367 238 202 351 Intracellular nitrogen 385 229 161 255 Chlorophyll 148 106 122 166 Water-soluble carbohydrates 413 382 260 411

* ** ns ns

* ns ns ns

ns ns ns ns

39.4 40.6 30.1 62.2

Size distribution of the chewed particles Median size d50 (mm)d 8.4 d15–d85 (mm)e 31.7 Part. <2 mm (g/kg dry part.) 149 Soluble (g/kg dry boli) 300

7.4 29.2 149 320

ns ns ns ns

0.06 ns 0.09 ns

ns ns ns ns

1.20 7.47 12.2 32.3

282 0.96 0.16 6.08

* * 0.05 **

ns * ns *

ns ns ns ns

39.3 0.080 0.010 0.628

10.4 43.5 115 325

dPR

10.3 46.5 128 318

Image analysis of the particles retained on the 16 mm sieve Number of particles 211 312 396 0.87 1.16 1.21 Length of the major axisf (cm) 0.17 0.15 0.14 Length of the minor axisf (cm) Maj/Ming 5.12 7.54 8.77

TF

a

WC: white clover, tPR: tetraploid perennial ryegrass, dPR: diploid perennial ryegrass, TF: tall fescue. *P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, S.E.: standard error of the means. c The proportions of IC released were expressed as proportions of individual IC consumed. d The median size was defined as the d50 fractile. e The difference between the d15 and d85 fractiles was considered as an index of the particle size dispersion in ingestive boli. f The data presented are the medians. g Ratio between the length of the major axis and the length of the minor axis. b

3.4. Effect of the animal on chewing behaviour, release of IC during ingestion and size distribution of the chewed particles in June An effect of cow on the median size of the chewed particles occurred, with the median particle size of the particles in the boli collected from cows 6249 and 7511 being twice as high as particles collected from the four other cows (13.5 versus 7.0 mm, P<0.01, Table 9). This was consistent with results obtained for the proportions of particles passing the 2 mm sieve, and the length of the minor axis of the particle retained on the 16 mm sieve (data not shown). The frequency of jaw movements was also strongly dependent on cow (P<0.01). None of the other chewing behaviour parameters (i.e., intake rate, DM content of the boli, dry weight of the boli) showed any effect of cow. The length of the jaw occupied by the molars was shorter for cows 6249 and 7511 compared with the other four suggesting that the median size of the chewed particles was negatively correlated with the length of the jaw occupied by the molars. The median size of the chewed particles also tended (P=0.06) to be positively related to the intake rate (r = 0.632). The space between the canines and the premolars on the lower jaw was also longer in both these cows.

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Table 9 Effect of the cow on the proportions of IC released during mastication and size distribution of the chewed particles in June Cows

Live weight (kg) Age (months) Lengths on the inferior jaw (mm) Of the incisive arcade Of the space between canines and premolars Of the molars

P-value**

4012

6247

6249

6372

7507

7511

618 97

620 72

704 72

612 70

536 36

544 36

110 142

115 137

123 150

125 138

97 142

97 146

89

95

72

92

83

55

Size distribution of the chewed particles Median size d50 (mm)b 7.1

6.7

11.4

6.0

Proportion of IC released during ingestive mastication (g/kg IC intake) Neutral detergent soluble 295 283 307 218 Chewing behaviour Intake rate (g DM/min) Frequency of jaw movements (/min) DM content of boli (g/kg) Dry weight of boli (g) a b

8.1 333

15.5 302

** ns

S.E.a

1.47 48.3

79.2 71.7

65.2 74.3

78.1 64.3

58.4 68.9

71.0 81.8

69.7 90.0

ns **

8.18 3.46

10.3 13.0

10.8 13.2

10.3 14.3

9.7 13.8

10.1 14.1

10.8 13.8

ns ns

0.55 1.35

*P<0.05, **P<0.01, ***P<0.001, ns: P>0.05, S.E.: standard error of the means. The median size was defined as the d50 fractile.

Among the six cows used in the study, one was 8 years old at the beginning of the experiment, three were 6 years old and two were 4 years old. The two youngest cows were lighter than the four others (540 versus 639 kg), and the width of their incisives was smaller (97 versus 118 mm). However, neither the length of the lower jaw occupied by the molars nor the space between the canines and the premolars on the lower jaw differed. The frequency of jaw movements was higher compared to the older cows. Neither proportions of ICs released during ingestion, nor proportions of soluble fractions in the boli were affected by cow.

4. Discussion 4.1. Effects of herbage species or variety on proportions of ICs released during ingestive mastication and the mechanical properties of herbages Proportions of NDS, intracellular N, chlorophyll and WSC released during ingestive mastication in this experiment were in the range of the proportions observed by Doyle (1967), Mangan et al. (1976), Waghorn and Shelton (1988), Boudon et al. (2002a). The average proportions of NDS released were 50 and 280 g/kg under those observed by Boudon and Peyraud (2001) and John and Ulyatt (1987), respectively. Proportions of chlorophyll

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released were 140 g/kg under those observed by Reid et al. (1962) and Boudon and Peyraud (2001) and proportions of WSC released were 150 g/kg under that observed by Boudon and Peyraud (2001). However, the relative proportions of the individual ICs released were consistent with previous studies (Reid et al., 1962; Mangan et al., 1976; Boudon and Peyraud, 2001; Boudon et al., 2002a). Herbage species or variety affected release of most of the ICs measured, and proportions of NDS and intracellular N released during ingestive mastication were higher for white clover compared with grasses in June. Over all, proportions of NDS, intracellular N and WSC released were higher for fescue versus ryegrasses, while the proportions of intracellular N released were lower for diploid compared with tetraploid ryegrass. Such an effect of plant species or variety on release of ICs has been rarely observed by others, at least in vivo and among the plant species most commonly used for grazing. It has been shown that release of chlorophyll or protein was lower in birdsfoot trefoil or sainfoin versus other species of legumes (Mangan et al., 1976; Lees et al., 1981; Howarth et al., 1982) or compared to perennial ryegrass (Mangan et al., 1976). However, these effects were related to the high tannin content of both species. Moreover, Mangan et al. (1976) did not observe any difference in the proportions of total N, proteins, Rubisco, chlorophyll or potassium released among lucerne, perennial ryegrass and red clover. Similarly, Howarth et al. (1982) failed to observe differences in the in vitro release of chlorophyll from leaves of lucerne, white clover or red clover. For that reason, there have been no investigations on mechanisms to explain effects of plant species, or variety, on release of ICs, except for the mechanisms linked to the tannin content of the plant. The values, and the range, among species in shear energy and shear tenacity in this experiment are consistent with previous studies. In the present study, the energy necessary to cut a leaf of diploid ryegrass perpendicularly to its main axis was estimated as 1.81 mJ. This is slightly higher than values reported by Inou´e et al. (1994), which ranged between 0.8 and 1.4 mJ, and Henry et al. (1997), which ranged between 0.3 and 0.8 mJ, but falls within the range of 1.4–2.2 mJ observed by MacKinnon et al. (1988). However, our values are much lower than results obtained by Wright and Illius (1995) for Festuca ovina L., of 2.6–13.9 mJ. These differences are probably largely due to the type of apparatus used on the texture analyser, which can be a guillotine (Henry et al., 1997), scissors (Wright and Illius, 1995) or a Warner–Bratzler device (Inou´e et al., 1994; MacKinnon et al., 1988), as well as to the angle and sharpness of the blades (Henry et al., 1996). As far as we know, only Henry et al. (1996, 1997) directly compared the shear properties of white clover, perennial ryegrass and fescue. Henry et al. (1997) observed that the shear tenacity of white clover folioles, (i.e., the ratio between the energy necessary to cut a leaf and the cross-sectional area of the leaf), was more than 100 times lower than the value for laminae of fescue or perennial ryegrass. However, they did not observe any systematic difference between the shear tenacity of laminae of perennial ryegrass and fescue (Henry et al., 1996, 1997). In our experiment, the difference between the white clover and the grasses was much lower, since we did not precisely measure the cross-sectional area of the organs cut and thus only obtained the index of shear tenacity and so calculated a mean index of shear tenacity integrating all organs. As there was no strong effect of herbage on chewing behaviour, most of the observed effect of herbage on the proportion of ICs released may be attributable to the histology of

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the species or variety used. However, it is possible that the absence of effects of herbages on chewing behaviour came from the large variability of the chewing behaviour among cows and the low number of cows in the study. For instance, the difference of 7.5 jaw movements per minute between WC and the ryegrasses in June could have been significant (i.e., P<0.05) if 11, instead of 6, cows had been used. 4.2. The higher release of some ICs from white clover compared with grasses was associated with a weaker plant tissue The higher release of NDS and intracellular N from white clover versus grasses in the present study was associated with the plant tissue having a lower index of shear tenacity. The lower shear tenacity of white clover, compared with grasses, is well known and demonstrated (Henry et al., 1997). Many other studies concluded that plant tissue is weaker in white clover versus grasses. Indeed, white clover contains much higher amounts of cell contents, and less cell wall, compared with ryegrass or fescue (Butler and Bailey, 1973; Jarrige et al., 1995). Moreover, the tissue of white clover contains a higher proportion of thin-walled cells (i.e., mesophyll or parenchyma) than either ryegrass or fescue (Reznavi Moghaddam and Wilman, 1998; Wilman and Reznavi Moghaddam, 1998). This lower shear tenacity of the cell walls may account for the finding that, under compression by teeth, more cells may have been ruptured and their membranes destroyed from white clover compared to grasses in the present study, even if the tissue was not more extensively sheared and the particle size was not more reduced. Along with lower mechanical resistance of the tissues, it may also be that membranes of white clover are less stable than in ryegrass. This could be inferred from the higher release of soluble proteins during in vitro incubations of white clover folioles for 2 h with no micro-organisms (Kingston-Smith et al., 2003), compared with incubations of ryegrass laminae under the same conditions (Beha et al., 2002). Given that the protease activity of the plant was just starting after 2 h of incubation, this disappearance of soluble protein could have been mainly due to leaching from the plant cells when their membranes were broken. However, the ICs most readily released from white clover, versus grasses, in the present study, were mainly those contained in chloroplasts or in high molecular weight molecules. These ICs require cell walls to be ruptured for their release (Boudon and Peyraud, 2001). Thus, the lower shear tenacity index of the white clover appears to be the most suitable explanation for the higher release of ICs from this legume compared with grasses. 4.3. The plant cell size may be a better synthetic criterion than the shear tenacity index to explain the variations in the proportions of ICs released from the grasses The shear tenacity index was not a good synthetic criterion to explain variation in the proportions of NDS and WSC released from the grasses used in this study. Indeed, proportions of NDS and WSC released were lower for fescue compared with ryegrasses in both May and June. While this result could be associated with a lower mean shear tenacity index and a lower proportion of stems in fescue compared with ryegrasses in June, this was not the case in May when fescue and ryegrasses were vegetative. Indeed, in our experiment, as observed previously (Henry et al., 1997), no differences in the shear tenacity index for the

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vegetative organs were observed between ryegrasses and tall fescue. Previous studies have generally failed to show any clear relationship between the shear properties of plant tissues and proportions of ICs released during ingestive mastication. In cases where such relationships were observed, proportions of ICs released were either weakly negatively correlated with shear tenacity of ryegrass at different stages of maturity (Boudon and Peyraud, 2001; Boudon et al., 2002a) or positively correlated with shear tenacity of red clover at different stages of maturity (Bryant, 1964). The size of the plant cells may be more relevant in explaining the higher release of NDS and WSC from fescue compared with grasses, and tetraploid compared with diploid ryegrass. Fescue can be distinguished from ryegrass, whatever the plant tissue considered, by having a higher cell volume than perennial ryegrass (Reznavi Moghaddam and Wilman, 1998). In the same way, the length of the cells seems to be higher in the tetraploid varieties of ryegrass versus the diploid varieties (Wilkins and Sabanci, 1990; Sugiyama et al., 2002). This means that, for a given shear length, and assuming the shear cuts across all cells, the volume of the cells sheared (hence the quantity of cell contents released) could be higher for fescue compared with ryegrass, and for tetraploid compared with diploid ryegrass. This hypothesis cannot be extrapolated to white clover since this species contains small cells (Reznavi Moghaddam and Wilman, 1998). However, as stated above, the weaker tissue, and possibly the lower stability of the membrane, could explain the higher release of NDS and intracellular N in white clover compared with grasses. 4.4. Strong effect of cow and absence of correlation between comminution of particles and proportions of ICs released during ingestive mastication It is noteworthy that ‘cow’ had a strong effect on the particle-size distribution in the boli without producing any impact on proportions of ICs released, whatever the IC considered. This implies that, even if compression of plant tissue between the teeth during chewing could rupture plant cells, it may not necessarily lead to shearing and reduction of particle size. Similarly, compression of plant tissue between the teeth could induce shearing of the tissue without rupturing plant cells. It is known from direct observation of the parenchyme of apples and potatoes that cells can be fractured by compression even though the tissue is not sheared (Vincent, 1990). Moreover, even when tissue is sheared, cells may not be ruptured depending on whether the cracks propagate between or across the cells (Vincent, 1990; Donald et al., 2003) and the actual resistance of the plant cells (Lees et al., 1981). Many authors have reported a strong effect of animal on the particle-size distribution in the boli among groups of adult ruminants of the same species (Gill et al., 1966; Lee and Pearce, 1984; Nelson, 1988; Luginbuhl et al., 1989; Perez-Barberia and Gordon, 1998). In our study, the cows producing the least reduction in particle size while chewing were those with a shorter molar length and thus possibly having a lower occlusion surface. This suggests that the occlusion surface-area was a determining factor in efficiency of ingestive mastication for comminution of particles in our experiment, as stated by Perez-Barberia and Gordon (1998). The cows with a presumed small occlusion surface did not try to compensate by increasing frequency of jaw movements, as shown by Perez-Barberia and Gordon (1998). Frequency of jaw movements was the only chewing behaviour parameter affected by cows, but its variation was unrelated to changes in the particle-size distribution of particles.

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We were unable to link the variability of the particle size in the boli to the age or body weight of the cows, even though some authors have observed a strong positive effect of body size on chewing behaviour (Bae et al., 1983), while others have noted an effect of age on efficiency of chewing (Gill et al., 1966; Perez-Barberia and Gordon, 1998). This may be because the group of cows in the present study was relatively homogenous in age and body weight, compared with the cited studies.

5. Conclusions The species or variety of grazed herbage can affect the proportions of cell contents released during ingestive mastication, even though only a low variation occurred in the chewing behaviour, and the proportion of cell contents released during ingestion is not linked to comminution of particles among different species or variety of herbage. In addition, shear tenacity of the plant tissue may not be a good synthetic parameter to predict the availability of plant cell nutrients (i.e., the comminution of particles or the release of cell contents). Consequences of these findings on the digestion process remain to be assessed. However, results illustrate the complexity of the interactions between the structures of plant tissue and the physical damage to the particles during chewing, demonstrating the difficulty in understanding kinetics of availability of nutrients to ruminal micro-organisms.

Acknowledgements The authors are very grateful to P. Lamberton and the farm staff for their involvement in cow welfare, feeding and sampling, to L. Finot, N. Huchet, T. Lemou¨el, M. Texier for chemical analyses and the physical measurements, to R. Delagarde for a review of the text and to M. Carpenter for post-edition of the English style.

References Association Franc¸aise de Normalisation, 1997. Aliments des animaux—Dosage de l’azote – M´ethode par combustion (DUMAS) – NF V18-120. AFNOR Editions, Saint-Denis La Plaine, France. Bae, D.B., Welch, J.G., Gilman, B.E., 1983. Mastication and rumination in relation to body size of cattle. J. Dairy Sci. 66, 2137–2141. Beha, E.M., Theodorou, M.K., Thomas, B.J., Kingston-Smith, A.H., 2002. Grass cells ingested by ruminants undergo autolysis which differs from senescence: implications for grass breeding targets and livestock production. Plant Cell Environ. 25, 1299–1312. Boudon, A., Mayne, C.S., Peyraud, J.L., Laidlaw, A.S., 2002a. Effects of stage of maturity and chop length on the release of cell contents of fresh ryegrass (Lolium perenne L) during ingestive mastication in steers fed indoors. Anim. Res. 51, 349–365. Boudon, A., Peyraud, J.-L., 2001. The release of intracellular constituents from fresh ryegrass (Lolium perenne) during ingestive mastication in dairy cows: effect of intracellular constituent, season and stage of maturity. Anim. Feed Sci. Technol. 93, 229–245. Boudon, A., Peyraud, J.-L., Faverdin, P., 2002b. The release of cell contents of fresh grass (Lolium perenne) during digestion in dairy cows: effect of intracellular constituents, season and stage of maturity. Anim. Feed Sci. Technol. 97, 83–102.

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