Effects of condensed tannins in white clover flowers on their digestion in vitro

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Animal Feed Science and Technology 142 (2008) 44–58

Effects of condensed tannins in white clover flowers on their digestion in vitro V. Burggraaf a,b,∗ , G. Waghorn b , S. Woodward b , E. Thom b a

AgResearch Limited, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand b Dexcel Limited, Private Bag 3221, Hamilton, New Zealand

Received 17 July 2006; received in revised form 24 June 2007; accepted 3 July 2007

Abstract Protein in white clover (Trifolium repens L.) is poorly utilised by ruminants because of its extensive degradation to ammonia in the rumen. However, white clover produces condensed tannins (CT) in its flowers, which can reduce rumen proteolysis. Effects of increasing proportions of clover dry matter (DM) as flowers (and therefore floral CT) on soluble protein, ammonia and volatile fatty acid (VFA) concentrations were determined with in vitro incubations. Minced mixtures of 0, 250, 500, 750 and 1000 g/kg of DM as white clover flower (F) with the remainder as white clover leaf, were incubated in vitro and sampled after 0, 2, 4, 8, 12 and 24 h. Treatments contained 0, 13, 26, 39 and 52 g CT/kg DM, respectively. A further treatment with 500 g/kg DM as flower and 500 g/kg DM as leaf had polyethylene glycol added to remove effects of CT. Increasing the proportion of white clover as flowers from 0 to 1000 g/kg DM reduced net conversion of plant N to ammonia N from 290 to 120 mM/M at least partly due to reduced solubility of the protein. Treatments with 750 g/kg DM or more as clover flowers reduced ammonia concentrations to levels likely to limit microbial growth. Total VFA production was not affected by flower content, although the proportion of acetate

Abbreviations: ADF, acid detergent fibre; A:P, acetate to propionate ratio; CHO, soluble carbohydrate; CP, crude protein; CT, condensed tannin; DM, dry matter; F, flower; ME, metabolisable energy; aNDF, neutral detergent fibre; NFC, non-fibre carbohydrate; OM, organic matter; OMD, OM digestibility; PEG, polyethylene glycol; SED, standard error of the difference; VFA, volatile fatty acid ∗ Corresponding author at: AgResearch Limited, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand. Tel.: +64 7 838 5806; fax: +64 7 838 5117. E-mail address: [email protected] (V. Burggraaf). 0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2007.07.001

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to propionate increased. The contribution of CT to treatment effects was small compared to effects attributed to difference in chemical composition between flowers and leaves. © 2007 Elsevier B.V. All rights reserved. Keywords: In vitro digestion; Nutritive value; Rumen; Trifolium repens

1. Introduction Increasing the proportion of white clover (Trifolium repens L.) in temperate pastures normally increases milk production of dairy cows (Harris et al., 1997). However, the practical value of high proportions of white clover in pastures is limited by the incidence of bloat, and also because of excessive protein degradation to ammonia, resulting in large ruminal losses and inefficient utilisation of dietary protein (Cohen, 2001). Dietary condensed tannins (CT) precipitate proteins in the rumen, prevent bloat and reduce protein degradation. CT bind to plant proteins when released from plant cells, lowering their solubility and ammonia concentrations in rumen fluid (Barry et al., 1986; Chiquette et al., 1989) and increasing the quantity of non-ammonia N and amino acids reaching the small intestine (Waghorn et al., 1987). This can lead to substantial improvement in milk production of ruminants fed legumes containing 20–40 g/kg dietary DM as CT (Wang et al., 1996; Woodward et al., 2000), although animal responses vary with type and concentrations of CT (Min et al., 2003). White clover forage consists mainly of leaves, with varying proportions of flowers which contain CT. A white clover variety has recently been selected for increased flowering in order to increase the CT concentration in clover forage (i.e. HT clover), although CT concentrations only reached a low maximum of 12 g/kg DM (Burggraaf et al., 2003). Dairy cows grazing HT clover had lower rumen ammonia concentrations than cows grazing Grasslands Huia white clover, but milk yields were similar (Burggraaf et al., 2004). In order for plant breeders to optimise the CT content of white clover through flowering, effects on digestion and animal responses to a range of floral CT contents needs to be determined. This experiment measured effects of increasing the proportion of clover flowers from 0 to 1000 g/kg of DM, relative to clover leaves, on plant protein degradation and production of volatile fatty acids (VFA), using in vitro incubations. White clover leaves and flowers were minced to resemble chewing, and incubated with buffered rumen fluid to simulate rumen digestion. Products of digestion were measured at intervals over a 24 h incubation period. Specific effects of CT were assessed by including polyethylene glycol (PEG) in one treatment, to bind with, and inactivate, the CT (Jones and Mangan, 1977).

2. Materials and methods 2.1. Experimental design Treatments comprised five proportions of white clover leaf:flower and one including PEG to remove the effects of CT. Samples of each mixture (18: see below) were incubated,

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and three replicates were removed for sampling after 0, 2, 4, 8, 12 and 24 h. White clover bred for increased floral condensed tannin (HT clover; Burggraaf et al., 2003) grown in pure swards at the Dexcel Lye Farm (latitude 37◦ 47 S, 175◦ 19 E, altitude 40 m above sea level) was used for all treatments as follows (DM basis): 1. 2. 3. 4. 5. 6.

0 g/kg DM as flower, 1000 g/kg DM as leaf (0F); 250 g/kg DM as flower, 750 g/kg DM as leaf (250F); 500 g/kg DM as flower, 500 g/kg DM as leaf (500F); 750 g/kg DM as flower, 250 g/kg DM as leaf (750F); 1000 g/kg DM as flower, 0 g/kg DM as leaf (1000F); 500 g/kg DM as flower, 500 g/kg DM as leaf + PEG (500F + PEG).

2.2. Collection and mincing of clover Approximately 800 g of clover leaves (i.e., leaflet plus petiole) and clover flowers (i.e., flower head plus stem) were harvested between 13:00 and 14:00 h in January 2003. The clover was 10–15 cm long and was cut to between 1 and 2 cm above ground level before immediate placement in a plastic bag on ice and transferral to a −18 ◦ C freezer. Plant material was maintained below 0 ◦ C throughout the preparation phase until placement in the incubator. Two days after collection, the clover was chopped into 2–3 cm lengths. Samples (60 g) of chopped clover flowers and chopped clover leaves were dried for 24 h at 95 ◦ C to determine DM content. Chopped clover components were mixed in appropriate treatment ratios. About 200 g of herbage for each treatment mixture was minced in a Kreft Compact meat mincer R70 (Kreft, GmbH, Gevelsberg, Germany) with 12 mm diameter holes in the sieve plate. Mincer components were placed in a freezer at −18 ◦ C prior to mincing to ensure the clover remained frozen while mincing, enabling the forage to be macerated, rather than squeezed, and preventing excessive cell wall rupture during mincing. This process is described by Barrell et al. (2000), who reported mincing to be the most effective method of preparing forages for in vitro incubations, as it results in a particle size distribution of DM similar to that of chewed forage (Waghorn et al., 1989). Minced material was stored frozen (−18 ◦ C) until incubation the following day. 2.3. Feed analysis A 50 g sub-sample of fresh clover leaves was dissected into leaflets and petioles and the same quantity of fresh clover flowers was dissected into flower heads and stems. Samples were dried at 95 ◦ C to determine their contribution to leaf and flower DM. A sample of minced clover flowers was freeze-dried and ground through a 1 mm sieve. Free, protein bound, and fibre bound CT concentrations were determined using the butanol–HCl colorimetric technique (Terrill et al., 1992a) with a lotus major (Lotus pedunculatus Cav.) standard. The CT concentration of each treatment was calculated by multiplying the proportion of clover flowers by their CT concentration. Minced leaves and flowers were oven dried at 60 ◦ C, ground through a 1 mm sieve and analysed for crude protein (CP), lipid, soluble carbohydrate (CHO), acid detergent

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fibre (ADF), neutral detergent fibre (aNDF), organic matter (OM) digestibility (OMD), and metabolisable energy (ME) content by near infrared spectroscopy (NIRS; Corson et al., 1999). Neutral detergent fibre was analysed with addition of a heat stable amylase and without sodium sulphite. The aNDF and ADF (acid detergent fibre) contents were expressed inclusive of residual ash. The composition of each treatment was calculated from the proportion of flower and leaf used in the mixtures. For each treatment, a sub-sample (15 g wet weight) of minced forage was used to measure particle size distribution by wet-sieving (Turner & Newall Ltd.) as described by Waghorn (1986). 2.4. Incubation Minced herbage samples were weighed into 50 ml Schott bottles with vented caps (0.5 g DM/bottle). Bottles were warmed and flushed with carbon dioxide to remove oxygen, and 12 ml of McDougall’s buffer (McDougall, 1948) and 0.5 ml of cysteine sulphide reducing agent were added before being capped and placed in an incubator at 39 ◦ C. The incubator was fitted with an orbital shaker, set at 90 oscillations/min. For the 500F + PEG treatment, a separate buffer containing 2 g/l PEG was used. The PEG had a molecular weight of 3350 and the amount added to each bottle was twice the weight of the CT, to ensure that all CT could bind with PEG (Barry and Forss, 1983). Once all bottles were in the incubator, 3 ml of rumen fluid was added to each. Rumen fluid was collected from three rumen fistulated dairy cows approximately 2 h after the start of morning feeding on mixed pastures of perennial ryegrass (Lolium perenne L.) and white clover. The fluid was filtered through two layers of cheese cloth and immediately transferred to a pre-warmed carbon dioxide gassed flask. The pH of the filtered fluid was measured and sub-samples collected for determination of ammonia, soluble protein and VFA concentrations. The rumen fluid inocula pH was 6.03 and it contained 387 mg/l soluble protein, 12.7 mM/l ammonia, 88 mM/l acetate, 23 mM/l propionate, 15 mM/l butyrate and 5 mM/l minor VFA (i.e. valerate, isovalerate, isobutyrate). 2.5. Incubation medium characteristics When the bottles were removed from the incubator, the pH was measured (Ecoscan series pH 5, Eutech Instruments Ltd., Singapore) and samples collected for ammonia, soluble protein and VFA analysis. Samples were centrifuged (HERMLE Z160M microlite centrifuge, Wehingen, Germany) for 15 min at 12,000 × g at 20 ◦ C and supernatant was either acidified (15 ␮l concentrated hydrochloric acid/ml) and frozen (−18 ◦ C) for later ammonia analysis, frozen (−18 ◦ C) for VFA determination or held at 4 ◦ C for soluble protein analysis measurement within 24 h of collection. Soluble protein concentrations were analysed using the colorimetric procedure of Bradford (1976) with a bovine serum albumin standard. VFA analysis was by gas chromatography (Playne, 1985) and ammonia by the procedure of Neely and Phillipson (1988). Net ammonia and VFA production were calculated after subtracting the 0 h concentration from values at 2, 4, 8, 12 and 24 h of incubation within treatments. Data are expressed in terms of plant N or DM incubated and plotted over time. Calculations involving sol-

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uble protein were adjusted to remove the contribution of soluble protein from the rumen inocula. 2.6. Statistical analysis Data were subjected to analysis of variance using the model for completely randomised experimental designs in Genstat 5 (Genstat 5 Committee, 1997). The standard error of the difference (SED) accounted for the variation in the 0 h samples as well as variation at each time period. Differences were treated as significant at P<0.05. Comparisons were made among treatments for each incubation period and among incubation times. Linear, quadratic and cubic regression analyses were performed on all data using Genstat 5 to define the response to increasing proportions of clover flower relative to leaf at each incubation time.

3. Results 3.1. Feed analysis Leaves comprised 710 g leaflet and 290 g petiole/kg DM, with flower head and stems accounting for 660 and 340 g per kg of flower DM, respectively. Minced clover flowers had 52.4 g CT/kg DM, of which 0.50 was free, 0.44 was bound to protein, and 0.06 was bound to fibre (Table 1). Particles larger than 2 mm accounted for 0.22 of minced flowers compared to 0.06 for minced leaf (Table 2). 3.2. Soluble protein At 0 h of incubation, soluble protein concentrations were highest for the 0F treatment (446 mg/l) and declined linearly as the flower content increased, to 188 mg/l for 1000F (Table 3). There was a linear relationship (P<0.001) between the clover leaf content and soluble protein concentrations at all times except at 2 h, when the relationship was a decline at a decreasing rate (Q:P = 0.005).

Table 1 Dry matter (g/kg) and chemical composition (g/kg DM) of minced white clover mixtures with different proportions (g/kg DM) of flower (F) and leaf Treatment

DM

CP

aNDF

ADF

Lipid

Ash

NFC

CT

0F 250F 500F 750F 1000F

168 173 182 189 196

239 218 197 176 155

240 245 249 254 258

205 218 231 243 256

41 39 37 34 32

139 131 123 115 107

341 354 368 382 396

0.0 13.1 26.2 39.3 52.4

Values represent duplicate assays of single samples. DM, dry matter; CP, crude protein; aNDF, neutral detergent fibre; ADF, acid detergent fibre; NFC, non-fibre carbohydrate; CT, condensed tannins.

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Table 2 Particle size distribution (g/kg DM retained on sieves) of minced white clover mixtures with different proportions (g/kg DM) of flower (F) and leaf Treatment

0F 250F 500F 750F 1000F

Particle size >4 mm

2–4 mm

1–2 mm

0.5–1.0 mm

0.25–1.0 mm

Residuea

Soluble

10 62 59 71 94

51 119 69 69 131

133 150 112 153 154

153 150 176 142 125

230 135 188 162 148

92 63 103 82 77

331 320 294 320 272

Values represent duplicate assays of single samples. a Residue is the non-soluble material that passed through a 0.25 mm sieve. Table 3 Net soluble protein and ammonia concentration after 0, 2, 4, 8, 12 and 24 h of in vitro incubation of white clover herbage with different proportions (g/kg DM) of flower (F) and leaf, with PEG included in one treatment to remove effects of condensed tannins. Treatment

SEDtime a Ptime b

Incubation time (h) 0

Soluble protein (mg/l) 0F 446 250F 379 500F 315 500F + PEG 290 750F 216 1000F 188 0.9 SEDtrt d <0.001 Plinear e Pquadratic f 0.586 PCT g 0.366 Ammonia (mM/l) 0F 25F 50F 50F + PEG 75F 100F SEDtrt d Plinear e Pquadratic f PCT g

0.00 0.00 0.00 0.00 0.00 0.00

2

4

8

12

24

316 247 219 273 201 191 0.6 <0.001 0.005 0.002

236 253 220 255 211 190 0.4 <0.001 0.086 0.006

226 223 216 218 212 210 0.3 0.028 0.772 0.726

229 264 246 252 231 209 0.6 0.024 0.005 0.642

303 14.8 296 293 295 265 254 0.4 <0.001 0.083 0.789

5.71 5.96 4.86 3.92 4.11 3.29 0.256 <0.001 0.021 0.003

6.55 5.91 4.85 4.24 3.70 2.85 0.455 <0.001 0.688 0.204

5.41 3.46 2.14 2.01 -0.69 -2.07 0.755 <0.001 0.853 0.864

9.18 7.70 2.61 3.79 0.74 -1.07 0.344 <0.001 0.016 0.005

21.81 16.40 12.82 13.40 6.86 7.01 0.776 <0.001 0.001 0.471

0.559

Ptreatment × time c

<0.001

<0.001

<0.001

<0.001

Values represent triplicate assays of three samples. a Standard error of the difference between different sampling times. b Probability value assessing the difference between sampling times. c Probability value assessing the interaction between treatment and sampling time. d Standard error of the difference between treatments at individual sampling times. e Probability value assessing the linear relationship between treatments at individual sampling times. f Probability value assessing the quadratic relationship between treatments for individual sampling times. g Probability value assessing the difference between 500F and 500F + PEG.

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Fig. 1. Proportion (g/kg) of plant CP in the soluble fraction of in vitro incubations of white clover herbage with different proportions (g/kg DM) of flower (F) and leaf, including one treatment containing PEG. (Error bars represent SED’s.)

Soluble protein concentrations in 0F, 250F and 500F declined rapidly over the first 2 h of incubation, and there were treatment differences at all times except 8 h, when values ranged from 210 to 226 mg/l (Table 3). Concentrations of soluble protein in 750F and 1000F gradually increased with incubation time, but at 24 h were still lower (P<0.001) than other treatments (Table 3). The CT in 500F only reduced soluble protein concentrations by 20% at 2 h and by 14% at 4 h of incubation relative to 500F + PEG. 500F + PEG had soluble protein concentrations intermediate to that of 250F and 0F at 2 h and similar to 250F at 4 h, after which concentrations were unaffected by CT (Table 3). Soluble protein in the incubation medium was always less than 70 g/kg of forage crude protein (Fig. 1). At 0 h, values were highest for treatments with a high leaf content, but from 8 to 24 h values were highest for treatments with a high flower content (Fig. 1). The concentration of soluble protein relative to plant crude protein was reduced by CT in the 500F treatment; by 26% at 2 h (P<0.001), and 17% at 4 h (P<0.01) of incubation (Fig. 1). 3.3. Ammonia Ammonia concentration in the incubation medium represents ammonia produced from CP degradation that is not incorporated into microbial protein. All treatments had net production during the first 2 h, and a decline in concentrations between 4 and 8 h, after which ammonia concentrations increased (Table 3). There was a linear relationship between net ammonia concentration and proportion of clover leaf at all incubation times (P<0.001). CT in the 500F treatment had little effect on ammonia concentration (Table 3). Net conversion of plant N to ammonia N (Fig. 2) followed the same trend as ammonia concentrations, with increasing proportions of flower decreasing the amount of plant N appearing in ammonia. Treatments with more than 500 g flowers/kg DM had very low ammonia concentrations at 8 and 12 h, suggesting a greater utilisation versus production by

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Fig. 2. Net conversion of plant-N to ammonia-N from in vitro incubations of white clover herbage with different proportions (g/kg DM) of flower (F) and leaf, including one treatment containing PEG. (Error bars represent SED’s.)

microbes. Net ammonia released by 24 h of incubation ranged from 120 to 290 mM/M of forage N, with highest values for the 0F treatment, but CT had little effect on net conversion of plant N to ammonia N in the 500F treatment (Fig. 2). 3.4. Volatile fatty acids Initial pH of all incubations was 7.1, declining to values of 5.8–6.1 over the first 12 h of incubation. All treatments maintained a pH over 5.7 for the 24 h of incubation, with no treatment effects. VFA production was rapid during the first 8 h of incubation, but concentrations (Fig. 3) were not affected by treatment in the first 12 h. At 24 h, VFA concentrations decreased

Fig. 3. Concentration of VFA after 2, 4, 8, 12 and 24 h from in vitro incubation of white clover herbage with different proportions (g/kg DM) of flower (F) and leaf, with PEG included in one treatment to remove effects of condensed tannins. (Error bars represent SED’s.)

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Fig. 4. Ratio of acetate to propionate after 2, 4, 8, 12 and 24 h of in vitro incubation of white clover herbage with different proportions (g/kg DM) of flower (F) and leaf, with PEG included in one treatment to remove effects of condensed tannins. (Error bars represent SED’s.)

linearly (P<0.05) as flower contents increased, with a range of 4.6–5.4 mM VFA/g DM. The concentration of acetate followed the same trend as total VFA, with maximum rates of production in the first 2 h, and a maximum yield of 3 mM/g DM at 24 h. There were no treatment effects on acetate concentrations. Propionate production was most rapid between 2 and 4 h of incubation, with a negative linear relationship between propionate concentration and flower content at each time (Table 4). Butyrate production was highest between 2 and 4 h for 0F, and between 4 and 8 h for other treatments (Table 4). Increasing flower contents caused a linear decrease in butyrate concentrations at each sampling time (P<0.001; Table 4). Concentrations of minor VFA differed after the first 2 h, decreasing linearly (quadratically at 8 h; P<0.001) with increasing clover flower contents (Table 4; P<0.001). The CT in the 500F treatment did not affect total VFA (Fig. 3) or acetate concentration, but it reduced propionate concentrations by 12% at 12 h and 17% at 24 h (P<0.01; Table 4). CT also increased butyrate concentration at 8 (P<0.01) and 12 h (P<0.05), and reduced concentrations of minor VFA by 27% at 12 h (P<0.05), and 16% at 24 h (Table 4). Acetate to propionate (A:P) ratios (Fig. 4) increased with increasing flower contents. At 4 h of incubation, A:P ratios ranged from 2.3 to 3.5, except when PEG was added to remove the CT effect of the 500F treatment, which resulted in a A:P ratio of 4.4 (P<0.001 for CT effect).

4. Discussion This research has defined effects of white clover leaf and flower, and combinations of each, as well as effects of the floral CT on fermentation end products. This information will aid clover breeders in setting targets for CT production and flowering in white clover.

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Table 4 Propionate, butyrate and minor (valerate, isovalerate, isobutyrate) volatile fatty acid (VFA) production after 2, 4, 8, 12 and 24 h of in vitro incubation of white clover herbage with different proportions (g/kg DM) of flower (F) and leaf, with PEG included in one treatment to remove effects of condensed tannins Treatment

Incubation time (h) 2

4

8

12

SEDtime a

Ptreatment × time b

24

Propionate (mM/g DM) 0F 0.18 25F 0.21 50F 0.15 50F + PEG 0.15 75F 0.14 100F 0.15 0.024 SEDtrt c 0.025 Plinear d 0.798 Pquadratic e 0.973 PCT f

0.65 0.49 0.49 0.38 0.44 0.41 0.061 0.003 0.213 0.089

0.90 0.86 0.76 0.83 0.76 0.69 0.039 <0.001 0.782 0.121

1.01 0.93 0.91 1.03 0.89 0.86 0.039 0.002 0.329 0.009

1.13 1.19 0.99 1.19 1.02 0.84 0.057 0.001 0.152 0.004

0.044

<0.001

Butyrate (mM/g DM) 0F 0.10 25F 0.11 50F 0.08 50F + PEG 0.06 75F 0.08 100F 0.07 0.009 SEDtrt c 0.104 Plinear d 0.862 Pquadratic e 0.140 PCT f

0.29 0.21 0.19 0.17 0.17 0.14 0.016 <0.001 0.029 0.296

0.60 0.57 0.50 0.43 0.43 0.35 0.018 <0.001 0.052 0.001

0.69 0.70 0.63 0.55 0.50 0.43 0.038 <0.001 0.066 0.046

0.84 0.79 0.75 0.67 0.59 0.55 0.050 <0.001 0.467 0.170

0.030

<0.001

Minor VFA (mM/g DM) 0F 0.03 25F 0.03 50F 0.03 50F + PEG 0.02 75F 0.02 100F 0.02 SEDtrt c 0.008 0.104 Plinear d Pquadratic e 0.598 0.735 PCT f

0.08 0.06 0.05 0.03 0.04 0.02 0.012 <0.001 0.780 0.158

0.13 0.09 0.07 0.08 0.06 0.05 0.008 <0.001 0.001 0.144

0.16 0.15 0.08 0.11 0.08 0.07 0.009 <0.001 0.034 0.032

0.27 0.23 0.16 0.19 0.13 0.11 0.008 <0.001 0.003 0.001

0.009

<0.001

Values represent triplicate assays of three samples. a Standard error of the difference between different sampling times. b Probability value assessing the interaction between treatment and sampling time. c Standard error of the difference between treatments at individual sampling times. d Probability value assessing the linear relationship between treatments at individual sampling times. e Probability value assessing the quadratic relationship between treatments for individual sampling times. f Probability value assessing the difference between 500F and 500F + PEG.

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Mincing of herbage before incubation was intended to mimic chewing by releasing cell contents and reducing particle sizes, which were similar to those reported previously for minced white clover, white clover from sheep boli, and the rumen contents of cows fed fresh lucerne (Medicago sativa L.) (Waghorn and Shelton, 1988; Waghorn et al., 1989; Burke, 2004). Clover flower particles were larger than that for leaves, probably because some florets passed through the mincer undamaged. This is also likely to occur in vivo. 4.1. Clover composition The composition of clover leaf and flowers was similar to previous reports (Wilman and Altimimi, 1984; Stockdale, 1999). The CT concentrations in herbage comprising 500 g/kg DM or more as flowers were similar to that in sulla (Hedysarum coronarium L.) and lotus species, which have been shown to affect digestion and animal performance (Min et al., 2003). Availability of free CT from flowers indicates good potential for binding with leaf proteins. 4.2. Protein digestion Soluble protein and ammonia concentrations indicate the balance between release by mincing and utilisation of readily fermentable N by microflora. The low proportion of white clover leaf CP in the soluble fraction at 0 h, compared to previous in sacco measurements (90–380 g/kg of total white clover leaf CP; Cohen and Doyle, 2001; Burke, 2004) is probably a function of measurement techniques. In our study measured soluble protein was truly dissolved, whereas in sacco studies determined soluble CP by washing plant material in bags with 35 ␮m pore apertures, thereby enabling organelles such as chloroplasts, which account for 750 g/kg of total leaf protein (Mangan, 1982), to also pass out of the bag. The lower proportion of soluble protein with increasing proportions of DM as flowers may be a result of less rupture of flower cells. Soluble protein is readily degraded by rumen bacteria and plant enzymes (Kingston-Smith et al., 2003) unless it reacts with condensed tannins (McNabb et al., 1996). The absence of a CT effect on the proportion of soluble protein (Fig. 2) at 0 h may have been due to a high pH (7.1) limiting stable CT–protein complex formation (Jones and Mangan, 1977) and because of the brief period (90 min) between thawing and sampling (Diaz-Hernandez et al., 1997). However, at 2 and 4 h the PEG treatment did have higher soluble protein concentrations than 500F. A high concentration of ammonia, and the presence of ‘minor’ VFA, are indicators of protein degradation, but interpretation is affected by ammonia utilisation for microbial growth and the CP concentration in plant material. At 24 h, 290 mM/M of clover leaf N was recovered as ammonia N (Fig. 3b), which is similar to reports of 210–400 mM/M by Caradus et al. (1995), but less than the 490 mM/M at 24 h by Barrell et al. (2000) and Burke (2004). Substitution of leaf by flower reduced soluble protein and ammonia concentrations and the net conversion of plant N to ammonia (Fig. 2). Satter and Slyter (1974) suggested a minimum ammonia N concentration of 3.6 mM/l) to maximise rumen bacterial growth, so

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when clover flowers exceeded 500 g/kg DM bacterial growth may have been limited at 8–12 h. However, such high flower contents are not likely to occur in pastures. The CT in 500F had little effect on protein digestion suggesting effects of flowers were due to composition and structure. CT are able to reduce proteolytic enzyme activity and proteolytic bacterial populations (Min et al., 2003), although effects depend on the type of CT. The CT concentration was low relative to the concentration of CP but the type of CT (all prodelphinidin; Jones et al., 1976) suggested good potential to inhibit proteolysis. Prodelphinidins are more inhibiting to proteolysis than the procyanidins dominant in birdsfoot trefoil (Lotus corniculatus L.) CT (McNabb et al., 1997). 4.3. Volatile fatty acids The rapid decline in pH over the first 12 h of incubation in all treatments implies rapid and extensive fermentation of plant material. Flower or CT content had minor effects on total VFA production. The low fibre and high non-fibre carbohydrate concentrations in both leaf and flower relative to most temperate grasses favour a rapid fermentation. Increasing the proportion of DM as flower in incubations reduced propionate yields and A:P ratios, which could reduce milk lactose synthesis and milk yield (Holmes et al., 2002). Effects of CT in flowers on VFA were minor and consistent with studies evaluating effects of CT in other forages. The reduction in the molar proportion of minor VFA has previously been reported for sheep fed birdsfoot trefoil and lotus (Waghorn et al., 1987, 1994), and in vitro incubations with sainfoin (Onobrychus viciifolia Scop.) have demonstrated similar negative effects of CT on propionate yields (McMahon et al., 1997). The increase in butyrate concentration due to CT in our experiment is consistent with increase reported in sheep fed sulla (Terrill et al., 1992b). High concentrations (i.e., >50 g CT/kg DM; Barry et al., 1986) or highly astringent CT (e.g., in erect dorycnium (Dorycnium rectum L.)) are able to reduce bacterial digestion in vivo (Waghorn and Molan, 2001) but CT in white clover flowers (52 g/kg DM) did not limit VFA production in our study. 4.4. Intensively flowering white clover for ruminants Increasing the proportion of white clover flowers in ruminant diets to provide sufficient CT to improve animal performance is unlikely to be practical because clover contents in New Zealand pastures rarely exceed 300 g/kg of DM. Grasses and non-tanniniferous forages will dilute and reduce the impact of CT, and seasonal variation in flower density (Burggraaf et al., 2003) will preclude use of white clover floral CT for improved dietary nutritive value throughout the year. The impact of CT per se was relatively minor, with most effects of flowers reported here attributable to the chemical and structural composition of flowers versus leaves. In vivo measurements (Burggraaf et al., 2004) have demonstrated a reduction in rumen ammonia concentrations in cows fed white clover with a high proportion of DM (up to 330 g/kg DM) as flowers. This effect was attributed to CT, and lower CP, in flowers versus leaves. Benefits of reduced ammonia detoxification requirements were offset by the higher A:P ratio produced by flowers, resulting in no effect of increased flowering on milk production (Burggraaf et al., 2004).

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5. Conclusion Increasing the ratio of white clover flowers to leaves in vitro reduced degradation of plant CP to ammonia, partly due to its reduced solubility of the protein, and CT in the flowers had a minimal impact on proteolysis. When flowers comprised more than 500 g/kg of clover DM, ammonia concentrations may have limited microbial growth. Treatments did not affect total VFA production, but the concentration of propionate and butyrate decreased with increasing flower contents. Increasing flowering is not likely to substantially improve the nutritive value of white clover fed to dairy cows.

Acknowledgements We thank Dairy InSight for research funding, the Bright Futures Enterprise Scholarship for financial support, Barbara Dow for statistical analysis and Scott Waghorn and Julia Lee for technical assistance.

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