J Sci Food Agric 1996,12,483-492

The Condensed Tannin Content of a Range of Subtropical and Temperate Forages and the Reactivitv of Condensed Tannin with Ribulose-

J

1,5=bis=phosphate Carboxylase (Rubisco) Protein Felicity S Jackson," Warren C McNabb,b* Tom N Barry," Yeap L Fooc and Jason S Petersb a Department of Animal Science, Massey University, Palmerston North, New Zealand

AgResearch Grasslands, Private Bag 11008, Palmerston North, New Zealand NZ Institute for Industrial Research and Development, PO Box 31-310, Lower Hutt, New Zealand

(Received 3 January 1996; revised version received 10 May 1996; accepted 28 June 1996)

Abstract: A series of subtropical grasses and temperate grasses, herbs and legumes were analysed for the presence of extractable and bound condensed tannin (CT) using colorimetric analysis by the butanol-HC1 method. Condensed tannins are routinely purified using affinity chromatography with Sephadex LH-20 as a matrix. Therefore, Sephadex LH-20 extracts were further analysed for the presence of CT by 13C nuclear magnetic resonance, for anthocyanidin forma- tion after butanol-HC1 treatment and for their ability to precipitate ribulose-1,5- bisphosphate carboxylase (Rubisco) protein from lucerne, at pH 7.0. Criteria for the presence or absence of CT were defined. Trace amounts of CT (0.2- 2.5 g kg-I dry matter; DM) were identified and confirmed in summer grass (Digiteria sanguinalis), perennial ryegrass (Lolium perenne) and red clover (Trifolium pretense), with chicory (Chicorium intybus), lucerne (Medicago sativa) and plantain (Plantago lanceolata) identified as probably containing CT. It was concluded that the subtropical grasses kikuyu (Pennisetum clandestinum), paspa- lum (Paspalum diatatum), smooth witchgrass (Panicum dichotomiforum) and crowfoot (Eleusine indica) and the temperate grass, Yorkshire fog (Holcus lanatus) probably did not contain CT. Analysis of the extractable fractions by vanillin- HCI gave higher values for CT than analysis by butanol-HC1 and wrongly iden- tified some forages as containing trace levels of CT. It was concluded that vanillin-HC1 was not specific enough for the detection of trace levels of CT in forages. These results raise the possibility of plant selection programmes to increase the level of CT in grazed forages to approximately 5 g kg-' DM, the suggested minimum level required to prevent bloat in cattle and to increase wool growth in sheep. It is suggested that this be considered for perennial ryegrass, chicory, red clover and lucerne.

Key words : condensed tannin, subtropical grasses, temperate forages.

INTRODUCTION metric procedures. Use of these methods has shown that CT is present in a restricted range of temperate forage

Extractable condensed tannin (CT) in forages are com- legumes including Lotus corniculatus, Lotus peduncu- monly determined by the vanillin-HC1 (Broadhurst and l a m and sainfoin (Onobrychis uiciifolia), but these Jones 1978) and butanol-HC1 (Porter et a1 1986) colori- methods have not detected CT in the leaves and stems

of most forages commonly used as temperate grazing * To whom correspondence should be addressed. plants, such as perennial ryegrass (Lolium perenne) and

J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain 483

484 F S Jackson et a1

red clover (Trifolium pratense). This has led to the wide- spread view that CT is probably absent in most of the forages used in temperate grazing systems (Barry 1989).

The sensitivity of plant CT analysis was improved with the development of procedures to measure protein- bound and fibre-bound CT in addition to extractable CT (Terrill et a1 1992). This resulted in the detection of small concentrations of CT in forages such as perennial ryegrass and Yorkshire fog (Holcus lanatus). The objec- tive of this study was to extend this methodology to the analysis of the total (extractable and bound) CT in sub- tropical grasses and in a range of temperate grasses, herbs and legumes consumed by grazing ruminants in New Zealand (NZ). In addition, the semi-purified CT extracts of these forages were also compared using 13C nuclear magnetic resonance ( 13C-NMR). The ability of each CT extract to reduce net ammonia formation during in uitro rumen fermentation of total plant protein and to precipitate ribulose- 1,5-bisphosphate car- boxylase (Rubisco) protein from lucerne (Medicago satiua) was also determined. These experiments were undertaken to confirm that any low CT concentrations obtained by colorimetric analysis were indeed due to the presence of CT. Rubisco was studied because it is the principal leaf protein, representing 30-50% of the total soluble protein present in plants (Mangan 1982).

MATERIALS AND METHODS

Experimental design

Leaves collected from a range of subtropical grasses and temperate grasses, herbs and legumes were analysed for CT using both the butanol-HC1 and vanillin-HC1 colorimetric analyses, and were also analysed for carbo- hydrate constituents and for total nitrogen (N). Con- densed tannins have routinely been purified using affinity chromatography with Sephadex LH-20 as a matrix (Porter et aE 1986; Terrill et al 1992). Therefore, Sephadex LH-20 extracts were prepared from all plants and analysed for the presence of CT using "C-NMR and thin-layer chromatography (TLC) of extracts heated with butanol-HC1 to detect anthocyanidins formed from CT. An in uitro incubation of each plant species in rumen fluid was used to determine if the net conversion of plant N to ammonia was effected by the presence or absence of polyethylene glycol (PEG). PEG binds to CT, preventing the CT binding to protein (Jones and Mangan 1977); hence the effect of CT on the net conversion of plant-N to ammonia can be deduced by comparing control flasks (CT active) with PEG flasks (CT inactivated). The in oitro conversion of plant-N to ammonia in rumen fluid represents the net outcome from several processes, including plant and microbial degradation to ammonia and ammonia uptake and biosynthesis into microbial protein. It is not

a direct measure for protein degradability and is likely to be affected by the amount of plant N, carbohydrate and CT added to the incubation. Therefore, it cannot be used to compare plant species, but within a plant species, it can be used to measure the effect of PEG addition. The ability of the Sephadex LH-20 extracts to precipitate Rubisco was also measured, with and without added PEG.

Plant materials

Leaves from a selection of subtropical and temperate grasses, herbs and legumes were harvested during mid- summer from various locations in the North Island of NZ. The subtropical grasses were harvested from pastures in three different geographical locations in the North Island (Manawatu, Waikato and North Auckland) and separated into the five subtropical grasses and also the temperate grass, perennial ryegrass. The species collected from different areas were analysed individually for CT, total N and carbohydrate fractions, but were pooled together to give one sample for each plant type for other analyses. The temperate forages (including a second reference sample of perennial ryegrass) were collected from test plots grown at AgRe- search Grasslands and Massey University (Palmerston North, New Zealand). Samples were transported on dry ice to AgResearch Grasslands and on arrival all root and dead or extraneous matter was removed. The remaining plant samples contained predominantly leaf material of varying vegetative maturity but did not contain any tissues indicative of a reproductive stage of growth such as older stems and seed heads. Leaf samples were then freeze-dried and ground to pass through a 1 mm sieve.

Plant analysis

All samples were analysed for total N by the Kjeldahl digestion procedure using a Kjeltec auto analyser (Tecator, Hoganas, Sweden) with crude protein calcu- lated as total N x 6-25. Condensed tannin was extracted from plant material using acetone/water/ diethyl ether (4.7 : 2.0 : 3.3, v/v/v) as described by Terrill et al(1992) and the concentration of extractable CT was then determined by both the butanol-HC1 (Porter et a1 1986) and vanillin-HC1 (Broadhurst and Jones 1978) methods. The residue from the acetone extractable frac- tion was then boiled (2 x 45 min) with sodium dodecyl sulphate (SDS) and 2-mercaptoethanol to extract protein-bound CT, which was then quantified with butanol-HC1. The concentration of fibre-bound CT was determined by boiling the residue with butanol-HC1 and SDS-2-mercaptoethanol for 75 min (Terrill et a1 1992). For all three fractions of the butanol-HC1 assay, sample blanks were prepared using plant extracts but

Condensed tannin concentration and reactivity 485

substituting butanol-H,O (95 : 5, v/v) for the butanol- HCl (95 : 5, v/v). In the vanillin-HC1 method, sample blanks were prepared using plant extracts but omitting vanillin from the vanillin/methanol reagent (4%, w/v in methanol). This effectively blanked out any interference due to plant coloration, as naturally occurring antho- cyanidins can also give a color reaction.

Neutral detergent fibre (NDF), acid detergent fibre (ADF) and lignin were determined using the method of Robertson and Van Soest (1981), with hemicellulose cal- culated as NDF - ADF and cellulose as ADF - lignin. Soluble sugars and pectins were determined by the method of Bailey (1967), with organic matter digest- ibility (OMD) determined using the method of Roughan and Holland (1977).

Animals and feed

Three adult male castrated sheep fitted with rumen can- nulae (55 mm id) were maintained on a basal diet of meadow hay (850 g day-') and lucerne chaff (150 g day- I), offered hourly from overhead belt feeders. After fasting for 16 h rumen fluid was collected at 08:OO h, strained through muslin cloth into a Dewar flask, flushed with CO, gas and warmed to 39°C in an atmo- sphere of CO,. This was used immediately for the in vitro incubations.

In vitro incubation of plants in rumen fluid

I n vitro rumen incubations were performed following the method described by McNabb et al (1994) to measure the net conversion of plant N to ammonia. Freeze-dried plant material were weighed (tropical grasses (6 g), with the exception of summer grass (3 g), temperate grasses (4 g), temperate herbs (4 g) and legumes (2 g)) into duplicate stoppered flasks containing 0.15 g D-( +)-cellobiose (Sigma, St Louis, USA) as an energy source for rumen microbes. PEG (0.32 g; MW 3350; Union Carbide, USA) was added to one of each duplicate flask. The highest concentration of CT in lotus species reported by Terrill et a1 (1992) using the butanol-HC1 technique is about 80 g kg-' dry matter (DM), therefore the addition of Lotus corniculatus (2 g DM) to in vitro rumen incubations would be unlikely to result in the addition of more than 0.16 g CT. Barry and Forss (1983) reported that 2 mg PEG g-' CT was required to inactivate CT so 0.32 g of PEG was added to all in uitro incubations. The addition of PEG did not interfere with protein degradation during in vitro rumen incubations (Yu et a1 1995b) so the weight of PEG added was held constant for all in vitro rumen incu- bations. Freshly prepared artificial saliva (60 ml, pH 6-8; McDougall 1948) was added to each flask along with 15 ml of strained rumen fluid, the flasks were then flushed with CO, , stoppered with one-way valves and

shaken (200 rpm) at 39°C for 8 h. Sub-samples (20 ml) were removed at 0 ,4 and 8 h from each flask and acid- ified with 1 M HISO,. Acidified 20 ml aliquots from the in vitro rumen incubations were stored at 4°C for ammonia analysis using a Cobas Fara automatic analyser (Cobas Faras, F Hoffmann-La Roche Ltd, Switzerland). Four replicates for each plant species were incubated with and without the addition of PEG over four consecutive days, with one replicate being done on each day.

Preparation of Sephadex LH-20 plant extracts

Sephadex LH-20 extracts were prepared using the method of Porter et al (1986), but with the following modifications. Fresh leaves of Lotus pedunculatus and freeze-dried samples of subtropical grasses and tem- perate grasses, herbs and legumes were extracted with acetone/H,O (70 : 30, v/v) containing ascorbic acid (1 g litre-'), followed by three extractions with dichloro- methane (industrial grade; Tergo Industries, Auckland, NZ) to remove all the chlorophyll and lipids. The aqueous defatted extracts were then freeze-dried and approximately 5 g redissolved in 50 ml of 1 : 1 methanol/H,O (v/v). The crude CT extracts were then semi-purified on a column containing 200 ml of Sepha- dex LH-20 (Pharmacia, Uppsala, Sweden) by washing with 1000 ml of 1 : 1 methanol/H,O (v/v) before eluting with 200 ml of acetone/H,O (70 : 30 v/v). The Sephadex LH-20 extracts were freeze-dried and stored in a desic- cator at -20°C until required. The Sephadex LH-20 extract from Lotus pedunculatus (analysis of this extract by 13C-NMR showed that it contained predominantly CT) was used as a standard for determining the CT concentration in the subtropical grasses and temperate grasses, herbs and legumes, whilst the Sephadex LH-20 extracts from the subtropical grasses and temperate grasses, herbs and legumes were used for in vitro protein precipitation assays.

Analysis of Sephadex LH-20 extracts

The Sephadex LH-20 extracts were analysed by 13C- NMR and also by TLC following reaction of the Sepha- dex LH-20 extracts with dilute HCl to generate antho- cyanidins. 3C-NMR spectra were obtained using a Bruker AC 300 NMR spectrometer with samples dis- solved in deuterated acetone-D,O. The spectra were examined for the presence of carbon signals character- istic of CT chemical structures (Porter et al 1982).

Anthocyanidin analysis were performed by treating Sephadex LH-20 extract (3 mg) with 5% HC1 in 2,2- dimethyl propanol (2 ml) and heating at 100°C for 1 h in sealed tubes. The TLC of anthocyanidins were per- formed on cellulose plates, developed with tert-butanoll acetic acid/water (60 : 20 : 20, v/v) and acetic acid/water

486 F S Jackson et a1

TABLE 1 Chemical composition (g kg-’ DM), and organic matter digestibility (OMD) of the leaves of subtropical grasses and the tem-

perate grasses, herbs and legumes (values represent the average of two replicates)

Species Total N Soluble Pectin NDF ADF Cellulose Hemicellulose Lignin Ratio OMD sugar (a) (a’) (b) (b‘) (a + a’)/(b + b’)

Subtropical grasses Kikuyu Paspalum Smooth witchgrass Crowfoot Summer grass

Temperate forages Perennial ryegrass” Perennial ryegrassb Yorkshire fog Chicory Plantain Red clover

27.4 53 5 581 249 234 25.0 71 8 615 264 242 15.9 91 4 558 271 249 13.0 69 10 640 303 290 25.0 60 6 483 253 234

36.8 117 6 384 196 184 45.2 14 10 406 194 184 34.4 12 4 468 210 194 19.7 111 98 168 124 104 17.5 170 60 231 166 115 46.9 95 39 181 127 115

332 351 287 337 230

188 212 258 44 65 54

15 22 22 13 19

12 10 16 20 51 12

0.10 0.14 0.18 0.13 0.14

0.33 0.43 0.36 1.69 1.39 1 -06

0.671 0.636 0.675 0.612 0.738

0.839 0.850 0.658 0.897 0.859 0.855

Dissected from the same pastures as the subtropical grasses. Grown in the same area as the other temperate forages.

(6 : 94, v/v) and visualised with 10 mM FeCl, ; 8 mM K,Fe(CN), according to the method described by Foo et a1 (1996).

Plant protein extraction

Total plant protein was extracted from fresh lucerne by grinding 10 g of leaves with 30 ml protein extraction buffer (0.1 M TES; N-tris(hydroxymethy1) methyl-2- aminoethanesulphonic acid), pH 7.8; 1.16% NaCl; 0.04% ethylenediaminetetra-acetic-acid disodium salt (EDTA); 0.5% phenyl methyl sulphonyl fluoride; 2% 2-mercaptoethanol; 1% ascorbic acid; 0.33 M sodium diethyldithiocarbamate) in a mortar and pestle held on ice. The extract was then centrifuged at 12000 x g for 20 min at room temperature to remove particulate plant material. The supernatant was then filtered through a 0.2 pm Minisart syringe filter (Sartorius, Germany) to clarify the total plant protein extract which was then stored at 4°C. The Bradford (1976) protein assay was used to determine total plant protein in the extract.

Precipitation of Rubisco protein by condensed tannin

Samples were weighed out to give 0, 0.1, 0.5, 1.0, 10, 50 and 100 pg of each Sephadex LH-20 extract per tube. Six tubes were prepared for each level of Sephadex LH-20 extract, with 50 pl McIlvaine’s buffer pH 7-0 (Elving et a1 1956) added to each tube and 200 pg PEG being added to three of the six tubes. Total soluble leaf protein (from lucerne; 12 pg) was added to each tube, after which all tubes were diluted to 100 pl with McIl-

vaine’s buffer pH 7.0. Tubes were then incubated at 39°C for 90 min. After incubation the tubes were centri- fuged at 12000 x g for 10 min and the resulting pellet washed three times in McIlvaine’s buffer pH 7.0 to remove all soluble leaf protein that was not precipi- tated. Finally, the pellet was resuspended in 20 pl protein loading buffer (64 mM Tris-HC1; Tris hydroxy- methyl aminomethane), pH 6.8; 2% SDS; 10% glycerol ; 0.005% bromophenol blue; 5% 2-p-mercap- toethanol) and stored at - 20°C for gel electrophoresis.

Analysis of samples by SDS-PAGE

Prior to fractionation by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE; using the method described by McNabb et a1 1994), samples were heated for 5 min at 95°C to denature protein and dissociate all protein complexes and then cooled on ice. After centrifugation at 12000 x g for 5 min, the supernatant was loaded into each well and electro- phoresis was carried out at a constant current of 80 V for approximately 3 h using mini-gels (0.75 x 75 x 100 mm; Bio-Rad, Hercules, CA, USA) as described by Yu et a1 (1995a).

After SDS-PAGE, the protein in the gels was fixed by washing the gels in methanol/acetic acid/water (40 : 10 : 30, v/v/v) for 30 min and total soluble protein was visualised by staining with Fast Green FCF (0.1% (w/v) Fast Green FCF in methanol/acetic acid/water (40 : 10 : 50, v/v/v)) for 30 min. Gels were de-stained in methanol/acetic acid/water (10 : 7.5 : 82.5, v/v) for 48 h

Condensed tannin concentration and reactivity 487

TABLE 2 Condensed tannin content (g kg-' DM) of the leaves of subtropical grasses and the temperate grasses,

herbs and legumes (values represent the average of two replicates)

Species Condensed tannin

Vanillin-HCl Butanol-HCI

Extractable Extractable Protein-bound Fibre-bound Total ~~~~~~~~~

Subtropical grasses Kikuyu Paspalum Smooth witchgrass Crowfoot Summer grass

Temperate forages Perennial ryegrass" Perennial ryegrassb Yorkshire fog Chicory Plantain Red clover Lucerne Lotus corniculatus

1.07 0.90 1.65 2.47 2.80

0.95 1 .00 3.10 3.30

25.10 1.40 4.20

36.80

0.60 0.50 1 .oo 0.23 0.45

0.8 0.50 2.30 1 .00

10.90 0.40 0.00

35.80

0.30 0.67 0.50 0.17 0.50

0.5 0.00 0.00 0.40 1.30 0.60 0.50 8.60

0.01 0.57 0.20 0.20 0.55

0.5 0.40 0.00 0.30 1.90 0.70 0.00 1.80

0.91 1.74 1.70 0.60 1.60

1.8 0.90 2.30 1.70

14.10 1.70 0.50

46.20

Dissected from the same pastures as the subtropical grasses. Grown in the same area as the other temperate forages.

to detect protein bands. Developed Fast Green FCF stained gels were quantified by imaging densitometry (Bio-Rad, Model GS-670 Imaging Densitometer, USA). The data was processed using image analysis software (Bio-Rad Molecular AnalystTM/" imaging analysis soft- ware, USA). RESULTS

hadex LH-20 extracts; and T is the density of Rubisco in the total plant protein added (12 pg).

Plant composition

CALCULATION OF DATA

Rubisco consists of eight large sub-units (LSU; MW 54000) eight small sub units (SSU; MW 16000; Kawa- shima and Wildman 1970). The LSU and SSU are readily detectable on stained SDS-PAGE gels as they represent the predominant proteins present. As the sub- units behaved identically, they have been grouped together and referred to as Rubisco.

The proportion of added Rubisco which was precipi- tated by Sephadex LH-20 extracts was calculated according to eqn (1).

YO precipitation = (x"; XO) x 100

where XCT is the density of the Rubisco precipitated at each level of added Sephadex LH-20 extracts; Xo is the density of the Rubisco precipitated with no added Sep-

Relative to perennial ryegrass (separated from the same pastures as the subtropical grasses), all the subtropical species had lower levels of total N, soluble sugars and OMD and a higher concentration of all fibre constitu- ents (cellulose, hemicellulose and lignin; Table 1). The ratio of readily fermentable carbohydrates : structural carbohydrates was significantly lower in the subtropical grasses, with a mean value of 0.14 compared with 0.33 for perennial ryegrass.

The temperate forages, with the exception of red clover (46.9 g kg-' DM), were all consistently lower in total N (17.5-34.4 g kg-' DM) compared with peren- nial ryegrass (45.2 g kg-' DM; grown in the same area as the temperate forages). The OMD values were high and showed little variation with the exception of a lower level in Yorkshire fog. The ratio of readily fer- mentable carbohydrate : structural carbohydrate was similar in the grasses Yorkshire fog and perennial rye- grass, but was much higher in the herbs chicory and

488 F S Jackson et a1

TABLE 3 Determination of the presence or absence of condensed tannins, anthocyanidin forma- tion (established using thin-layer chromatography (TLC) of Sephadex LH-20 extracts subjected to butanol-HC1 treatment for 1 h at l O O T ) , flavanoid glycosides and other phenolic glycosides in Sephadex LH-20 extracts of subtropical and temperate forages

using I3C-NMR"

Condensed Anthocyanidins Flavanoid Other tannin formation, after glycosides phenolic

butanol-HC1 glycosides ~~~~~~~ ~

Subtropical grasses Kikuyu Paspalum Smooth witchgrass Crowfoot Summer grass

Temperate forages Perennial ryegrass Yorkshire fog Chicory Plantain Red clover Lucerne Lotus corniculatus

ND ND ND ND -k

+ ND ND ND + + ND

+ + +

ND ND ND ND

+ (D)

a Abbrevs: (ND), not detectable and ( + ), trace; (+ +), low, (+ + +), medium concen- trations of condensed tannins (CT) and anthocyanidins present in Sephadex LH-20 extracts. The concentration of CT, anthocyanidin, flavonoid and other phenolic glyco- sides, could not be quantified by I3C NMR and TLC, therefore these values represent qualitative estimates only. D, delphinindin; C, cyanidin. P, present and A, absent from Sephadex LH-20 extracts.

plantain and in the legume, red clover. The lignin con- centration in plantain was unusually high.

Condensed tannin

For this study trace levels of CT have been defined as 0.2-2-5, low as 2.5-10.0 and medium as 10.0-50.0 g CT kg-' DM. The concentration of CT in the plants are shown in Table 2.

Butanol-H CI Condensed tannin was detected in all plants species using the butanol-HC1 method (Table 2). The sub- tropical grasses all contained trace amounts of total CT, with the CT being present in both the extractable and bound forms. Of the temperate forages, plantain and Lotus corniculatus contained medium concentrations of total CT. All other temperate forages contained trace concentrations of CT, with the levels in lucerne being extremely low. All species contained extractable CT, with the exception of lucerne. All species contained bound CT, with the exception of Yorkshire fog.

Vanillin-HC1 The concentration of extractable CT (determined by vanillin-HC1) was consistently higher for all plants than the concentration of extractable CT determined by butanol-HCI. Lucerne was the only plant where extractable CT was not detectable by butanol-HC1 but 4.2 g kg- DM was detected by vanillin-HC1. Similar values for the two methods were obtained for extract- able CT in Lotus corniculatus.

I3C-NMR and thin-layer chromatography The presence or otherwise of CT in the Sephadex LH-20 extracts was established by examination of I3C- NMR carbon resonances which were similar to those expected for the chemical structure of CT. In particular the characteristic carbon signals associated with the interflavonoid bond on the heterocyclic ring must be present together with the A- and B-ring carbons of a flavan-3-01 structure before a sample was judged to contain CT. Being a polymer, the 13C carbon signals of CT tended to be broad and in samples where CT occurred at low levels, its presence was obscured by background resonance. In addition, the much larger

Condensed tannin concentration and reactivity 489

TABLE 4 Ammonia accumulation after 8 h in vitro rumen fermentation, expressed as a percentage of the total forage nitrogen added. The incubations were carried out with and without the addition of

polyethylene glycol (PEG; MW 3350) ~~ ~~

Species Total N per $ask PEG Level of signijicance" + -

(ms)

Subtropical grasses Kikuyu Paspalum Witchgrass Crowfoot Summer grass

Temperate forages Perennial ryegrassb Yorkshire fog Chicory Plantain Red clover Lucerne Lotus corniculatus

160.0 150.0 95.4 78.0 75.0

147.2 73,2 39.4 35.0 93.8 92.2 77.0

4-7 f 0-32 4.6 f 0.25 1.3 * 0.61 1.7 f 0-31 0.3 f 0.30 0.4 f 0.35

0.2 _+ 1.17 0.2 1.29 - 2.2 f 0.26 - 2.1 f 0.29

5.3 f 0.60 7.5 f 0.50

5.3 f 0.71 8.0 f 0.68

-0.2 0.62 - 1.2 f 0.60 -3.7 & 1.23 -3.5 f 1.26

7.7 f 0.50

4.7 f 0.38

8.3 f 0.38

10.7 f 1.56 -2'2 f 0.26 -2.1 f 0.29

NS NS NS NS NS

NS NS NS NS NS NS **

NS, P > 0.05; **, P < 0.01. Dissected from the same pastures as the subtropical grasses.

neighbouring 13C signals of other components such as flavonoid glycosides also interfered with and obscured the much weaker CT signals. Therefore, the I3C NMR analysis of CT erred on the conservative side and may explain the differences observed between the spectro- scopic method and the anthocyanidin analysis.

The 13C-NMR and TLC analysis of Sephadex LH-20 extracts are shown in Table 3. Examination of the Sep- hadex LH-20 extracts by ' 3C-NMR confirmed the pres- ence of CT in perennial ryegrass and red clover, while anthocyanidin production with butanol-KCl established the presence of low levels of CT in all the temperate herbs and legumes studied. Trace amounts of CT were also confirmed in Yorkshire fog and summer grass, but all the other subtropical grasses tested negative for CT by both ' 3C-NMR and anthocyanidin formation. Fla- vonoid glycosides were identified by their characteristic chemical shifts in all the Sephadex LH-20 extracts that were studied. However, it was apparent that for all the Sephadex LH-20 extracts examined, except that from Lotus corniculatus, considerably more flavonoid glyco- sides were present than CT. Hydrolysable tannins were not detected in any Sephadex LH-20 extract.

In vitro rumen incubations

With Lotus corniculatus the addition of PEG signifi- cantly increased net ammonia accumulation ( P < 0.01)

indicating that the action of CT reduced the net conver- sion of plant N to ammonia (Table 4). Ammonia forma- tion in oitro was not affected by PEG in all the other species studied. The negative values observed for net ammonia accumulation with crowsfoot, chicory, plan- tain and lucerne indicates that the ammonia yield at the completion of these incubations was less than the ammonia content of the rumen fluid added to the in vitro incubations. Clearly, unlike Lotus corniculatus, all other plants tested appeared to contain insufficient CT to effect net ammonia accumulation.

Precipitation of Rubisco protein by Sephadex LH-20 extracts

The amount of Sephadex LH-20 extract required to precipitate all the Rubisco when total plant protein was incubated with Sephadex LH-20 extracts from Lotus corniculatus, red clover, lucerne, chicory and perennial ryegrass varied with each extract (Figs 1 and 2) with 1-10 pg of Lotus corniculatus Sephadex LH-20 extract required, compared with 10-50 pg from red clover, 50- 100 pg from lucerne, 10-50 pg from chicory and 50- 100 pg from perennial ryegrass.

Plantain and summer grass both demonstrated slight precipitation (approximately 15%) with 50-100 pg of added Sephadex LH-20 extract, whilst Sephadex LH-20 extracts from Yorkshire fog, smooth witchgrass, kikuyu,

F S Jackson et a1 490

l a) Lotus corniculatus

8

20 " z s g

0 0 c

v - * -

ug Sephsdex LH-20 oxbrct

b) Lucerne

3 60

= 20 " 0

2 2 - Z S 8 v

ug Sephrdex LH-20 exboct

I I

c) Red clover

40 20

0 80 c

d

- u ! Y Z O O 0 0

ug Sephadex LH-20 extract I 1

Fig 1. Precipitation of Rubisco from lucerne leaves by con- densed tannin extracted from (a) Lotus corniculatus, (b) lucerne and (c) red clover. 0, Incubations done in the pres- ence of polyethylene glycol (PEG); m, incubations done in the

absence of PEG (MW 3350).

crowsfoot and paspalum all gave no precipitation of Rubisco. In all instances where protein precipitation occurred it was eliminated by the addition of PEG, showing the reaction between Rubisco protein and the Sephadex LH-20 extracts was reversible.

DISCUSSION

From the measurements made, criteria used to establish the presence of CT were; detection by butanol-HC1 during colorimetric analysis of plant samples and sub- sequent confirmation of the presence of CT in Sephadex LH-20 extracts by 13C-NMR and precipitation of Rubisco by Sephadex LH-20 extracts and its reversal by PEG. Positive results in all three categories were inter- preted as confirmation of CT being present. Those

plants with only a positive colorimetric result but nega- tive results in all other categories were regarded as probably not containing CT. If a Sephadex LH-20 extract does not precipitate Rubisco, it may still contain CT, but at a concentration or reactivity insufficient to precipitate protein. Although, Hagerman and Robbins (1987) reported that 1.0 mg of CT precipitated about 2-0 mg of bovine serum albumin, therefore the addition of 0-100 pg of Sephadex LH-20 extract to 12 pg of total plant protein should have been sufficient to indicate if a Sephadex LH-20 extract contained CT.

The most important results in this study were the detection and positive identification of CT in red clover, summer grass and perennial ryegrass and the probable presence of CT in chicory, lucerne and plantain. The subtropical grasses kikuyu, paspalum, smooth witch- grass and crowfoot and the temperate grass, Yorkshire fog also contained trace levels of CT according to the butanol-HC1 analysis, but their presence could not be confirmed by either "C-NMR or by the protein binding assay, indicating that the low readings in the colorimetric assay were probably artefactual.

Summer grass was the only subtropical grass to have the colorimetric presence of CT confirmed by I3C- NMR and by a rather weak protein binding activity. This species also had the highest digestibility, lowest NDF and hemicellulose and among the highest N content, indicating that nutritionally it is the most favourable of the subtropical grasses.

An extremely interesting finding from this study was that all the Sephadex LH-20 extracts contained flavo- noid glycosides and some contained other phenolic gly- cosides Unfortunately, when examining Sephadex LH-20 extracts by I3C-NMR it was not possible to get an accurate estimate of the concentration of the consti- tutents present. However, in all the Sephadex LH-20 extracts examined, except that obtained from Lotus cor- niculatus, the flavonoid glycosides and other phenolic glycosides when present, exceeded the CT. This was rather unusual as these compounds have previously been thought to elute with the methanol-H,O wash. In the present study this was not the case and all Sephadex LH-20 extracts that we have examined to date con- tained flavonoid and other phenolic glucosides. However, the flavonoid glycosides present in the Sepha- dex LH-20 extracts from kikuyu, paspalum, smooth witchgrass and crowfoot, which did not contain CT, did not precipitate Rubisco or react with butanol-HCI to yield anthocyanidins. Plantain appeared to contain medium levels of CT (14 g kg-' DM) with analysed using the butanol-HC1 method yet I3C-NMR and anthocyanidin production suggested only trace levels of CT were present. This was further substantiated by the low Rubisco precipitation. This may indicate that the very high content of flavonoids and other phenolic gly- cosides which were present in the plantain Sephadex LH-20 extract may have interfered with the colorimetric

Condensed tannin concentration and reactivity 49 1

a) Perennial ryegrass I

c) Plantain 100 T

2oc 0 T - L q Y O O O 0 0 T r n O

ug Sephadex LH-20 extract ,-

b) Summer grass 100 T

a

* 2 o L - Q - # s 0 F r r > F O O O 0 0 y m o

ug !hphadex LH-20 exW8d .-

I - q T - o o o 0 0 Y

7 0 0 ug Sephadex LH-20 extracl

Fig 2. Precipitation of Rubisco from lucerne leaves by condensed tannin extracted from (a) perennial ryegrass, (b) summer grass, (c) plantain and (d) chicory. 0, Incubations done in the presence of PEG; ., incubations done in the absence of PEG (MW 3350).

analysis of this plant. Further research is needed to confirm what compounds contribute to Sephadex LH-20 extracts and what role, if any, these flavonoids and phenolic glycosides have when examining effects attributed to CT which has been semi-purified by Sep- hadex LH-20.

Horigome et a1 (1988) showed that the protein pre- cipitating capacity of CT was directly proportional to the degree of polymerisation in the CT molecule. The weak precipitation by Sephadex LH-20 extracts from plantain and summer grass could indicate that these extracts contained CT of low molecular weight or highly polymerised CT or CT was present in extremely low concentrations.

Terrill et al (1992) concluded that similar low colori- metric CT values as those obtained in this study for perennial ryegrass were a result of background inter- ference. However the present data were more consistent with those of Li et a1 (1995) which confirmed the pres- ence of trace amounts of CT in lucerne and red clover using histochemical techniques. It seems that CT con- centrations (2-5 g kg-' DM in the three step pro- cedure of Terrill et a1 (1992) may need confirmation by other procedures such as 13C-NMR and the protein binding assay used in the present study.

Hagerman and Butler (1991) concluded that determi- nation of CT by the butanol-HC1 and vanillin-HC1 procedures involved different chemical reactions and that the butanol-HC1 procedure was the more specific. The results obtained by the vanillin-HC1 method can be misleading. Positive values were obtained in the extract- able fraction for kikuyu, paspalum, smooth witchgrass, crowfoot and especially for lucerne, when no CT was present in this fraction. Whilst vanillin-HC1 is reliable for detecting medium on high CT concentrations, it is not suitable for detecting low values, probably due to a reaction of vanillin with other plant phenolic com- pounds.

While CT at medium concentrations such as in Lotus corniculatus reduced in the in oitro net conversion of plant N to ammonia, at low and trace levels, CT did not influence in uitro ammonia formation (Table 4). This contrasts with in vivo studies where trace amounts of CT have been shown to reduce rumen ammonia con- centration in deer fed perennial ryegrass and chicory (Kusmartono, pers comm) and in sheep fed perennial ryegrass (Montossi 1995). These experiments were con- ducted with and without the addition of PEG which suggests that the effect may have been due to CT. The overall results of the present in vitro experiment suggest

492 F S Jackson et a1

that it maybe a useful technique for studying the effects of large CT concentrations upon the net conversion of plant N to ammonia but it is not sensitive enough to confirm or deny the presence of low or trace levels of CT in the forages studied.

Li et a1 (1995) concluded that levels of 5 g total CT kg-' DM were sufficient to prevent bloat in cattle, whilst Montossi (1995) found that 4 g total CT kg- ' D M was the minimum CT concentration required to increase wool production (10%) in sheep grazing peren- nial ryegrass. The identification and confirmation of trace amounts of CT in some forages raises the possi- bility of initiating plant selection and breeding pro- grammes to increase CT concentration to the level which is nutritionally desirable. With the new data available from this study the possibility of plant selec- tion and breeding programs to increase the level of CT in the temperate forages perennial ryegrass, chicory, red clover and lucerne to 5 g CT kg- DM, would make a very valuable contribution to forage grazing systems in NZ.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the help of Bruce Campbell, Bruce Cooper and David Wardle (AgResearch Grasslands) and Maggie Zou, Mirian Hen- driks and Joseph Bateson of Massey University for their valuable technical assistance.

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