The concentration of phenolic acids are low in winter and spring but ..... to 4-hydroxybenzoic acid at the same molar concentration. This is consistent with the ...
Oecologia 9 Springer-Verlag1985
Oecologia (Berlin) (1985) 65:314-318
Seasonal variation of phenols, crude protein and cell wall content of birch (Betula pendula Roth.) in relation to ruminant in vitro digestibility R. Thomas Palo 1, Kerstin Sunnerheim z, and Olof Theander 2 1 Department of Animal Physiology, 2 Department of Chemistry and Molecular Biology, Swedish University of Agricultural Sciences, S-75007 Uppsala, Sweden
Summary. Birch twigs of diameter < 1.5 mm exhibit seasonal trends in ruminant in vitro organic matter digestibility (IVOMD), and in the contents of crude protein, cell walls (neutral detergent fibre, NDF), and phenolic compounds. The IVOMD is low in winter twigs, increases in spring, and reaches a maximum in early summer. Crude protein behaves similarly. On the other hand, the proportion of hydrophilic phenols and cell walls (cellulose, hemicellulose, and lignin) to dry weight decreases dramatically in spring when leaves start emerging and growth is initiated. This reduction of phenols is reflected by concomitant changes in concentration of catechin, a major phenolic compound of birch. The concentration of phenolic acids are low in winter and spring but increase after leafing. The biological activity of an extract containing the phenolic compounds, measured as reduction of IVOMD, also decreases concomitantly with the decline of the total phenolic concentration and catechin. It is notable that catechin when tested alone at natural concentrations does not depress IVOMD. It is possible, however, that the amount of catechin reflects the level of condensed tannins, which may be responsible for IVOMD depression. The results strongly indicate that the decline of NDF and phenolic constituents is important for an improved food quality. Phenols may constitute the major chemical defense of birch in winter against browsing vertebrates by reducing digestibility and having toxic properties.
Birch (Betula spp.) plays an important part in the feeding ecology of many boreal vertebrate herbivores (Le Resche and Davis 1973; Lindl6f et al. 1978; Oldmeyer 1982; Cederlund et al. 1980). It is regarded as of intermediate preference in a continuum of food plants from evergreens to deciduous shrubs and trees (Lindl6f et al. 1978; Hjeljord et al. 1982; Bryant et al. 1983; Pehrson 1983). However, different animals exhibit different feeding behavior in regard to birch. For instance, the mountain hare (Lepus timidus) rejects fine diameter winter twigs of birch ( < 1.5 mm) in spite of higher crude protein content and apparent digestibility of this fraction compared to thicker birch twigs (Pehrson 1980, 1981). Mature growth forms are also preferred to juvenile ones (Pehrson 1981; Bryant 1981b; Sinclair and Smith 1984). Bryant (1981 a, b) and Bryant et al. (1983) suggest that resinous components of the birch twigs are responsible for
Offprint requests to: R.T. Palo
selective feeding by snowshoe hares (L. amerieanus) on this genus. Palo et al. (1983) showed that the phenolic content of winter birch twigs (B. pubescens) is correlated with body weight losses and reduced food consumption by the mountain hare. Furthermore, Palo (unpubl.) found that extracts containing high amounts of water-soluble phenols from B. pendula reduced the ruminant in vitro organic matter (IVOMD), protein, and cell wall digestibilities. Thus birch seems to allocate a large portion of its carbon resources into secondary metabolites which possess high biological activity (Niem~il~i et al. 1979; Bryant et al. 1983; Prudhome 1983 ; Palo 1984). Most vertebrate herbivores show seasonal changes in the utilization of food plants (Cederlund et al. 1980; Kuropat and Bryant 1979, 1983). For example, deer increase their food intake in spring when their nutritional requirement and food quality are both high (Moen 1973, 1978; Kaletskii 1967; Verme 1970; Palo 1981). Boreal deciduous trees are postulated to be highly selected for rapid leaf production at the beginning of the growing season at the expense of chemical defense (Bryant et al. 1983). The higher proportion of birch in the diet of, for example, moose (Alees alees) and reindeer (Rangifer spp.) in spring and summer suggests lack of major defenses during this time (Kuropat and Bryant 1979; Cederlund et al. 1980; Haukioja and Heino 1974). This paper deals with seasonal variation in birch from the aspect of food quality for vertebrate herbivores. Seasonal trends in allocation of resources into growth and defense are studied by determination of the contents of crude protein, cell walls, and phenolic compounds, and the relationship of these factors to in vitro ruminant digestibility. Materials and methods
Plant material Twigs of Betula penduIa Roth. with a diameter at clipping point of 1.5 mm were collected on six occasions from midApril to late July 1982, north-east of Uppsala, Sweden. On each occasion 10 mature birches were chosen at random and twigs were collected from 1-2.5 m above ground. When leaves appeared they were included in the twig fraction. The plant material was dried at 40~ for 72 h and milled to pass a 1 mm sieve.
315 PROTEIN(%)
Nutritional and chemical analyses Nitrogen was analysed by the Kjeldahl technique using 3 g of dried milled plant material. The values are expessed as crude protein (N x 6.25). Cell walls were measured as neutral detergent fibre (NDF) according to Van Soest and Wine (1967), which is an estimate of hemicellulose, cellulose, and lignin (see Theander and ~ m a n 1980). The procedure was modified to suit glass filter tubes. In vitro organic matter digestibility (IVOMD) was measured by incubation with sheep rumen inoeula for 96 h (den Braaver and Eriksson 1967). The sheep were previously accustomed to a browse diet composed of twig and leaves of birch, poplar (Populus spp.) and willow (Salix spp.). Thin layer chromatography (TLC) was performed on Mercks silica gel plates, layer thickness 0.25 mm, with fluorescent indicator. Mobile phases: (1) chloroform: methanol: acetic acid, 80:15 : 1 (2) chloroform: methylethylketone: acetic acid 10:7:2. The spots were visualized by spraying with diazotized sulfanilic acid dissolved in 10% sodium carbonate, followed by 50% sulfuric acid. HPLC analyses were performed on a Waters Ass. apparatus. The UV detector was a Waters Model 440 absorbance detector, and the wave-length used was 280 nm. The fluorescence detector used was a Shimadzu RF-530, excitation wave-length 273 nm, emission wave-length 617 nm. The recorder and integrator was a Hewlett-Packard model 3385 A. The column was a Chrompac CP tm sphere C18, 25 cm. The mobile phase was a linear gradient from methanol:water: formic acid, 20:80:1 to methanol: water: formic acid, 70: 30:1. Time for the gradient was 20 min and flow rate was 1 ml/min. Reference substances were all commercially available. The enzyme used for hydrolysis was pectinase No P-4625 available from Sigma.
Preparation of extracts One gram of twig-flour was extracted with 10 ml acetone in an ultrasonic bath for 30 min. The acetone was decanted and filtered into a round-bottomed flask. The twig-flour was extracted once more with 50% aqueous acetone as above. The liquid was decanted and filtered and the twigresidue was washed with 5 ml aqueous acetone. The combined liquid phases were evaporated under reduced pressure below 40~ until no acetone remained. The residue was diluted with water to 10 ml and extracted with 3 x 10 ml light petroleum (bp. 40~176 5 ml of the water phase was used in the biological experiments. The remaining water extract was further extracted with 3 x 10 ml ethyl acetate. The combined organic extracts were dried over anhydrous sodium sulfate and evaporated below 40 ~ C. The ethyl-acetate extract was analysed with two-dimensional TLC systems 1 and 2 and with HPLC equipped with a fluorescence detector. The remaining water-phase, after extraction with organic solvents, was analysed for phenols by the Folin-Ciocalteau method as described by Singleton and Rossi (1965). The colour reaction was measured after 2 h at 500 nm. The concentrations are expressed as mg phenol equivalent/g twig dry matter (D.M.). The accuracy of the method was tested by reaction with compounds according to increased hydroxylation of the ring and increased molecular weight. The following substances were tested: 2.3-dimethoxybenzoic acid, veratric
10
i
16,4
3,5 14,5
i
2.6
_ _ L
18.5
23,7
DATE
Fig. l. Increase in the level of crude protein of fine birch twigs from April to July. n= 10. Mean and S.D. n=number of individuals S.D. = Standard deviation acid, vanillic acid, protocatechuic acid, p-hydroxybenzoic acid, naringenin, tannic acid, quercetin, and phenol. Catechin was isolated from aliquots of the ethyl acetate extracts with preparative TLC, eluent 2. Plates used were of the same kind as above.
Biological analyses The effect of phenolic extracts from different seasons on IVOMD was tested. Briefly, phenolic extract was mixed with hay or coarse birch twig (1.5-5 mm) fluor to mimic natural concentrations of 1.5 mm twigs. The treated plant material was incubated with sheep rumen liquor in glass-filter tubes for 96 h. Organic matter digestibility of controls and treated samples was measured in the traditional way (den Braaver and Eriksson 1967). Reduction of IVOMD was calculated by IVOMDc~ IVOMDtreat x 100%. IVOMDoo~t Results
Nutritional and chemical analyses Crude protein content of fine birch twigs increases from winter to summer. The protein content is about 52% higher in early June than in mid-April. After a maximum of about 16% of D.M. in early June the level declines to about 12% of D.M. in mid-June and remains at this level for the rest of June and July (Fig. 1). The reverse pattern is shown by the level of N D F (neutral detergent fibre). A rapid decline occurs in April and still lower levels are observed in the middle of May as the leaves develop. From this time and to late July the N D F level remains fairly constant (Fig. 2). Figure 3 shows the seasonal variation of IVOMD of fine birch twigs. It is positively related to the protein curve (Fig. 1) but negatively related to the N D F curve (Fig. 2). The IVOMD reaches its maximum at the same time as
316 % NDF
MG PHENOL EQV./G TWIG D.M.
53 30
52
51
20
50
49
48
o 47
i
16.4
3.5 14,5
25.7
2,6
16.4
DATE
Fig. 2. Decline of NDF in spring. Mean of two replicates from a gross sample of 10 individuals
3,5 14.5
2.6
18,8
23.2
DATE
Fig. 4. Changes in the levels of water-soluble phenols with season measured by the Folin reaction, n = 10. Mean and S.D. CATECHIN(MG/G)
% IVOMD
1.5
A
1.0
8.5
T
16.4
i
i
3.5 1q.5
i
2.6
i
18,6
~
i
23.7
DATE
16.4
3.5 14.5
2.6
18.5
23.7
DATE
Fig. 3. The seasonal change in ruminant in vitro digestibility of fine birch twigs, n = 10. Mean and S.D.
Fig. 5. Concentration of catechin at different occasions. Mean of two replicates
the crude protein and declines correspondingly. At h i g h IVOMD values, high protein and low N D F values are characteristic. This is true except for early May twigs, which show low IVOMD, low protein, and low N D F values. Phenolic analyses by the Folin-Ciocalteau method on water extracts of fine birch twigs showed high absorbance in winter twigs. As the buds break in the middle of May there is a rapid decline in absorbance. It falls to a minimum in early June and remains at about this level for the rest of the summer period studied (Fig. 4). The Folin-Ciocalteau reagent shows different absorbance values for different phenolic compounds at the same molar concentration. Tannic acid (TA) shows the lowest absorbance per mole followed by quercetin (3.2 x absorbance of TA), p-hydroxybenzoic acid (4.2 x ), protocatechuic acid (5 x ), phenol (7 x ) and vanillic acid (8.5 x ) in order of increasing absorbance. Naringenin, 2,3-dimethoxyben-
zoic acid, and veratric acid did not give any measurable absorbance with the Folin-Ciocaltean reagent. One major component of fine birch twig extract is catechin, which shows the most pronounced seasonal change of all the phenolic constituents. It is high in winter twigs (1.5 mg/g) but declines as leaves start emerging and growth is initiated. The concentration in early June is about 0.06 mg/g and it remains at about this level for the rest of the period studied (Fig. 5). The extracts contain glycosidically-bonded phenolic acids. They were studied as aglycones after enzymatic hydrolysis. The following acids were present: p-coumaric acid, p-hydroxybenzoic acid, and trace amounts of vanillic, protocatechuic, and gallic acids. Only low concentrations of these components were detectable at any season. However, except for vanillic acid, the concentration of acids increased from April to June.
317 REDUCTION OF
IVOMD(~)
3O
2O
10
~q'.5 3',5 ~
7
DATE
Fig. 6. Effect of addition of phenolic extract from fine birch twigs
on the IVOMD of coarse birch twigs and hay. Phenols were added to mimic natural concentrations in fine birch twigs collected at different times, n = 3 9 = Birch o = Common Timothy
Biological effects Test of water extracts from fine birch twigs on the IVOMD of common timothy (Phleum pratense L.) and coarse birch twigs showed that extracts reduced the IVOMD significantly on both substrates. The reduction of IVOMD was positively correlated with the level of Folin reactive phenols and catechin in the extracts. The highest inhibition of IVOMD occurred with extracts from winter twigs and with coarse twigs as substrate. Using common timothy as a substrate in the incubations, the effect was similar to that with birch, but the reduction of IVOMD was much less with summer extracts (Fig. 6). However, commercial catechin did not show any measurable effects on the IVOMD when added in the range of natural concentrations. Discussion
Rapid and large seasonal changes occur in several birch components which are important determinants of food quality for vertebrate herbivores. It is commonly believed that high cell-wall content of browse is negatively related to digestibility while the reverse is true for protein (Fox and Macauley 1977; Schultz et al. 1982; Milchunas etal. 1978; Robbins 1983). In general these conclusions seem to be valid for the results presented here. However, the changes in the different food components are not in phase with each other. For example IVOMD is 4 weeks delayed compared to the decline of N D F and 2 weeks delayed compared to the crude protein.
This suggests that complex biochemical changes and interactions between plant constituents may occur at this time. The rapid decline of N D F occurs before any visible changes in the plant is apparent. It is possible that carbon reserves located in the wood or bark are mobilized to prepare for leaf development (Bryant et al. 1983; Chapin et al. 1980). This mobilization of cell wall components may explain the rise in phenol content and increased inhibition of IVOMD in early May twigs. Phenols are generally associated with negative effects on both digestibility and nutrient assimilation for many different herbivorous animals (Niemfil/i et al. 1979; Haukioja 1980; Bernays et al. 1980; Feeny 1970; Watermann et al. 1980, 1983; Kuropat and Bryant 1983; Palo et al. 1983; Palo 1984; Lindroth and Batzli 1984). The rapid decline of phenolic concentration of birch twigs in spring may thus be important for the improved food quality at this time. Especially a decline of phenolic compounds with digestibility-reducing or toxic properties may be determining factors for superior food quality. The antagonism between the crude protein and phenoI curves may reflect different allocation priority of resources into growth or defense. Growth has priority over defense during summer when damage by herbivores may be less than the gain by growth and photosynthesis (Prudhome 1983 ; Westcott and Henshaw 1976). The depression of IVOMD by added birch extracts is well correlated with the phenolic concentration measured by the Folin-Ciocalteau procedure. Furthermore, the concentration of catechin correlates well with the Folin reaction. However, the response factor of this reagent varied considerably between different phenols. For example, vanillic acid had a response factor about 2.0 times relative to 4-hydroxybenzoic acid at the same molar concentration. This is consistent with the results reported by Morita (1980). Despite the unspecific reaction by the Folin method, it gives a reasonably good view of the seasonal and quantitative changes of biologically active phenols. The method may thus be useful as a rapid screening test of total phenols of defined extracts of approximately the same composition. Seasonal changes of catechin correspond very well with the biological effects on IVOMD. Thus this compound could be assumed to be involved in the depression of IVOMD. One hypothesis explaining the lack of effect on IVOMD of (+)-catechin is that this compound merely reflects the level of condensed tannins. It may thus be an indirect measure of the amount of this type of tannin. Further experimental analyses are in progress to shed light on this relationship. The more pronounced effects of phenols on the IVOMD of coarse birch twig compared to hay may be due to stress
Table 1. Some characteristic data for the plant material used as substrate in the IVOMD experiments. The phenol concentration refers to the level before organic extraction Species
Timothy Birch (1.5-5 mm twigs collected on 3 May)
Percent of dry matter (D.M.)
Mg/g D.M.
Organic matter
NDF
Crude protein
IVOMD
Total phenols
94.7 98.0
57.1 69.70
11.1 4.8
76.0 23.0
5.4 31.3
318 on the microbial p o p u l a t i o n is stressed due to the poorer quality of the former. Moreover, the endogenous level of phenols in coarse birch twigs is six times higher t h a n in hay (Table 1). Thus addition of phenols to birch twigs is cumulative to the endogenous level, giving the higher depression of I V O M D .
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Moen AN (1973) Wildlife Ecology, an analytical approach. Freeman Co. San Fransisco Moen AN (1978) Seasonal changes in heart rates, activity, metabolism and forage intake of white-tailed deer. J Wildl Manage 42:71~738 Morita H (1980) Total phenolic content in the pyrofosfate extracts of two peat soil profiles. Can J Soil Sci 60:291-297 Niemfil/i P, Aro EM, Haukioja E (1979) Birch leaves as resources for herbivores. Damage-induced increase in leaf phenols with trypsin-inhibiting effects. Rep Kevo Subarctic Res Stat 15 : 37-40 Oldemeyer JL (1982) Estimating production of paper birch and utilization by browsers. Can J For Res 12:52-57 Palo RT (1981) A review of deer nutrition and energetics with special reference to the moose. Report no. 7, Department of Wildlife Ecology. The Swedish University of Agricultural Sciences Palo RT (1984a) Distribution of birch (Betula spp.),willow (Salix spp.) and poplar (Populus spp.) secondary metabolites and their potential role as chemical defense against herbivores. J Chem Ecol I0 (3):499-520 Palo RT, Pehrson ,~, Knutsson P-G (1983) Can birch phenolies be of importance in the defense against browsing vertebrates ? Finnish Game Research 41 : 75-80 Pehrson A (1980) Intake and utilization of winter food in the mountain hare (Lepus timidus L.). A laboratory investigation. Ph.D. Thesis. Department of zoology, University of Stockholm, Sweden Pehrson ,~ (1981) Winter food consumption and digestibility in caged mountain hares. In: Myers K, MacInnes CD (eds) Proceedings of the World Lagomorph Conference (1979) Guelph, Ontario, pp 732-742 Pehrson A_(1983) Maximal winter browse intake in captive mountain hares. Finnish Game Res 41:45-55 Prudhome TI (1983) Carbon allocation to antiherbivore compounds in a deciduous and an evergreen subarctic shrub species. Oikos 40 (3) : 344-356 Robbins CT (1983) Wildlife feeding and nutrition. Academic Press, New York Schultz JC, Nothnagle PJ, Baldwin IT (1982) Seasonal and individual variation in leaf quality of two northern hardwood species. Amer J Bot 69 (5):753-759 Sinclair ARE, Smith JNM (1984) Do plant secondary compounds determine feeding preferences of snowshoe hares? Oecologia 61:403-410 Singleton VL, Rossi Jr JA (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagens. Am J Enol Viticult 16:144-158 Theander O, Aman P (1980) Chemical composition of some forages and various residues from feeding value determinations. J Sci Food Agric 31:31-37 Van Soest PJ, Wine RH (1967) Use of detergents in the analysis of fibrous feeds. Determination of plant cell wall constituents. J Assoc Agric Chem 50:50-55 Verme LJ (1970) Some characteristics of captive Michigan moose. J Mamm 51 : 403-405 Watermann PG, Mbi CN, Mckey DB, Gartlan JS (1980) African rain forest vegetation and rumen microbes; Phenolic compounds and nutrients as correlates of digestibility. Oecologia 47 : 22-23 Watermann PG, Choo GM, Vedder AL, Wotts D (1983) Digestibility, digestion inhibitors and nutrients of herbaceaus foliage and green stems from an African montane flora and comparison with other tropical flora. Oecologia 60: 244-249 Westcott RJ, Henshaw GG (1976) Phenolic synthesis and phenylalanine ammonia-lyase activity in suspension cultures of Acer., pseudoplatanus L. Planta (Berl) 131:67-73
Received August 15, 1984
