Journal of Chemical Ecology, Vol. 26, No. 1, 2000

0098-0331/ 99/ 0100-0293$18.00/ 0 2000 Plenum Publishing Corporation

293

SEASONAL VARIATION IN CONCENTRATIONS OFFIBER, CRUDE PROTEIN, AND PHENOLIC COMPOUNDS

IN LEAVES OF RED ALDER (Alnus rubra):NUTRITIONAL IMPLICATIONS FOR CERVIDS

M. P. GONZALEZ-HERNANDEZ,1,* E. E. STARKEY,2

and J. KARCHESY3

1Department of Crop Production,Santiago de Compostela University

EPS, Campus University 27002-Lugo, Spain2USGS-Forest and Rangeland Ecosystem Science Center

Corvallis, Oregon 973313Department of Forest Products,

Oregon State UniversityCorvallis, Oregon 97331

(Received March 23, 1999; accepted September 9, 1999)

Abstract—We sought to determine whether changes in chemical compositioncould be a factor in increased utilization of red alder (Alnus rubra) by the twocervids, the Columbian black-tailed deer (Odocoileus hemionus columbianus)and Roosevelt elk (Cervus elaphus roosevelti), during the fall in parts of theDouglas fir region of the Pacific Northwest. We found that concentrationsand astringency of phenolic compounds decreased from spring through fall,while crude protein content remained high. We conclude that red alder leavesprovide a significant source of digestible protein for cervids during fall, whenavailability of nutrients in most forage species is generally declining.

Key Words—tannin, condensed tannin, phenolic compounds, astringency,diarylheptanoid glycoside, oregonin, red alder, deer, elk, crude protein,nutrition

INTRODUCTION

Red alder (Alnus rubra Bong.) is a common deciduous tree widely distributedthroughout much of the Douglas fir region of the Pacific Northwest, but isnot generally considered to be an important food resource for cervids during

*To whom correspondence should be addressed.

GONZALEZ-HERNANDEZ, STARKEY, AND KARCHESY294

most seasons of the year. However, in some areas, black-tailed deer (Odocoileushemionius columbianus) and Roosevelt elk (Cervus elaphus roosevelti) consumesignificant quantities of red alder leaves during the late summer and fall (Crouch,1968; Jenkins and Starkey, 1991; Radwan et al., 1978). Leslie et al. (1984) foundthat black-tailed deer diets contained increased proportions of red alder fromspring (0%), through summer (22%), to autumn (50%), but diets of Rooseveltelk only contained significant proportions of red alder in the autumn (37%).During the autumn, Leslie (1983) observed Roosevelt elk feeding heavily uponabscissing leaves.

Radwan et al. (1978) suggested that the major factor influencing increasedconsumption of red alder during the fall was the changing chemical composi-tion of the leaves during the growing season. They concluded that increasedenergy content (fat) and decreased levels of total phenols might be responsiblefor increased utilization in late summer and autumn.

Phenolics influence forage consumption by reducing digestibility or act-ing as toxins (McArthur et al., 1993). Tannins are phenolic compounds thatare widely recognized as digestibility reducers for cervids feeding upon woodyplants (Robbins et al., 1987; Happe et al., 1990; Bryant et al., 1992; Hagermanet al., 1992). Condensed tannins are commonly retained in the digestive tractsof cervids and are bound to protein and excreted in feces, but hydrolyzable tan-nins are degraded to low-molecular-weight phenolics that do not interact withprotein (Hagerman et al., 1992). Robbins et al. (1987) found that green leavesof red alder collected during summer contained levels of condensed tannin thatsignificantly reduced digestibility of protein for deer.

In contrast to tannins, small nontannin phenolics are readily absorbed fromthe gut and may function as metabolic toxins (McArthur et al., 1993). Nontanninphenolics contained in extracts of birch (Betula sp.) depress ruminant in vitrodigestibility, and phenols in birch may be involved in metabolic disturbancesin hares (Palo et al., 1983, 1985; Palo 1985). Palo (1984) reported distributionof phenolic glycosides in different tissues of Salix spp., some of which haveherbivore deterrent properties. The same study suggested that low-molecular-weight phenolics of Scandinavian deciduous trees may be of importance as anti-nutritional agents in mountain hare and in ruminants. Sunnerheim et al. (1988)reported that the diarylheptanoid glycoside, platyphylloside, inhibits digestibilityof birch (Betula pendula).

McArthur et al. (1993) found that concentrations of nontannin phenolics inred alder leaves were greater than those of nine other shrub species from the PacificNorthwest. Oregonin, a diarylheptanoid glycoside that is structurally similar toplatyphylloside, has been found in bark and wood of red alder (Karchesy andLaver, 1974), and our preliminary analysis utilizing thin-layer chromatography(TLC) confirmed that it is present also in leaves. Because of the structural simi-larity to platyphylloside, oregonin could have significant antiherbivore properties.

RED ALDER LEAF CONTENT AND CERVIDS 295

Our objective was to determine if seasonal increases in the use of red alderleaves by cervids could be related to changes in the relative concentrations ofnutrients and phenolic compounds. Specifically, we focused on neutral deter-gent fiber (NDF), acid detergent fiber (ADF), lignin, and crude protein as indi-cators of nutritional content. Concentrations of condensed tannins and oregoninwere measured to determine if there were seasonal changes in concentrations ofdigestion-reducing and potentially toxic phenolics. Additionally, we measuredthe astringency (capacity of tannins to form insoluble tannin–protein complexes)of leaves to determine if their potential for reducing digestibility varied season-ally.

METHODS AND MATERIALS

Study Areas. Our study was conducted in two locations in the central CoastRange of Oregon (USA), Keller Creek (44816′N, 123858′W) and Missouri Bend(44820′N, 128843′W). The red alder stand at Keller Creek was established asa plantation in 1992 following clear-cut harvest of a mature Douglas fir standin 1991. Elevation of the site ranges from 180 to 240 m MSL with slopes of40–80%. Annual precipitation is about 2000 mm. At the Missouri Bend site,the red alder stand developed through natural seeding following harvest of asecond-growth Douglas fir stand in about 1950. Elevation is 150 m MSL andprecipitation is about 2200 mm.

Methods. Study areas were divided into quarters within which a single tran-sect was randomly selected and sampled during each season. For each transect,10 green leaves were collected at a height of approximately 1.3 m from each of10 trees. These leaves were combined into a single sample. In summer and fall10 abscized leaves were collected from the ground below these same trees andalso pooled into a single sample. Thus for each season and leaf condition (greenor abscized), four pooled samples were collected from a total of 40 trees at eachstudy site. Transects were only sampled once, new transects were selected foreach season.

The leaves were transported immediately to the laboratory where they wereweighed, lyophilized, and stored frozen until analysis. Before analysis, sampleswere ground through a Wiley mill with 1-mm mesh screen. Acid detergent fiber(ADF), neutral detergent fiber (NDF), and lignin were determined with sequen-tial detergent analysis (Goering and Van Soest, 1970). Crude protein was mea-sured using the Kjeldahl method (Harris, 1970).

Tannins were extracted with aqueous acetone at room temperature. One-half gram of ground leaves was stirred with 10 ml of acetone–water (7 : 3) for15 min. The mixture was then centrifuged for 5 min in a clinical centrifuge(5000g). The supernatant was saved, and the sample extracted for 15 min again

GONZALEZ-HERNANDEZ, STARKEY, AND KARCHESY296

with fresh solvent. That process was repeated four times. Acetone was evapo-rated from the supernatant with a rotary evaporator in a 308C water bath. Theremaining aqueous phase was rinsed twice with an equal quantity of diethylether to remove extraneous materials (e.g., waxes and chorlorophyll) (Broad-hurst and Jones, 1978). The aqueous phase, containing proanthocyanidin dimersand oligimers as well as hydrolyzable tannins (Scalbert, 1992), was retained foranalysis and the ether phase was discarded. Residual diethyl ether in the aqueousphase was evaporated with a rotary evaporator and distilled water was added toresult in a total volume of 25 ml. Extracts were placed in polypropylene tubesand stored in a freezer at −108C. Condensed tannins were analyzed using thevanillin method of Broadhurst and Jones (1978) with modifications of Swainand Hillis (1959). Astringency, or the capacity of tannins to precipitate proteins,was determined using the radial diffusion technique of Hagerman (1987).

Oregonin concentrations were determined with high-pressure liquid chro-matography (HPLC), conducted with a Waters HPLC using a Maxima 820 datasystem with UV detection at 280 nm. Analyses of extracts were carried out ona reversed-phase 150- × 4.6-mm 5mm C18 Luna (Phenomenex) column usingisocratic elution with acetonitrile–water (25 : 75) with a flow rate of 0.6 ml/ min.Authentic oregonin was used as an external calibration standard. Known con-centrations of oregonin in methanol were injected onto the HPLC for detectionand were used to obtain a calibration curve equation. This was done by graphingthe area against the concentration of the sample solution (r2

c 0.99). We usedthis equation to quantify the unknown amount of oregonin in each sample.

For the analysis of data, we treated sites as blocks in a completely random-ized block design. Abscissed leaves were only available in summer and fall; thus,a one-way ANOVA was used to determine if nutrient availability or tannin con-tent of green leaves varied seasonally. Seasonal changes in abscized leaves aswell as differences between green and abscized leaves were tested with a two-way ANOVA.

RESULTS AND DISCUSSION

Nutrient Availability. NDF, ADF, and lignin content of green leavesincreased significantly from spring through summer to fall (Table 1). NDF, ADF,and lignin content of abscized leaves did not change significantly between sum-mer and fall. Abscized leaves contained significantly (P < 0.05) more ligninthan did green leaves. Although NDF did not differ between abscized and greenleaves, ADF differences approached significance (P < 0.06), likely as a resultof the greater lignin content. Crude protein content of green and abscized leavesdid not change seasonally, nor were there significant differences between greenand abscized leaves.

RED ALDER LEAF CONTENT AND CERVIDS 297

TABLE 1. SEASONAL VARIATION IN NEUTRAL DETERGENT FIBER (NDF), ACID

DETERGENT FIBER (ADF), LIGNIN, CRUDE PROTEIN OF GREEN AND ABSCIZED RED

ALDER LEAVES

Percent dry weight (mean ± SD)Nutritional variable

and leaf condition Spring Summer Fall

NDFGreena 23.42 ± 2.45 34.03 ± 1.34 40.25 ± 1.48Abscized 39.55 ± 4.66 39.53 ± 2.93

ADFGreena 14.08 ± 0.76 22.33 ± 0.69 25.69 ± 1.46Abscized 28.52 ± 2.34 27.87 ± 3.21

Ligninb

Greena 7.20 ± 1.29 8.60 ± 1.21 11.98 ± 2.26Abscized 13.59 ± 3.32 14.26 ± 3.08

Crude proteinGreen 15.62 ± 1.00 14.68 ± 1.41 14.08 ± 2.36Abscized 12.68 ± 2.10 14.20 ± 3.31

aSeasonal effect significant P < 0.05 (one-way ANOVA).bLeaf condition significant P < 0.05 (two-way ANOVA).

In the coastal Pacific Northwest, forage quality is usually highest in spring,declines through summer, and reaches lowest levels in fall and winter. Leaf fiber(NDF, ADF, lignin) of deciduous shrubs and trees is highest and crude proteinis lowest in the autumn (Happe et al., 1990). We found that fiber content of redalder leaves followed this pattern, but crude protein did not decline significantly,perhaps because red alder is a nitrogen fixer. Red alder continued to be a goodsource of crude protein, even after leaves were abscized.

Phenolic Concentrations and Astringency. Condensed tannin content ofgreen leaves was highest in spring and declined (P < 0.05) during summer andfall (Table 2). Condensed tannin levels of abscized leaves did not vary betweensummer and fall, nor did they differ significantly from those of green leaves.Astringency of green leaves declined from spring through summer and fall.

Availability of protein is influenced by astringent tannins, which reduce pro-tein digestibility. Happe et al. (1990) found that astringency of understory shrubleaves in western Washington was highest in the spring and significantly lowerin summer and fall. We found that astringency of green leaves of red alder fol-lowed a similar seasonal pattern of decline (P < 0.06), decreasing by about 50%from spring through fall, and conclude that protein digestibility of these leaves isgreatest in the fall. Seasonal differences were not significant for abscized leaves(Table 2).

Oregonin content varied seasonally in red alder leaves (P < 0.001). Con-

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TABLE 2. SEASONAL VARIATION IN ASTRINGENCY AND CONCENTRATIONS OF CONDENSED

TANNINS AND OREGONIN OF GREEN AND ABSCIZED RED ALDER LEAVES

Variation (mean ± SD)Nutritional variable

and leaf condition Spring Summer Fall

Condensed tannin(mg catechinequivalent/ gdry weight)

Greena 65.18 ± 7.12 43.17 ± 4.20 25.44 ± 4.34Abscized 42.73 ± 13.62 42.36 ± 11.90

Astringency(mg tannic acidequivalent/ gdry weight)

Green 74.22 ± 6.00 62.50 ± 2.21 29.69 ± 17.90Abscized 62.50 ± 9.11 48.44 ± 20.31

Oregoninb

(mg oregonin/ gdry weight)

Greena 96.40 ± 10.10 70.20 ± 10.60 47.60 ± 6.56Abscized 15.20 ± 7.50 37.50 ± 10.50

aSeasonal effect significant P < 0.05 (one-way ANOVA).bLeaf condition significant P < 0.05 (two-way ANOVA).

centrations of oregonin in green leaves decreased from spring through summerto fall (Table 2). Levels during the fall were about 50% lower than in spring.Abscized leaves contained significantly lower levels of oregonin than did greenleaves (P < 0.001). However, oregonin content of abscized leaves did not changesignificantly between summer and fall.

Nutritional Implications for Cervids. Unlike many understory shrubs(Happe et al., 1990), red alder leaves maintain relatively high levels of crudeprotein from spring until abscission in the fall, when concentrations and astrin-gency of phenolic compounds are at their lowest seasonal levels. Thus, seasonalavailability of protein in red alder leaves is likely to be greatest in the fall, whenthey are very abundant. Annual leaf litter production of red alder stands mayexceed 4000 kg/ ha, much of which occurs in September and October (Gesseland Turner, 1974). Red alder leaves provide a significant source of protein in aseason when forage quality is an important limiting factor for cervid populations(Nelson and Leege, 1982).

Diet selection by cervids is complex and reflects animal preferences, nutri-tional quality, and availability (Putman, 1988). Individuals seek to optimize quan-tity and quality of forage, and diets will often be a compromise between preference

RED ALDER LEAF CONTENT AND CERVIDS 299

and availability. We were not able to determine if cervids actively select red alderleaves in the fall, nor if the increased consumption is a response to increased avail-ability. Selection would require a perception of nutritional requirements as wellas the ability to identify those foods that would meet those requirements (Rob-bins, 1983). Changes in condensed tannin concentrations and astringency likelyresult in changes in taste and may provide cues by which cervids can select foragewith relatively greater digestibility of protein. However, McArthur et al. (1993)found that neither condensed tannins nor astringency were primary factors influ-encing dietary selection by mule and black-tailed deer. They suggest that low-molecular-weight, nontannin phenolics may significantly constrain consumptionof some shrubs by deer in the Pacific Northwest. McArthur et al. (1993) also foundthat dietary choices by deer were influenced by the relative balance between nutri-ent content and concentration of allelochemicals.

Tahavanainen et al. (1985) reported that condensed tannins, as well as phe-nolic glycosides, influence diet selection by hares. They also suggested that phe-nolic glycosides and catechins or their volatile derivatives provide importantolfactory stimulae, with food selection not being explained solely by the nutri-tional content and/ or total phenolic concentration. According to Rowell-Rahierand Pasteels (1982) and Rowell-Rahier (1984), phenolic glycosides are essentialin directing the food selection and species composition of willow-feeding insects.Sunnerheim et al. (1988) suggest that deterrence by woody deciduous plantsagainst vertebrate herbivores is related to specific substances rather than groupsof compounds. They reported that the polyphenolic fraction of birch (Betula pen-dula) does not exhibit any measurable effect on digestibility and found a par-ticular phenolic glycoside to be responsible for the main antinutritional effect.Other observations on green alder (A. crispa), willows (Salix sp.), and birchhave shown that phenolic glycosides and isoprenoids are responsible for defen-sive properties against hares in these species (Bryant et al., 1983; Reichart etal., 1984; Tahvanainen et al., 1985).

We hypothesize that decreased concentrations of tannins and the phenolicglycoside, oregonin, result in increased palatability of red alder leaves for cervidsduring the fall. Additional research will be required to determine if cervids areselecting red alder leaves because of increased palatability or are respondingto a seasonally abundant forage resource. Regardless of the cause of increasedconsumption, fall diets of cervids in the coastal Pacific Northwest commonlyinclude significant proportions of red alder leaves (Crouch 1968; Jenkins andStarkey 1991; Leslie et al., 1984; Radwan et al., 1978), and we conclude thatthese leaves represent an important source of digestible protein during a periodwhen forage quality is generally declining.

Acknowledgments—We thank D. Hibbs and M. Newton for helping us in the selection of thestudy areas. Ruben Gonzalez and Yvette Karchesy provided helpful suggestions and assistance in

GONZALEZ-HERNANDEZ, STARKEY, AND KARCHESY300

the laboratory. This research was conducted while the first author was a research associate with theForest and Rangeland Ecosystem Science Center of the U.S. Geological Survey and the Departmentof Forest Science, Oregon State University. She received financial support from the Ministry ofEducation and Culture of the Spanish Government (Programa de Perfeccionamiento de Doctores yTecnologos en el Extranjero).

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