selectiot by red-backed voles

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1 Acta Theriologica W3): ,2004. PLISSN Diet selectiot by red-backed voles ~lekhrionom~~r Kristine KASPARIAN and John S. MILLAR Kasparian K. and Millar J. S Diet selection by red-backed voles Clethrionomys gapperi. Acta Theriologica 49: Food supplementation studies often assume that animals will select artificial diets in their natural environment, and that high-protein, high-energy foods are the most appropriate supplement. These assumptions were tested in red-backed voles Clethrionomys gapperi Vigors, 1830 using food-choice experiments with sunflower seeds, oats, and commercial diets in the laboratory and field. Preferred level of dietary protein was also examined using isocaloric diets that varied only in protein content (14%, 20% and 30%). Preferences exhibited in the abovetrials were subsequently examined relative to natural forage. Voles demonstrated a strong preference for sunflower seeds over oats, dried alfalfa, and rabbit, guinea pig, rat and cat food. Voles preferred the 14% protein diet over the 20% and 30% protein diets. Although sunflower seeds contain more than 20% protein, voles consistently preferred this food over natural forage, perhaps because of their high fat and energy content. This indicates that tradeoffs in protein content may be made to maximize energy. We suggest that red-backed voles will select sunflower seeds in their natural environment, and that their preference for low protein likely reflects their herbivorous diet. This study highlights the importance of an a priori understanding of species-specific preferences and requirements when designing food supplementation studies. Department of Biology, University of Western Ontario, London, ON, Canada, N6A 5B7, k-kasparian@hotmail.com Key words: Clethrionomys gapperi, food choice, food preference, protein, supplement Introduction Experimental manipulation of food availability has been widely used to examine the effects of food resources on individual behaviour, life histories, and population or community dynamics (for review see Boutin 1990). Such manipulations may involve the addition or reduction of available food, although there has been a strong bias towards food addition because food removal studies are difficult to conduct. Foods that are not naturally available are commonly used in supplementation studies because they are easily obtained, cost effective and nutritional content is known and easily adjusted. Generally, supplemental foods meet nutritional requirements for maintenance in the laboratory. However, an effective supplement must also offer the dietary components that are potentially limiting in the natural environment. For example, growth and maturation of deer mice Peromyscus maniculatus in the Kananaskis Valley, Alberta are constrained by food (McAdam and Millar 1999a). While several

2 290 K. Kasparian and J. S. Millar diets were adequate for maintenance and reproduction in the laboratory (McAdam and Millar 1999b), only cat food, which contained high levels of animal protein, was effective in enhancing growth and maturation in the field. Hence, studies that do not show a response to food supplements may not be providing an appropriate quality of food (eg Gilbert and Krebs 1981, Teferi and Millar 1993, Prkvot-Julliard et al. 1999). The period of lactation may be of particular importance in mammals because protein requirements are thought to reach their peak during this phase (Clutton-Brock et al. 1989, Chan-McLeod et al. 1994). Inadequate protein during lactation reduces growth rates of nestlings in many small mammals (National Research Council 1995). Protein content is often used to indicate the quality of a diet, although foods with the same protein content can have different amounts of various amino acids. While high quality foods should have the essential amino acids that most closely approximate the needs of the animal (Maynard et al. 1979), essential amino acid requirements are unknown for most species. Consequently, high-energy, high- -protein foods tend to be used as supplements (eg Cole and Batzli 1978, Desy and Thompson 1983). High-protein foods may not be appropriate for herbivorous species that traditionally feed on low-protein foods (Robbins 1993) because they may incur costs associated with the catabolism and excretion of excess protein (Maynard et al. 1979, Lewis et al. 2001). Using commercial diets as supplements assumes that animals will select a novel food in their natural environment. The alternative involves augmenting foods that are part of the natural diet, but this may have other consequences. For example, fertilizer can increase natural forage over large areas (eg Holmes 1991), but concurrently changes the plant community, habitat structure (Holland et al. 1992), and the chemistry of plants, with possible effects on herbivore preference (Bryant and Chapin 1983). Similarly, seeds that are naturally available can be supplied in excess (eg Eccard and Ylonen 2001), but may not be favoured when present in large amounts because of deterrent compounds such as resin (Grodzihski and Sawicka- -Kapusta 1970). Foods eaten by red-backed voles Clethrionomys gapperi Vigors, 1830 in their natural environment have been well documented (Schloyer 1977, Perrin 1979, Martell 1981, Merritt 1981, Norrie and Millar 1990), and include vegetative portions of plants, nuts, seeds, berries, mosses, lichens, ferns, fungi and occasionally insects. Supplementation experiments with related Clethrzonomys species have commonly used sunflower seeds, oats, or rabbit chow as an additional food source. Such diets are sufficient to maintain voles in the laboratory but pqeferences for particular foods were not established. We conducted food-choice experiments in the laboratory and field to determine the preferences of red-backed voles for commonly used commercial diets. We then used isocaloric laboratory diets varying only in protein content to investigate the preferred level of dietary protein during different stages of reproduction. We predicted that lactating females would prefer higher levels of dietary protein than

3 Diet selection by Clethrionomys 291 immature voles and scrotal males due to the high protein requirements associated with lactation (Clutton-Brock et al. 1989, Chan-McLeod et al. 1994). Finally, we examined the preferences exhibited in the first two studies relative to natural forage. Material and methods Red-backed voles were live-trapped throughout the Kananaskis Valley, Alberta (51 N, 115"W) from 3 June to 17 August At capture, all voles were uniquely tagged for identification, weighed, and breeding condition was recorded. Males were classified as having testes scrotal or abdominal, and females as perforate, non-perforate, pregnant (as determined by body weight and swelling of the abdomen), and/or lactating (nipples prominent and free of hair). Lactating females were released upon capture to prevent death of young in the field. All other voles were returned to the Kananaskis Field Station (University of Calgary) and held in plastic cages (0.29 x 0.18 x 0.12 m) containing corn-cob bedding, cotton batting, and provided with sunflower seeds, oats and water ad libitum. The daily photoperiod in the laboratory was 16 light : 8 dark, and laboratory temperature was maintained at 20 C. Commercial food selection Voles were acclimated in the laboratory 0-5 days before being transferred to plexi-glass enclosures (0.8 x 0.4 x 0.6 m) with corn-cob bedding and a metal nest box containing cotton batting. Water was provided ad libitum. Voles were weighed daily (k 0.5 g) using a Pesola spring balance. Trials included 3 days of acclimation to the enclosure followed by 3 days of food selection. During the 3-day acclimation period, voles were initially provided with sunflower seeds and oats. However, after five replications, natural forbs and peanuts were used during the acclimation period of the remaining twelve replicates because sunflower seeds and oats were among the test foods. Voles were allowed to choose among eight experimental diets, including shelled sunflower seeds, whole sunflower seeds, whole oats, Purina rat chow 5010, rabbit chow, cat food (Co-op No. 320C), guinea pig food, and dried alfalfa (Table 1). The sunflower seeds represented high-energy food, while the oats were low-energy food. Shelled and whole sunflower seeds were used to determine if voles had a handling preference between these foods. Rat and rabbit chow represented diets used in the laboratory and in vole supplementation experiments (Cole and Batzli 1978, Bronson 1998, Koskela et al. 1998, Eccard and Ylijnen 2001). Cat food was the only diet composed almost entirely of animal protein. Guinea pig food was selected as high-protein and low-energy while the alfalfa most closely resembled the herbs upon which these voles naturally forage. On the first day of feeding trials, g of each diet, with the exception of 2.00 g of alfalfa due to the low density of this food, was weighed on an Table 1. Composition of diets used in the laboratory and field food selection experiments with Clethrionomys gapperi. Sh - shelled sunflower seeds, Wh - whole sunflower seeds, Oa - whole oats;ra - Purina rat chow 5001, Ca - CO-OP cat food No. 320C, Gu - Hartz guinea pig food, A1 - Hartz alfalfa, Rab - Hagan rabbit chow. "utrient composition from Scherz and Senser (1994), modified from McAdam and Millar (1999b), carbqhydrate component of energy values was estimated by the difference method (Scherz and ~enser 1994). Nutrient Units Sh a Wha Oa a Rac cab Gu" Al" Rab c Energy Protein kcallg % Fat %

4 292 K. Kasparian and J. S. Millar electronic balance ( g), and randomly assigned to sections of a feeding tray. At 24-h intervals, for 3 consecutive days, all food was removed from the enclosure (including husks and pieces of food in the bedding) separated by diet, and weighed. Food was replaced after weighing, and at the end of each trial, all bedding was removed and replaced with fresh material. To account for moisture absorbed by the food, mock trials were conducted in which food was left in enclosures along with a water dish. Each diet was weighed daily over three consecutive days. Rates of water absorption were estimated as the slope of the linear regression of mass on hours for each diet. Total amounts of each diet consumed per day were calculated, correcting for water absorption. Field food selection Shelled sunflower seeds and oats were tested against one another in the field from 1 August to 11 August Longworth live traps (n = 95) that consistently caught voles on three monitored grids were used as test sites. Sunflower seeds and oats were dried to a constant weight, and 5.00 g of each diet were weighed (20.01 g). Traps were cleaned, supplied with fresh bedding, and baited with the sunflower seeds and oats for a total of g ( g) in each trap. Traps were set in the evening and checked the following morning. Traps containing voles were emptied of all contents, which were then dried, weighed, and consumption of oats and sunflower seeds was calculated. Voles were marked with ear tags, weighed, and breeding condition was assessed prior to their release at the site of capture. Traps were set for six nights at each site, and consumption was averaged for all voles caught more than once. Protein selection Protein selection experiments were conducted at the Kananaskis Field Station from 7 June to 6 September Protein selection experiments were conducted in metal enclosures (0.6 x 0.2 x 0.1 m) lined with wood shavings and provided with a nest can containing cotton batting. Water was provided ad libitum.trials involving immature and scrota1 voles were conducted during five consecutive days following two days of acclimation to sunflower seeds and oats. Trials involving pregnant and lactating Table 2. Nutritional composition of isocaloric diets with different protein content, used in the laboratory food selection experiments with Clethrionomys gapperi (modified from McAdam and Millar ). Nutrient Protein content Units 14% 20% 30% Energy Fat Protein Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Minerals Vitamins

5 Diet selection by Clethrionomys 293 females were conducted during twelve consecutive days (approximately 60% of the lactation period) following two days of acclimation to sunflower seeds and oats. Three isocaloric diets containing 14, 20, or 30% casein based protein (Purina Test Diets, Nos. 5773C-F, 5773C-G, 5773C-H; Table 2) were dried to a constant weight. Immature voles, scrota1 males, and pregnant females received 5.00 & 0.01 g of each diet daily, while lactating females received g of each diet daily. To identify the different diets, pellets were dyed with food colouring and colours were rotated daily. Diets were randomly assigned to sections of a feeding tray. Total trial time was recorded to the nearest five minutes each day, and all food was removed from the enclosure, separated by diet, dried, and weighed. New food was added daily and at the end of each trial, all bedding was removed and replaced with fresh material. Food selection using natural forage Food selection experiments were conducted at the Kananaskis Field Station from 27 July to 13 August Natural food selection experiments were conducted in plastic cages (0.46 x 0.25 x 0.21 m). Breeding condition was assessed, but only males and non-reproductive females were used because adequate numbers of breeding females were not available. Trials were conducted during 3 consecutive days following 2 days of acclimation to rabbit chow and oats. Isocaloric diets (14, 20 and 30% protein) and shelled sunflower seeds were dried to a constant weight, and natural forbs were picked daily. The forbs used in the experiment were known to be highly palatable for the same population of red-backed voles (Norrie and Millar 1990), and included: Astragalus spp., Campanula rotundifolia, Cornus canadensis, Hedysarum sulphurescens, Lathyrus ochroleucus, and Linnaea borealis. Protein content was known to vary between 6 and 21% (Millar and Zwickel 1972). Five grams of each isocaloric diet, g of fresh forbs and 5.00 g of shelled sunflower seeds were weighed (k 0.01 g). Isocaloric diets were dyed with food colouring and colours were rotated daily. Diets were randomly assigned to sections of a feeding tray. A control sample of fresh forbs (control-forbs) was weighed and placed in a wire chamber that allowed for water loss but prevented eating to account fdr moisture loss. Each day, total trial time was recorded to the nearest 5 minutes and all food, as well as the control-forbs, was removed from the enclosure. Isocaloric diets and sunflower seeds were dried, and weighed. Remaining forbs and the control-forbs were immediately weighed. Rate of moisture loss was estimated as the slopb of the linear regression of water loss over time. New food was added daily and all bedding was removed and replaced with fresh material at the end of each trial. Statistical analyses were performed using SPSS 11.0 (SPSS Inc 2001). Post hoc tests (Tukey's HSD) were performed using Statistica (Statsoft Inc 1995). Differences among groups were considered statistically significant at p = Data are presented as means + 1 standard error. Results With the exception of lactating females, voles gained an average of g (range ) during experiments. Lactating females used in the protein selection experiments had a mean change in body mass of : 0.1 g during lactation, and supported an average of 5.0 +_ 0.5 young (range 4-8). No diet was consumed solely on any day and no diet was completely depleted over the course of the trials. Commercial food selection Data were pooled for voles acclimated on sunflower seeds and oats with those acclimated on peanuts and natural forbs because there was no difference in growth rates between them (t = 0.16, df = 15, p = 0.88). Simple transformations did not remove the heterogeneity of variance in consumption of commercial diets, so Friedman's analysis of variance by ranks was used to test for differences among +

6 K. Kasparian and J. S. Millar Wh Oa Aal Rab Ra Gu Ca -I Kind of food Fig. 1. Mean (+ SE) amounts of food consumed by Clethrionomys gapperi during 3-day commercial food choice trials. For an explanation of abbreviations of foods see Table 1. Bars with different letters are significantly different (Tukey-type multiple comparison: p < 0.05). total consumption of the eight diets followed by Tukey-type multiple comparison tests for ranked data (Zar 1999). Amounts of food consumed per trial differed significantly among diets k: = 95.46, df = 8,17,p < 0.001; Fig.1).Sunflower seeds were preferred over all other diets, and only negligible amounts of cat food were consumed. Concordance analysis, using Kendall's coefficient of concordance (W) (Siege1 1956), was performed to test whether voles consistently preferred particular food items, and top down concordance was used to verify that the order of the most preferred foods was constant among voles (Zar 1999). There was a high degree of 2 concordance by individual voles among diet rankings (W = 0.79, xr = 94.05, df = 7, p < 0.001). In addition, individual voles were consistent in their order of most preferred foods (%' = 41.84, df = 7, p < 0.001). Field food selection Food choice in the field was compared using a paired t-test. Food preferences exhibited by red-backed voles in the field paralleled that observed in the laboratory. Voles consumed nearly four times more shelled sunflower seeds (1.98 r 0.30 g) than oats (0.55 k 0.19 g; t = 6.40, df = 17, p < 0.001). To determine if voles consistently ate the same proportion of oats and sunflower seeds, the ratio of ingested oats to sunflower seeds was compared between the laboratory and the field

7 Diet selection by Clethrionomys 295 using an independent sample t-test. The proportion of oats and sunflower seeds ingested by red-backed voles in the laboratory ( , n = 17) and in the field ( , n = 18) was not significantly different (t= 0.80, df = 33,p = 0.43). Protein selection Immature voles and scrotal males were pooled because there was no significant difference in daily consumption rates of isocaloric diets (ANOVA: F = 0.64, df = 8,184, p = 0.74). One non-scrota1 male was excluded from the analysis because his total consumption rate and weight gain fell more than two standard deviations above the mean change in body mass of immature voles and scrotal males. Daily consumption rates of pooled data and of breeding females were analyzed separately with a two-way (diet and trial-day as within-subject factors), repeated measures ANOVA. There was a significant main effect of diet (F = 86.03, df = 2,48, p < 0.001), but no main effect of trial-day (F = 2.03, df = 4,96, p = 0.10), and no diet x trial-day interaction for consumption rates of isocaloric diets by immature voles and scrotal males (F = 0.96, df = 8,192, p = 0.47). Multiple comparisons showed that 14% protein was preferred over 20% and 30% protein (Fig. 2a). For breeding females, there was a significant main effect of diet (F = 33.41, df = 2,18,p < 0.001), and a significant main effect of trial-day (F = 7.83, df = 5,45, p < 0.01), but no diet x trial-day interaction for consumption rates of isocaloric diets (F = 1.7, df = 10,90, p = 0.09). Breeding females also consumed more of the low (14%) protein diet than the medium (20%) or high (30%) protein diets (Fig. 2b). There were no differences in consumption rates of breeding females over 2-day intervals (Tukey HSD: p > 0.05) and consumption rate was averaged over this period to simplify comparisons among days. Trial day affected food consumption by lactating females such that consumption rate was significantly lower during the first 2 trial-days than trial days 3 through 12 (Fig. 3). Protein diet (O/O) Fig. 2. Mean (+ SE) consumption rates of three isocaloric diets by Clethrionomys gapperi during protein seleckion trials with (a) immature voles and scrotal males and (b) lactating females. Bars with different letters are significantly different (Tukey HSD: p < 0.001).

8 296 K. Kasparian and J. S. Millar Trial day Fig. 3. Mean (+ SE) consumption rates of iso- Fig. 4. Mean (+ SE) consumption rates of three caloric diets by lactating Clethrionomys gapperi isocaloric diets (14, 20 and 30% protein), natural during 12-day protein selection trials. Days with forbs (Forbs) and shelled sunflower seeds (Sh) different letters are significantly different (Tukey by immature and scrota1 Clethrionomys gapperi HSD: p < 0.05). during 3-day food selection trials. Bars with different letters are significantly different (Tukey HSD: p < 0.001). Food selection using natural forage Food consumption varied among food types (F = 17.66, df = 4,32,p < 0.001), but there was no effect oftrial-day (F = 2.58, df = 2,16,p = 0.11) and no diet x trial-day interaction (F = 1.92, df = 8,64, p = 0.07). Voles consumed shelled sunflower seeds at twice the rate of the 14% protein diet and three times higher than the 20% and 30% protein diets and natural forbs (Fig. 4). There were no differences among the consumption rates of the less preferred diets. Discussion The basis for food choice by herbivorous animals involves numerous dietary and sensory factors, including nutritional content, secondary compounds, palatability, and texture (Ramos 1996). While the relative importance of these factors was not clear from this study, the maintenance of body mass by lactating females, and the consistent weight gained by all other voles indicated that voles were selecting foods that were nutritionally adequate. Food selection in all experiments was partial rather than absolute, although voles could have met daily energy requirements (approximately 20 Kcal x day-'; McManus 1974) through the consumption of any one of the experimental diets, Tests of diet selection and optimal foraging theory consistently falsify the all-or- -nothing hypothesis (Krebs and Kacelnik 1991), and have suggested that small mammals may benefit from sampling novel foods to assess their nutritional value

9 Diet selection by Clethrionomys 297 (Vickery 1984). In addition, selection of a variety of foods may ensure a balanced intake of nutrients not provided in a single diet (Naim et al. 1986). Red-backed voles consistently selected sunflower seeds over the six other commercial diets. Oats and alfalfa were consumed to a lesser degree, but only small quantities of the remaining diets (guinea pig, rat, rabbit and cat food) were eaten. There was donsiderable variation in the degree to which less desirable foods were eaten. Significantly more oats were consumed than guinea pig, rat, rabbit and cat food, suggesting that voles were not simply sampling the oats, but the remaining foods may have been sampled to assess their nutritional value (Vickery 1984). Findings from the field choice experiments were consistent with laboratory results. Voles preferred sunflower seeds to oats and consumed each food in similar proportions to voles in the lab, suggesting that diet selection in the laboratory reflects choices made in the field. Voles were able to differentiate among foods based on protein content, and they exhibited a preference for the lowest protein content (14%) offered. This level of dietary protein was higher than the minimum daily nitrogen requirements (4.4%) of captive prairie voles Microtus ochragaster (Ditchkoff et al. 1998), the 8% dietary protein that appears sufficient for free ranging field voles Microtus agrestis (Spears and Clarke 1987), and the 2 13% casein required for normal growth rates of weanling meadow voles Microtus pennsyvanicus (Shenk 1976). Protein requirements should vary with species and diet composition (Robbins 1993), but because the lowest protein content available in this experiment was higher than that known to be required by other voles, the desired protein content of foods eaten by red-backed voles is likely lower than 14%. Selection by all reproductive classes, including lactating females, for the lowest protein diet likely indicates that 14% protein may exceed the requirements of even breeding females. In this study, females met the additional cost of lactation by increasing the quantity rather than the quality of food consumed. This suggests that protein requirements during lactation increase in proportion to energy requirements, and therefore lactating females were able to meet both needs through increased consumption of the 14% protein diet. Preferences exhibited in the natural food selection experiments were consistent with those from the commercial and protein selection experiments. Preference for sunflower seeds was maintained when offered natural foods. The 14% protein diet also tended to be consumed at a rate 1.5 to 2 times greater than the natural forage although the difference was not significant. This suggests that red-backed voles will select sunflower seeds and potentially the 14% isocaloric diet over natural foods and that familiarity of visual cues such as shape and texture may be of less importance than nutritional value in food selection. In additiori, maximizing energy intake appears to be of greater importance than optimizing protein intake. Red-backed voles may prefer a quality of food that was not satisfied by any of the choices offered in the trials, and voles may have been forced to optimize their diet through some combination of what was available. For example, the high fat content of sunflower seeds (49.0%) may have been desirable while the protein content

10 298 K. Kasparian and J. S. Millar (22.5%) was not. High-fat foods are generally high in energy and numerous studies have shown that small mammals forage in a manner that maximizes energy intake (eg Smith and Folmer 1972, Kerley and Erasmus 1991), rather than protein intake (Vickery et al. 1994, Lewis et al., 2001). This implies that small mammals can acquire sufficient protein by choosing high-fat, energy-rich foods (Vickery et al. 1994). In addition, amounts of fat consumed beyond requirements are readily stored in the form of body reserves (Maynard et al. 1979). In contrast, excess protein provides no such benefit and is associated with higher costs of catabolism and excretion (Maynard et al. 1979). While it is important to note that cafeteria trials are constrained because consumption of food is dependent on the choices offered, which rarely reflect natural diversity, we suggest that sunflower seeds are a palatable and nutritionally appropriate food supplement for red-backed voles in Kananaskis, Alberta. In addition, voles prefer low-protein to high-protein diets regardless of age, sex, or reproductive condition. This is most likely a reflection of their herbivorous diet. High-protein diets are not necessarily the most appropriate quality of food, and species-specific preferences should be investigated prior to carrying out food supplementation studies. Acknowledgements: We thank the staff at the Kananaskis Field Station for their support during the summers. V. Pilorz was most helpful in the laboratory, and we thank E. J. Herdman and D. T. J. Sare for valuable discussion on early drafts. L. Zanette's comments greatly improved the manuscript. Animal care and treatment protocols were approved by the University Council on Animal Care and were in accordance with the principles outlined by the Canadian Council on Animal Care. This study was supported by an NSERC operating grant to J.S.M. References Boutin S Food supplementation experiments with terrestrial vertebrates: patterns, problems, and the future. Canadian Journal of Zoology 68: Bronson F. J Do small voles require less food than large voles? Canadian Journal of Zoology 76: Bryant J. P. and Chapin 111 F. S Carbonlnutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40: Chan-McLeod A. C. A., White R. G. and Holleman D. F Effects of protein and energy intake, body condition, and season on nutrient partitioning and milk production in caribou and reindeer. Canadian Journal of Zoology 72: Clutton-Brock T. H., Albon S. D. and Guinness F. E Fitness costs of gestation and lactation in wild mammals. Nature 337: Cole F. R. and Batzli G Influence of supplemental feeding on a vole population. Journal of Mammalogy 59: Desy E. A. and Thompson C. F Effects of supplemental food on a Microtus pennsylvanicus population in central Illinois. Journal of Animal Ecology 52: Ditchkoff S. S., Boyd C. S., Welch E. R. Jr, Raglin J. B. and Lochmiller R. L Nitrogen requirements of the adult prairie vole (Microtus ochragaster). American Midland Naturalist 140: Eccard J. A. and Ylonen H Initiation of breeding after winter in bank voles: effects of food and population density. Canadian Journal of Zoology 79:

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