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Sequence of food presentation influences intake of foods containing tannins and terpenes
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Applied Animal Behaviour Science 113 (2008) 57–68 www.elsevier.com/locate/applanim
Sequence of food presentation influences intake of foods containing tannins and terpenes Travis E. Mote, Juan J. Villalba *, Frederick D. Provenza Department of Wildland Resources, Utah State University, 5230 Old Main Hill, Logan, UT 84322-5230, USA Accepted 9 October 2007 Available online 26 November 2007
Abstract Interactions among nutrients and secondary compounds in plants can influence the kinds and amounts of different forages herbivores ingest, but little is known about how the sequence of plant ingestion may influence these interactions. The physiological pathways and rates of nutrient and secondary compound metabolism in the body influence food intake by herbivores. On this basis, we predicted the sequence in which foods that vary in nutrients and secondary compounds are ingested would influence food intake and preference. In a 2 2 factorial experiment, we evaluated the relationship between the sequence of presenting two foods, one with terpenes and the other with tannins, and the time when lambs ate a nutritious food (alfalfa–barley), either before or after eating foods with tannins and terpenes. When alfalfa–barley was fed prior to the terpenes, intake of the terpene-containing food was lower than when alfalfa–barley was fed after terpenes (P < 0.05). The sequence when alfalfa–barley was fed did not influence intake of the tannin-containing food (P > 0.10). Lambs ate more total foods with terpenes + tannins when fed tannins ! terpenes ! alfalfa/barley than when fed alfalfa/barley ! tannins ! terpenes (P < 0.10). During preference tests, when lambs were offered all three foods simultaneously, lambs previously conditioned with the sequence tannins ! terpenes ! alfalfa–barley preferred alfalfa–barley > terpenes > tannins (P < 0.05), whereas lambs in other treatments preferred alfalfa–barley > tannins > terpenes (P < 0.05). During preference tests when lambs were fed only foods with secondary compounds, lambs previously conditioned with the sequence tannins ! terpenes ! alfalfa–barley showed equal preference for foods with tannins and terpenes, whereas lambs in other treatments preferred food with tannins > terpenes (P < 0.05–0.10). All of these results are consistent with the hypothesis that the sequence in which foods are consumed affects both food intake and preference. Understanding the importance of sequencing when herbivores consume foods that vary in nutrients and secondary compounds may help managers create new grazing strategies
* Corresponding author. Tel.: +1 435 797 2539; fax: +1 435 797 3796. E-mail address: [email protected] (J.J. Villalba). 0168-1591/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.applanim.2007.10.003
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that include sequential foraging patterns to optimize food intake and more evenly use all plant species in a community, a practice used by herders in France. Published by Elsevier B.V. Keywords: Foraging sequence; Diet mixing; Sheep; Intake; Tannins; Terpenes
1. Introduction Most plants contain secondary compounds that, at too high concentrations, limit how much of any particular food a herbivore can consume (Cheeke and Schull, 1985; Foley et al., 1999). Thus, herbivores must regulate intake of plants with secondary compounds in order to ingest adequate levels of nutrients and avoid toxicosis. Any mechanism that increases rates of elimination of secondary compounds can increase the ability of herbivores to consume plants that contain these chemicals (Dziba et al., 2006). Eating a variety of foods is one way to accomplish this objective (Freeland and Janzen, 1974; Provenza, 1996), as different kinds of secondary compounds are processed at different rates and through different metabolic pathways, thereby providing multiple avenues for detoxification (Freeland and Janzen, 1974; Boyle et al., 1999; Marsh et al., 2006). Thus, varied diets enable herbivores to ingest more nutrients from plants that contain secondary compounds, provided the secondary compounds are complementary (Dearing and Cork, 1999; Burritt and Provenza, 2000; Villalba et al., 2004). Diversity in the chemical composition and availability of plants on landscapes also provides the context for herbivores to learn about the consequences of eating different foods in succession, which may provide another opportunity to ingest plants with secondary compounds. The time when herbivores consume different foods in a sequence may influence how much of different foods they can consume. This is because food intake and preference are influenced by rates of absorption of secondary compounds and their interactions in the gastrointestinal tract. For instance, terpenes are small, fat-soluble molecules (Foley and McArthur, 1994) that are absorbed quickly through the rumen epithelium such that they can quickly decrease food intake (Dziba et al., 2006). Conversely, tannins are high-molecularweight compounds (2000–4000) that remain in the gut where they can interact with other chemicals (Kumar and Singh, 1984; Min and Hart, 2003). Tannins form complexes with several molecules including proteins, polysaccharides, nucleic acids and minerals mainly through hydrophobic/hydrogen interactions (Min and Hart, 2003). Thus, a meal of tannins followed by a meal of terpenes may lead to higher levels of intake than the reverse, if tannins reduce absorption of terpenes. Nutrients in the gut may further enhance these responses. Dietary proteins bind to tannins, mitigating the negative effects of tannins on tissues and cellular processes (Kumar and Singh, 1984), and energy and protein can enhance intake of foods high in secondary compounds (Provenza et al., 2003). We hypothesized that food intake and preference can be influenced by the aforementioned physiological processes, whereby nutrients and secondary compounds may interact in different ways as they are absorbed and metabolized at different rates. On this basis, we predicted the sequence in which foods that vary in nutrients and secondary compounds are ingested influences food intake and preference. We determined whether the temporal sequence in which foods with secondary compounds (tannins and terpenes) and a high-quality food (alfalfa–barley mix) were ingested affected food intake and preference by lambs.
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2. Materials and methods The experiment was conducted at the Mote Farm in Willard, UT, USA (41.48N, 112.08W). Weaned commercial Rambouillet-Columbia-Finn-Targhee crossbred lambs, averaging 35 kg (S.E.M. = 0.9), were individually penned with free access to trace mineralized salt blocks and fresh water. The lambs were kept outdoors, under a protective roof in individual, adjacent pens measuring 2.4 m 3.6 m. All procedures were approved by the Utah State University Institutional Animal Care and Use Committee (IACUC #1207). 2.1. Test foods Test foods were formulated to be similar in secondary compound and nutrient contents to the current year’s growth of shrubs such as sagebrush (Artemisia tridentata) and bitterbrush (Purshia tridentata), which are common forages for domestic and wild herbivores in the sagebrush steppe ecosystem during autumn and winter. We used two isocaloric (12.1 MJ DE/kg DM) and isonitrogenous (126 g CP/kg DM) (National Research Council (NRC), 1985) foods, similar to those used by Villalba et al. (2004). The tannin-containing food was 76% beet pulp, 9% soybean meal, and 15% quebracho tannin powder. The terpene-containing food was 52.8% beet pulp, 26% grape pomace, 14% soybean meal and 7.2% terpene solution (24% camphor, 16% 1-8-cineole, 1.0% p-cymene and 59% vegetable oil). Terpene volatilization was reduced by mixing terpenes with vegetable oil, which also improved terpene homogeneity in the diet (Kimball et al., 1998). The high-quality food was a (50:50) mix of alfalfa and barley. All food ingredients were ground to 2–3 mm particle size to facilitate mixing. 2.2. Conditioning Thirty-two lambs were stratified by weight and randomly assigned to four treatments (eight lambs/ treatment) in a 2 2 factorial design that included the sequence of presenting the tannin- and terpenecontaining foods relative to the basal diet (Table 1). The first food in the sequence was offered at 06:00 h each morning, and each component of the sequence was available to lambs for 1 h. Lambs were fed their respective sequences for a 14-day conditioning period. Lambs in different treatments were randomly distributed in adjacent pens. In previous studies, we found that the likelihood of animals influencing one another is minimal because they all eat at the same time and the type of food on offer is more important than observing neighbors eat (Villalba and Provenza, 1997c). The alfalfa–barley mix was restricted to 250 g/d, whereas the secondary compound-containing foods were offered ad libitum. Sheep learn best to ingest foods with secondary compounds when the availability of nutritious alternatives is limited (Shaw et al., 2006a), and they could meet nutritional needs by eating a combination of the foods with tannins and terpenes used in our study, especially given the additional 250 g/d of alfalfa–barley (Villalba et al., 2004). In rangeland settings, sheep are unlikely to learn to eat shrubs such as sagebrush, which is high in terpenes, unless the availability of more palatable and nutritious grasses and forbs is restricted (Shaw et al., 2006b). Thus, we restricted the availability of the palatable alfalfa–barley mix to encourage consumption of the tannin- and terpene-containing foods (Shaw et al., 2006a). 2.3. Preference tests After conditioning, all animals were fed alfalfa pellets ad libitum from 06:00 to 09:00 h for 3 d to allow tannins and terpenes to be eliminated from the body. The retention of quebracho tannin in the gastrointestinal tract ranges from 48 h (free and soluble) to 72 h (bound to protein and fiber) (Silanikove et al., 1994, 1996). The average first pass elimination of plasma terpenes occurs within minutes after terpenes are infused into the rumen or blood stream (Dziba et al., 2006). Preference tests were performed at 06:00 h. Test foods were offered simultaneously, in separate plastic containers that fitted tightly into a wooden food box attached to each pen. The placement (left vs. right) of specific foods was random across pens and days. During the first period of preference tests, lambs were offered all three test foods –terpene, tannin, alfalfa–barley – ad libitum for 30 min, whereas during the
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Table 1 Treatments for the 2 2 factorial design for the sequence of presentation to lambs of secondary compounds relative to the alfalfa–barley basal diet
second period of preference tests, lambs were offered only the tannin- and terpene-containing foods ad libitum for 30 min. Refusals were collected and weighed. Intake was determined as the difference between the amount of food presented and the amount of food refused for each food offered during the 30-min preference test. Thus, we determined food choice when animals could avoid secondary compounds and consume a high-quality food (alfalfa–barley) (Period 1), and when they could chose between two foods of similar nutritional composition that both contained secondary compounds (terpenes and tannins) (Period 2). During both periods, preference tests were conducted on 2 consecutive days (Table 1). Lambs showed a consistent pattern of selection during these 2 d (see Section 3 for details), so no further tests were performed. In tests such as these, sheep typically manifest consistent patterns of preference, such that once preference is established, they continue to prefer/avoid specific foods (Villalba and Provenza, 1997a,b,c, 1999a). 2.4. Statistical analyses Data were analyzed as a 2 2 factorial design with two factors: (1) sequence of presentation of secondary compounds (level 1: first terpenes, then tannins; level 2: first tannins, then terpenes), and (2) time
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when the basal diet was fed (level 1: prior to offering the secondary compounds, level 2: after offering the secondary compounds) (Table 1). Day was the repeated measure in the analyses. When significant effects were detected (P < 0.10), differences among means were determined using Tukey’s multiple comparison procedure (Hayter, 1984). Intake of individual secondary compound-containing foods and total food consumption were the response variables for the conditioning and preference trials. Daily refusals were subtracted from the amount offered (as-fed basis) to estimate intake, which was calculated based on body weight as grams of food ingested/kg body weight. For preference tests, percentage preference was also used as a response variable. Preference was computed as: [intake of a single plant secondary compoundcontaining food/total intake of plant secondary compound-containing foods] 100%. ANOVAs were performed using the MIXED procedure in SAS 9.1 for Windows (SAS, Littell et al., 1996).
3. Results 3.1. Conditioning Total consumption of the foods with secondary compounds during conditioning differed for lambs fed in different sequences (P = 0.006, Fig. 1). When the high-quality diet of alfalfa–barley was fed after the secondary compounds, intake of terpene-containing food was higher (6.60 g/kg
Fig. 1. Consumption of foods with tannins and terpenes and total consumption of food with secondary compounds (tannins + terpenes) by four groups of lambs during conditioning. Lambs in Treatment 1 were offered three foods in the sequence alfalfa–barley (AB) ! terpene (Ter) ! tannin (Tan). Lambs in Treatment 2 were fed Ter ! Tan ! AB. Those in Treatment 3 were fed AB ! Tan ! Ter. Lambs in Treatment 4 were fed Tan ! Ter ! AB.
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BW; S.E.M. = 0.42) than when the high-quality diet was fed before the secondary compounds (4.73 g/kg BW; S.E.M. = 0.42; P < 0.05). Conversely, the time when the high-quality diet was fed did not influence intake of the tannin-containing food (P > 0.10). Though the pattern of food intake was consistent among treatments (Fig. 1), there was a treatment day interaction (P = 0.02). Intake of the terpene-containing food was reduced relative to the other treatments when the sequence was alfalfa–barley ! tannins ! terpenes (Treatment 3) (P < 0.05; Fig. 1). In contrast, intake of the terpene-containing food was highest when the sequence was tannins ! terpenes ! alfalfa–barley (Treatment 4) (P < 0.05; Fig. 1). Averaged across the 14-d period, consumption of the food with terpenes for Treatments 1, 2, 3, and 4 was 6.26, 6.17, 3.20, and 7.04 g/kg BW, respectively. Averaged across the 14-d conditioning period, total consumption of foods with secondary compounds was higher (P < 0.10) for Treatments 1 (alfalfa–barley ! terpenes ! tannins) (13.99 g/kg) and 4 (tannins ! terpenes ! alfalfa–barley) (14.46 g/kg) than for Treatment 3 (alfalfa–barley ! tannins ! terpenes) (11.06 g/kg) (S.E.M. = 1.35). None of the treatments differed from Treatment 2 (terpenes ! tannins ! alfalfa–barley) (12.97 g/kg; P > 0.10; Fig. 1). 3.2. Preference tests During Preference Test 1, when all three foods were offered simultaneously, lambs in Treatment 4, previously conditioned with the sequence tannins ! terpenes ! alfalfa–barley, preferred alfalfa–barley > terpenes > tannins (P < 0.05), whereas lambs in the other three treatments preferred alfalfa–barley > tannin > terpene (P < 0.05) (Fig. 2). Treatments did not differ in total intake of foods with secondary compounds (sequence of secondary compound presentation time alfalfa–barley was fed, P = 0.32), and outcomes of preference tests did not differ across days (day effect; treatment day interaction; P > 0.10) (Fig. 2). During Preference Test 2, when lambs were offered only foods with secondary compounds, lambs in Treatments 1, 2, and 3 preferred tannin > terpene, whereas those in Treatment 4 showed similar preference for terpenes and tannins (Fig. 3). Intake of foods with secondary compounds also differed by treatments during this preference test (P = 0.006; Fig. 3). Lambs ate more food with terpenes when alfalfa–barley was fed last as opposed to first (4.88 vs. 2.63 g/kg BW;
Fig. 2. Intake of alfalfa–barley, terpene-, and tannin-containing foods offered ad libitum during Preference Test 1. Lambs were conditioned to eat alfalfa–barley before or after eating one of two possible sequences of tannin- and terpenecontaining foods. Lambs in Treatment 1 were fed three foods in the sequence alfalfa–barley ! terpene ! tannin. Lambs in Treatment 2 were fed terpene ! tannin ! alfalfa–barley. Those in Treatment 3 were fed alfalfa–barley ! tannin ! terpene. Lambs in Treatment 4 were fed tannin ! terpene ! alfalfa–barley.
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Fig. 3. Intake of terpene- and tannin-containing foods offered ad libitum during Preference Test 2. Lambs were previously conditioned to eat alfalfa–barley before or after eating one of two possible sequences of tannin- and terpene-containing foods. Lambs in Treatment 1 were fed three foods in the sequence alfalfa–barley ! terpene ! tannin. Lambs in Treatment 2 were fed terpene ! tannin ! alfalfa–barley. Those in Treatment 3 were fed alfalfa–barley ! tannin ! terpene. Lambs in Treatment 4 were fed tannin ! terpene ! alfalfa–barley.
S.E.M. = 0.47, P < 0.05). Conversely, they tended to eat less food with tannin when alfalfa– barley was fed last rather than first in the meal (5.95 vs. 7.46 g/kg BW; S.E.M. = 0.47, P < 0.15). Lambs in Treatment 3 previously conditioned with the sequence alfalfa–barley ! tannins ! terpenes ate much less terpene-containing food (2.05 vs. 5.50 g/kg BW; S.E.M. = 0.90; P < 0.05; Fig. 3) and more tannin-containing food (7.92 vs. 5.14 g/kg BW; S.E.M. = 0.90; P < 0.10; Fig. 3) than lambs in Treatment 4, previously conditioned with the sequence tannins ! terpenes ! alfalfa–barley. Total intake of foods with secondary compounds was similar among treatments (sequence of secondary compound presentation time basal diet was fed, P = 0.95), and preference tests were consistent across days (day effect; treatment day interaction; P > 0.10) (Fig. 3). 4. Discussion We determined if the temporal sequence in which foods with tannins and terpenes and a nutritious food were offered affected food intake and preference of lambs. We hypothesized that the physiological pathways whereby nutrients and secondary compounds are metabolized and the rates of metabolism influence food intake. On this basis, we predicted the sequence in which foods that vary in nutrients and secondary compounds are ingested influences food intake and preference. We found that when the alfalfa–barley food was offered in the sequence had the greatest impact on intake during conditioning and preference tests, but the sequence in which foods with tannins and terpenes were offered also affected intake and preference. 4.1. Conditioning When the nutritious alfalfa–barley was fed first, sheep did not eat much food with terpenes, and the effect was greatest for lambs in Treatment 3 fed terpenes last during conditioning. This finding is consistent with the observation that sheep learn to eat foods with terpenes best when alternative foods are restricted (Villalba et al., 2004; Shaw et al., 2006a,b), and that total intake of tannin-, terpene-, and oxalate-containing foods by sheep is greater when these foods are eaten
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prior to nutritious foods than when they are eaten after the nutritious foods (Papachristou et al., 2007). Thus, lambs are more highly motivated to consume foods with terpenes when somewhat food deprived than after eating a large meal of a nutritious food. French herders use this principle to enhance consumption of less palatable foods in grazing circuits (Meuret, 1997). Low consumption of terpene-containing food by lambs fed alfalfa–barley ! tannins ! terpenes (Treatment 3) was likely due to (1) the strong flavor of terpenes, which can deter sheep from eating foods with terpenes for several weeks (Banner et al., 2000); (2) eating alternative foods prior to terpenes in the foraging sequence (Shaw et al., 2006a,b); and (3) the lack of synergism between terpenes and tannins when food with terpenes was eaten first. Tannins are large molecules that interact with other compounds as they move slowly through the GI tract (Kumar and Singh, 1984; Min and Hart, 2003). Eating food with tannins first in a meal increases the likelihood of interaction, and possible deactivation, of food with terpenes eaten subsequently in the meal. For example, the sequential supply of tannins followed by rumen degradable protein increases the likelihood of dietary protein interacting with tannins, whereas the reverse sequence causes protein degradation and ammonia absorption before tannins can bind to proteins (Ben Salem et al., 2005). In contrast, terpenes are small non-polar molecules highly soluble in membranes; they are absorbed readily through the GI walls (Dziba et al., 2006). As opposed to sheep in Treatment 3 (alfalfa–barley ! tannins ! terpenes), sheep in Treatment 4 (tannins ! terpenes ! alfalfa–barley) began to include terpenes in their diet from the onset of conditioning, and they showed equal preference for terpenes and tannins, an effect likely linked to previous tannin consumption. Tannins and terpenes can interact synergistically to increase food intake, though no one has studied how sequence affects the outcome. Lambs (Villalba et al., 2004) and brushtail possums (Dearing and Cork, 1999) eat more when allowed to ingest tannin- and terpene-containing foods than when offered either food individually. Moreover, lambs fed 100 g of tannin-containing food and ad libitum amounts of terpene-containing food eat more terpenes and have greater preferences for food with terpenes than lambs fed tannins and terpenes ad libitum or fed tannins ad libitum and 100 g of terpene-containing food (Mote et al., 2007). These findings are consistent with landscape-level studies that show ewes with a high preference for sagebrush, a shrub high in terpenes, also consume more bitterbrush, a shrub high in tannins, compared with ewes that have a lower preference for sagebrush (Seefeldt, 2005). While further studies are required to assess how the tannin ! terpene sequence affects food consumption, all these data indicate there is a strong effect. While the synergistic effects of the sequence tannins ! terpenes may be due to tannins decreasing absorption of terpenes, lambs in Treatment 3 that also ate tannins ! terpenes never manifested this synergism, likely because they ate the alfalfa–barley prior to tannins ! terpenes. Proteins from the alfalfa–barley likely bound with tannins in the rumen (Jones and Mangan, 1977), such that tannins were less available to interact with terpenes eaten last in the sequence alfalfa–barley ! tannins ! terpenes. Foraging sequences can positively affect food intake for three reasons. First, herbivores satiate on single foods before they have necessarily met their needs for all nutrients, especially energy. Animals satiate on energy protein, micronutrients, and secondary compounds (Provenza et al., 2003). Provided intake is not limited by forage structure, satiation will reinforce the change in consumption from one food to another (Provenza, 1996). Animals fed a diet high in protein subsequently prefer foods high in energy and vice versa (Villalba and Provenza, 1999b). Second, different secondary compounds are metabolized via different pathways (Freeland and Janzen, 1974). By increasing the number of forage species consumed, particularly if their secondary
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compounds are complementary, herbivores can increase their total intake of food (Dearing and Cork, 1999; Burritt and Provenza, 2000; Villalba et al., 2004). Finally, some combinations of foods can have ameliorating and possible synergistic effects when fed in the right order and proportions. For instance, mice mix tannin and saponins in ways that nullify the effects of both compounds (Freeland et al., 1985), and lambs form preferences for medicinal supplements that ameliorate the effects of illness-inducing foods (Villalba et al., 2006). Our conditioning protocol simulated the potential contrasting sequences herbivores may face while grazing on pastures or when guided through a grazing circuit by herders (see Meuret, 1997). The protocol provided ample energy intake for animals, including a reasonable period of fast consistent with herding situations. Previous research has described 24 h as a moderate fast for sheep (Jung and Koong, 1985), and restrictions to intake of 12–24 h have been proposed as a realistic reflection of the experience of sheep grazing cool temperate pastures (Newman et al., 1994). Such restrictions in nutrient supply create different degrees of food deprivation in grazing ruminants, as well as those created by periods of vigilance at the expense of foraging effort (Laundre´ et al., 2001) or imposed by herders to reduce the risk of predation. With high stock densities commonly used in management-intensive and short-duration grazing (Savory and Butterfield, 1999; Gerrish, 2004), livestock eat foods lower in quality and higher in secondary metabolites as they deplete forage resources on a daily basis (e.g., Treatments 1 and 3). Such conditions likely challenge herbivores to learn to ‘‘mix the best with the rest’’ rather than ‘‘eat the best and leave the rest’’ (Provenza, 2003a,b) Under these conditions, some mixes of foods and sequences of eating plants are likely to be better than others at mitigating the negative effects of secondary compounds. Unfortunately, other than this study, we are not aware of research on how foraging sequences influence food intake or preference in chemically diverse pastures or rangelands. 4.2. Preference tests The objective of preference tests was to determine whether different sequences of exposure to foods during conditioning influenced subsequent preference of lambs. Preferences were conducted for a short period of time after conditioning and they were consistent. In Preference Test 1, all lambs preferred alfalfa–barley to the foods with secondary compounds. In Preference Test 2, when only foods with secondary compounds were offered, lambs in Treatments 1 (alfalfa–barley ! terpenes ! tannins), 2 (terpenes ! tannins ! alfalfa/ barley), and 3 (alfalfa–barley ! tannin ! terpene) preferred tannins to terpenes. However, lambs in Treatment 4 (tannins ! terpenes ! alfalfa/barley) ate equal amounts of terpenes and tannins, suggesting tannins affected intake of, and preference for food with terpenes. Terpene consumption was higher during conditioning for treatments fed alfalfa–barley after secondary compounds, whereas tannin consumption was higher for treatments fed alfalfa–barley before secondary compounds during conditioning. During Preference Test 2, the increased preference for food with tannins by lambs first fed alfalfa–barley was likely due to interactions between protein and tannins, experienced previously during conditioning, which attenuated the negative effects of tannins. Alfalfa and barley can induce bloat in ruminants due to high protein degradability (70–80%) (NRC, 1996). Tannins alleviate bloat by binding to proteins in the rumen (Waghorn, 1990). They also provide protein to the small intestines by binding to degradable protein in the rumen, making the protein unavailable for digestion and absorption until it reaches the more acidic abomasum (Barry et al., 2001).
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5. Conclusions Herbivores encounter an array of foods that vary in primary and secondary compounds. While the role of variety in food intake is appreciated by researchers, little is known about how the sequence of food ingestion affects food intake and preference. Our results suggest eating foods that differ in primary and secondary chemistries in different temporal arrangements creates different degrees of intake and preference for foods with secondary compounds. Thus, the sequence in which plants with different secondary compounds are consumed may affect patterns of plant use, which in turn may influence the structure of plant communities. When herbivores consume only a small suite of plant species, they can shift the balance of plant competition and reduce plant species diversity (Krueger et al., 2002; Villalba et al., 2004). In contrast, herbivores can be trained to use landscapes more evenly and to increase intake of invasive or unpalatable plant species (Provenza, 2003a,b; Provenza et al., 2003). Elucidating how sequencing affects intake of foods with nutrients and secondary compounds will enable ecosystem managers to better use grazing animals to influence the diversity of plant communities on pastures and rangelands. If people and animals learn how to use the diversity of plants in a landscape, in many areas plant diversity and wildlife habitat could be enhanced. Acknowledgements This research was supported by grants from the Utah Agricultural Experiment Station and the Initiative for the Future of Agriculture and Food Systems, USDA (Agreement No. 2001-5210311215). This paper is published with the approval of the Director, Utah Agricultural Experiment Station, and Utah State University, as paper Number 7794. We thank two anonymous reviewers for valuable comments to improve the manuscript. References Banner, R.E., Rogosic, J., Burritt, E.A., Provenza, F.D., 2000. Supplemental barley and activated charcoal increase intake of sagebrush by lambs. J. Range Manage. 53, 415–420. Barry, T.N., McNeill, D.M., McNabb, W.C., 2001. Plant secondary compounds: their impact on nutritive value and upon animal production. In: Proc. XIX Int. Grass. Conf, Sao Paulo, Brazil, pp. 445–452. Ben Salem, H., Makkar, H.P.S., Nefzaoui, A., Hassayoun, L., Abidi, S., 2005. Benefit from the association of small amounts of tannin-rich shrub foliage (Acacia cyanophylla Lindl.) with soya bean meal given as supplements to Barbarine sheep fed on oaten hay. Anim. Feed Sci. Technol. 122, 173–186. Boyle, R., McLean, S., Foley, W.J., Davies, N.W., 1999. Comparative metabolism of dietary terpene, p-cymene, in generalist and specialist folivorous marsupials. J. Chem. Ecol. 25, 2109–2127. Burritt, E.A., Provenza, F.D., 2000. Role of toxins in intake of varied diets by sheep. J. Chem. Ecol. 26, 1991–2005. Cheeke, P., Schull, L.R., 1985. Natural Toxicants in Feeds and Poisonous Plants. Avi Publishing Co., Westport, CT, pp. 64–87. Dearing, M.D., Cork, S., 1999. Role of detoxification of plant secondary compounds on diet breadth in a mammalian herbivore, Trichosurus vulpecula. J. Chem. Ecol. 25, 1205–1219. Dziba, L.E., Hall, J.O., Provenza, F.D., 2006. Feeding behavior of the lambs in relation to kinetics of 1.8-ceneole dosed intravenously or into the rumen. J. Chem. Ecol. 32, 391–408. Foley, W.J., McArthur, C., 1994. The effects and costs of allelochemicals for mammalian herbivores: an ecological perspective. In: Chivers, D.J., Langer, P. (Eds.), The Digestive System in Mammals: Food, Form and Function. Cambridge University Press, Cambridge, U.K., pp. 370–391. Foley, W.J., Iason, G.R., McArthur, C., 1999. Role of plant secondary metabolites in the nutritional ecology of mammalian herbivores: how far have we come in 25 years? In: Jung, H.G., Fahey, Jr., G.C. (Eds.), Nutritional Ecology of Herbivores. Proc. Vth Int. Symp. Nutr. Herb. Am. Soc. Anim. Sci., Illinois, pp. 130–209.
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Freeland, W.J., Janzen, D.H., 1974. Strategies of herbivory by mammals: the role of plant secondary compounds. Am. Nat. 108, 269–286. Freeland, W.J., Calcott, P.H., Anderson, L.R., 1985. Tannins and saponin: interaction in herbivore diets. Biochem. Syst. Ecol. 13, 189–193. Gerrish, J., 2004. Management-Intensive Grazing: The Grassroots of Grass Farming. Green Park Press, Ridgeland, MS, pp. 87–156. Hayter, A.J., 1984. A proof of the conjecture that the Tukey–Kramer method is conservative. Annu. Stat. 12, 61–75. Jones, W.T., Mangan, J.L., 1977. Complexes of condensed tannins of sainfoin (Onobrychis viciifolia Scop.) with fraction1 leaf protein and with submaxillary mucoprotein, and their reversal by polyethylene-glycol and pH. J. Sci. Food Agric. 28, 126–136. Jung, H.G., Koong, L.J., 1985. Effects of hunger satiation on diet quality by grazing sheep. J. Range Manage. 38, 302–305. Kimball, B.A., Nolte, D.L., Engenman, R.M., Johnston, J.J., Stermitz, F.R., 1998. Chemically mediated foraging preference of black bears (Ursus americanus). J. Mammal. 79, 448–456. Krueger, W.C., Sanderson, M.A., Cropper, J.B., Miller-Goodman, M., Kelley, C.E., Pieper, R.D., Shaver, P.L., Trlica, M.J., 2002. Environmental impacts of livestock on U.S. grazing lands. Council for Agricultural Science and Technology issue paper No. 22. CAST, Iowa. Kumar, R., Singh, M., 1984. Tannins: their adverse role in ruminant nutrition. J. Agric. Food Chem. 32, 447–453. Laundre´, J.W., Herna´ndez, L., Altendorf, K.B., 2001. Wolves, elk, and bison: reestablishing the ‘‘landscape of fear’’ in Yellowstone National Park. U.S.A. Can. J. Zool. 79, 1401–1409. Littell, R.C., Milliken, G.A., Stroup, W.W., Wolfinger, R.D., 1996. SAS System for Mixed Models. SAS Institute, Inc., Cary, NC, pp. 31–86. Marsh, K.J., Wallis, I.R., McLean, S., Sorensen, J.S., Foley, W.J., 2006. Conflicting demands on detoxification pathways influence how common brushtail possums choose their diets. Ecology 87, 2103–2112. Meuret, M., 1997. How do I cope with that bush? Optimizing on less palatable feeds at pasture using the MENU model. In: Lindberg, J.E., Gonda, H.L., Ledin, I. (Eds.), Recent Advances in Small Ruminant Nutrition, A-34. Options Mediterraneennes, pp. 53–57. Min, B.R., Hart, S.P., 2003. Tannins for suppression of internal parasites. J. Anim. Sci. 81 (E. Suppl. 2), E102–E109. Mote, T.E., Villalba, J.J., Provenza, F.D., 2007. Relative availability of tannin- and terpene-containing foods affects food intake and preference by lambs. J. Chem. Ecol. 33, 1197–1206. Newman, J.A., Penning, P.D., Parsons, A.J., Harvey, A., Orr, R.J., 1994. Fasting affects intake behaviour and diet preference of grazing sheep. Anim. Behav. 47, 185–193. NRC, 1985. Nutrient Requirements of Sheep, 6th ed. National Academy Press, Washington, DC, pp. 54–77. NRC, 1996. Nutrient Requirements for Beef Cattle, 7th ed. National Academy Press, Washington, DC, pp. 16–101. Papachristou, T.G., Dziba, L.E., Villalba, J.J., Provenza, F.D., 2007. Patterns of diet mixing by sheep offered foods varying in nutrients and plant secondary compounds. Appl. Anim. Behav. Sci. 108, 68–80. Provenza, F.D., 1996. Acquired aversions as the basis for varied diets of ruminants foraging on rangelands. J. Anim. Sci. 74, 2010–2020. Provenza, F.D., 2003a. Foraging Behavior: Managing to Survive in a World of Change. Utah State Univ., Logan, pp. 35– 49. Provenza, F.D., 2003b. Twenty-five years of paradox in plant-herbivore interactions and ‘‘sustainable’’ grazing management. Rangelands 25, 4–15. Provenza, F.D., Villalba, J.J., Dziba, L.E., Atwood, S.B., Banner, R.E., 2003. Linking herbivore experience, varied diets, and plant biochemical diversity. Small Rum. Res. 49, 257–274. Savory, A., Butterfield, J., 1999. Holistic Management: A New Framework for Decision-Making. Island Press, Covalo, CA, pp. 167–258. Seefeldt, S.S., 2005. Consequences of selecting Rambouillet ewes for Mountain Big Sagebrush (Artemisia tridentata ssp. Vaseyana) dietary preference. Rangeland Ecol. Manage. 58, 380–384. Shaw, R.A., Villalba, J.J., Provenza, F.D., 2006a. Resource availability and quality influence patterns of diet mixing with foods containing toxins by sheep. J. Chem. Ecol. 32, 1267–1278. Shaw, R.A., Villalba, J.J., Provenza, F.D., 2006b. Influence of stock density and rate and temporal patterns of forage allocation on the diet mixing behavior of sheep grazing sagebrush steppe. Appl. Anim. Behav. Sci. 100, 207–218. Silanikove, N., Nitsan, Z., Perevolotsky, A., 1994. Effect of a daily supplementation of polyethylene glycol on intake and digestion of tannin-containing leaves (Ceratonia siliqua) by sheep. J. Agric. Food Chem. 42, 2844–2847. Silanikove, N., Gilboa, N., Nir, I., Perevolotsky, A., Nitsan, Z., 1996. Effect of a daily supplementation of polyethylene glycol on intake and digestion of tannin-containing leaves (Quercus calliprinos, Pistacia lentiscus, Ceratonia siliqua) by sheep. J. Agric. Food Chem. 44, 199–205.
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Villalba, J.J., Provenza, F.D., 1997a. Preference for wheat straw by lambs conditioned with intraruminal infusions of starch. Br. J. Nutr. 77, 287–297. Villalba, J.J., Provenza, F.D., 1997b. Preference for flavored foods by lambs conditioned with intraruminal administrations of nitrogen. Br. J. Nutr. 78, 545–561. Villalba, J.J., Provenza, F.D., 1997c. Preference for flavored wheat straw by lambs conditioned with intraruminal infusions of acetate and propionate. J. Anim. Sci. 75, 2905–2914. Villalba, J.J., Provenza, F.D., 1999a. Effects of food structure and nutritional quality and animal nutritional state on intake behaviour and food preference of sheep. Appl. Anim. Behav. Sci. 63, 145–163. Villalba, J.J., Provenza, F.D., 1999b. Nutrient-specific preferences by lambs conditioned with intraruminal infusions of starch, casein, and water. J. Anim. Sci. 79, 2066–2074. Villalba, J.J., Provenza, F.D., Han, G., 2004. Experience influences diet mixing by herbivores: implications for plant biochemical diversity. Oikos 107, 100–109. Villalba, J.J., Provenza, F.D., Shaw, R., 2006. Sheep self-medicate when challenged with illness-inducing foods. Anim. Behav. 71, 1131–1139. Waghorn, G.C., 1990. Beneficial effects of low concentrations of condensed tannins in forages fed to ruminants. In: Akin, D.E., Ljungdahl, L.G., Wilson, J.R., Harris, P.J. (Eds.), Microbial and Plant Opportunities to Improve Lignocellulose Utilization by Ruminants. Elsevier Science Publishing, New York, p. 137.
Applied Animal Behaviour Science 113 (2008) 57–68 www.elsevier.com/locate/applanim
Sequence of food presentation influences intake of foods containing tannins and terpenes Travis E. Mote, Juan J. Villalba *, Frederick D. Provenza Department of Wildland Resources, Utah State University, 5230 Old Main Hill, Logan, UT 84322-5230, USA Accepted 9 October 2007 Available online 26 November 2007
Abstract Interactions among nutrients and secondary compounds in plants can influence the kinds and amounts of different forages herbivores ingest, but little is known about how the sequence of plant ingestion may influence these interactions. The physiological pathways and rates of nutrient and secondary compound metabolism in the body influence food intake by herbivores. On this basis, we predicted the sequence in which foods that vary in nutrients and secondary compounds are ingested would influence food intake and preference. In a 2 2 factorial experiment, we evaluated the relationship between the sequence of presenting two foods, one with terpenes and the other with tannins, and the time when lambs ate a nutritious food (alfalfa–barley), either before or after eating foods with tannins and terpenes. When alfalfa–barley was fed prior to the terpenes, intake of the terpene-containing food was lower than when alfalfa–barley was fed after terpenes (P < 0.05). The sequence when alfalfa–barley was fed did not influence intake of the tannin-containing food (P > 0.10). Lambs ate more total foods with terpenes + tannins when fed tannins ! terpenes ! alfalfa/barley than when fed alfalfa/barley ! tannins ! terpenes (P < 0.10). During preference tests, when lambs were offered all three foods simultaneously, lambs previously conditioned with the sequence tannins ! terpenes ! alfalfa–barley preferred alfalfa–barley > terpenes > tannins (P < 0.05), whereas lambs in other treatments preferred alfalfa–barley > tannins > terpenes (P < 0.05). During preference tests when lambs were fed only foods with secondary compounds, lambs previously conditioned with the sequence tannins ! terpenes ! alfalfa–barley showed equal preference for foods with tannins and terpenes, whereas lambs in other treatments preferred food with tannins > terpenes (P < 0.05–0.10). All of these results are consistent with the hypothesis that the sequence in which foods are consumed affects both food intake and preference. Understanding the importance of sequencing when herbivores consume foods that vary in nutrients and secondary compounds may help managers create new grazing strategies
* Corresponding author. Tel.: +1 435 797 2539; fax: +1 435 797 3796. E-mail address: [email protected] (J.J. Villalba). 0168-1591/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.applanim.2007.10.003
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that include sequential foraging patterns to optimize food intake and more evenly use all plant species in a community, a practice used by herders in France. Published by Elsevier B.V. Keywords: Foraging sequence; Diet mixing; Sheep; Intake; Tannins; Terpenes
1. Introduction Most plants contain secondary compounds that, at too high concentrations, limit how much of any particular food a herbivore can consume (Cheeke and Schull, 1985; Foley et al., 1999). Thus, herbivores must regulate intake of plants with secondary compounds in order to ingest adequate levels of nutrients and avoid toxicosis. Any mechanism that increases rates of elimination of secondary compounds can increase the ability of herbivores to consume plants that contain these chemicals (Dziba et al., 2006). Eating a variety of foods is one way to accomplish this objective (Freeland and Janzen, 1974; Provenza, 1996), as different kinds of secondary compounds are processed at different rates and through different metabolic pathways, thereby providing multiple avenues for detoxification (Freeland and Janzen, 1974; Boyle et al., 1999; Marsh et al., 2006). Thus, varied diets enable herbivores to ingest more nutrients from plants that contain secondary compounds, provided the secondary compounds are complementary (Dearing and Cork, 1999; Burritt and Provenza, 2000; Villalba et al., 2004). Diversity in the chemical composition and availability of plants on landscapes also provides the context for herbivores to learn about the consequences of eating different foods in succession, which may provide another opportunity to ingest plants with secondary compounds. The time when herbivores consume different foods in a sequence may influence how much of different foods they can consume. This is because food intake and preference are influenced by rates of absorption of secondary compounds and their interactions in the gastrointestinal tract. For instance, terpenes are small, fat-soluble molecules (Foley and McArthur, 1994) that are absorbed quickly through the rumen epithelium such that they can quickly decrease food intake (Dziba et al., 2006). Conversely, tannins are high-molecularweight compounds (2000–4000) that remain in the gut where they can interact with other chemicals (Kumar and Singh, 1984; Min and Hart, 2003). Tannins form complexes with several molecules including proteins, polysaccharides, nucleic acids and minerals mainly through hydrophobic/hydrogen interactions (Min and Hart, 2003). Thus, a meal of tannins followed by a meal of terpenes may lead to higher levels of intake than the reverse, if tannins reduce absorption of terpenes. Nutrients in the gut may further enhance these responses. Dietary proteins bind to tannins, mitigating the negative effects of tannins on tissues and cellular processes (Kumar and Singh, 1984), and energy and protein can enhance intake of foods high in secondary compounds (Provenza et al., 2003). We hypothesized that food intake and preference can be influenced by the aforementioned physiological processes, whereby nutrients and secondary compounds may interact in different ways as they are absorbed and metabolized at different rates. On this basis, we predicted the sequence in which foods that vary in nutrients and secondary compounds are ingested influences food intake and preference. We determined whether the temporal sequence in which foods with secondary compounds (tannins and terpenes) and a high-quality food (alfalfa–barley mix) were ingested affected food intake and preference by lambs.
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2. Materials and methods The experiment was conducted at the Mote Farm in Willard, UT, USA (41.48N, 112.08W). Weaned commercial Rambouillet-Columbia-Finn-Targhee crossbred lambs, averaging 35 kg (S.E.M. = 0.9), were individually penned with free access to trace mineralized salt blocks and fresh water. The lambs were kept outdoors, under a protective roof in individual, adjacent pens measuring 2.4 m 3.6 m. All procedures were approved by the Utah State University Institutional Animal Care and Use Committee (IACUC #1207). 2.1. Test foods Test foods were formulated to be similar in secondary compound and nutrient contents to the current year’s growth of shrubs such as sagebrush (Artemisia tridentata) and bitterbrush (Purshia tridentata), which are common forages for domestic and wild herbivores in the sagebrush steppe ecosystem during autumn and winter. We used two isocaloric (12.1 MJ DE/kg DM) and isonitrogenous (126 g CP/kg DM) (National Research Council (NRC), 1985) foods, similar to those used by Villalba et al. (2004). The tannin-containing food was 76% beet pulp, 9% soybean meal, and 15% quebracho tannin powder. The terpene-containing food was 52.8% beet pulp, 26% grape pomace, 14% soybean meal and 7.2% terpene solution (24% camphor, 16% 1-8-cineole, 1.0% p-cymene and 59% vegetable oil). Terpene volatilization was reduced by mixing terpenes with vegetable oil, which also improved terpene homogeneity in the diet (Kimball et al., 1998). The high-quality food was a (50:50) mix of alfalfa and barley. All food ingredients were ground to 2–3 mm particle size to facilitate mixing. 2.2. Conditioning Thirty-two lambs were stratified by weight and randomly assigned to four treatments (eight lambs/ treatment) in a 2 2 factorial design that included the sequence of presenting the tannin- and terpenecontaining foods relative to the basal diet (Table 1). The first food in the sequence was offered at 06:00 h each morning, and each component of the sequence was available to lambs for 1 h. Lambs were fed their respective sequences for a 14-day conditioning period. Lambs in different treatments were randomly distributed in adjacent pens. In previous studies, we found that the likelihood of animals influencing one another is minimal because they all eat at the same time and the type of food on offer is more important than observing neighbors eat (Villalba and Provenza, 1997c). The alfalfa–barley mix was restricted to 250 g/d, whereas the secondary compound-containing foods were offered ad libitum. Sheep learn best to ingest foods with secondary compounds when the availability of nutritious alternatives is limited (Shaw et al., 2006a), and they could meet nutritional needs by eating a combination of the foods with tannins and terpenes used in our study, especially given the additional 250 g/d of alfalfa–barley (Villalba et al., 2004). In rangeland settings, sheep are unlikely to learn to eat shrubs such as sagebrush, which is high in terpenes, unless the availability of more palatable and nutritious grasses and forbs is restricted (Shaw et al., 2006b). Thus, we restricted the availability of the palatable alfalfa–barley mix to encourage consumption of the tannin- and terpene-containing foods (Shaw et al., 2006a). 2.3. Preference tests After conditioning, all animals were fed alfalfa pellets ad libitum from 06:00 to 09:00 h for 3 d to allow tannins and terpenes to be eliminated from the body. The retention of quebracho tannin in the gastrointestinal tract ranges from 48 h (free and soluble) to 72 h (bound to protein and fiber) (Silanikove et al., 1994, 1996). The average first pass elimination of plasma terpenes occurs within minutes after terpenes are infused into the rumen or blood stream (Dziba et al., 2006). Preference tests were performed at 06:00 h. Test foods were offered simultaneously, in separate plastic containers that fitted tightly into a wooden food box attached to each pen. The placement (left vs. right) of specific foods was random across pens and days. During the first period of preference tests, lambs were offered all three test foods –terpene, tannin, alfalfa–barley – ad libitum for 30 min, whereas during the
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Table 1 Treatments for the 2 2 factorial design for the sequence of presentation to lambs of secondary compounds relative to the alfalfa–barley basal diet
second period of preference tests, lambs were offered only the tannin- and terpene-containing foods ad libitum for 30 min. Refusals were collected and weighed. Intake was determined as the difference between the amount of food presented and the amount of food refused for each food offered during the 30-min preference test. Thus, we determined food choice when animals could avoid secondary compounds and consume a high-quality food (alfalfa–barley) (Period 1), and when they could chose between two foods of similar nutritional composition that both contained secondary compounds (terpenes and tannins) (Period 2). During both periods, preference tests were conducted on 2 consecutive days (Table 1). Lambs showed a consistent pattern of selection during these 2 d (see Section 3 for details), so no further tests were performed. In tests such as these, sheep typically manifest consistent patterns of preference, such that once preference is established, they continue to prefer/avoid specific foods (Villalba and Provenza, 1997a,b,c, 1999a). 2.4. Statistical analyses Data were analyzed as a 2 2 factorial design with two factors: (1) sequence of presentation of secondary compounds (level 1: first terpenes, then tannins; level 2: first tannins, then terpenes), and (2) time
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when the basal diet was fed (level 1: prior to offering the secondary compounds, level 2: after offering the secondary compounds) (Table 1). Day was the repeated measure in the analyses. When significant effects were detected (P < 0.10), differences among means were determined using Tukey’s multiple comparison procedure (Hayter, 1984). Intake of individual secondary compound-containing foods and total food consumption were the response variables for the conditioning and preference trials. Daily refusals were subtracted from the amount offered (as-fed basis) to estimate intake, which was calculated based on body weight as grams of food ingested/kg body weight. For preference tests, percentage preference was also used as a response variable. Preference was computed as: [intake of a single plant secondary compoundcontaining food/total intake of plant secondary compound-containing foods] 100%. ANOVAs were performed using the MIXED procedure in SAS 9.1 for Windows (SAS, Littell et al., 1996).
3. Results 3.1. Conditioning Total consumption of the foods with secondary compounds during conditioning differed for lambs fed in different sequences (P = 0.006, Fig. 1). When the high-quality diet of alfalfa–barley was fed after the secondary compounds, intake of terpene-containing food was higher (6.60 g/kg
Fig. 1. Consumption of foods with tannins and terpenes and total consumption of food with secondary compounds (tannins + terpenes) by four groups of lambs during conditioning. Lambs in Treatment 1 were offered three foods in the sequence alfalfa–barley (AB) ! terpene (Ter) ! tannin (Tan). Lambs in Treatment 2 were fed Ter ! Tan ! AB. Those in Treatment 3 were fed AB ! Tan ! Ter. Lambs in Treatment 4 were fed Tan ! Ter ! AB.
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BW; S.E.M. = 0.42) than when the high-quality diet was fed before the secondary compounds (4.73 g/kg BW; S.E.M. = 0.42; P < 0.05). Conversely, the time when the high-quality diet was fed did not influence intake of the tannin-containing food (P > 0.10). Though the pattern of food intake was consistent among treatments (Fig. 1), there was a treatment day interaction (P = 0.02). Intake of the terpene-containing food was reduced relative to the other treatments when the sequence was alfalfa–barley ! tannins ! terpenes (Treatment 3) (P < 0.05; Fig. 1). In contrast, intake of the terpene-containing food was highest when the sequence was tannins ! terpenes ! alfalfa–barley (Treatment 4) (P < 0.05; Fig. 1). Averaged across the 14-d period, consumption of the food with terpenes for Treatments 1, 2, 3, and 4 was 6.26, 6.17, 3.20, and 7.04 g/kg BW, respectively. Averaged across the 14-d conditioning period, total consumption of foods with secondary compounds was higher (P < 0.10) for Treatments 1 (alfalfa–barley ! terpenes ! tannins) (13.99 g/kg) and 4 (tannins ! terpenes ! alfalfa–barley) (14.46 g/kg) than for Treatment 3 (alfalfa–barley ! tannins ! terpenes) (11.06 g/kg) (S.E.M. = 1.35). None of the treatments differed from Treatment 2 (terpenes ! tannins ! alfalfa–barley) (12.97 g/kg; P > 0.10; Fig. 1). 3.2. Preference tests During Preference Test 1, when all three foods were offered simultaneously, lambs in Treatment 4, previously conditioned with the sequence tannins ! terpenes ! alfalfa–barley, preferred alfalfa–barley > terpenes > tannins (P < 0.05), whereas lambs in the other three treatments preferred alfalfa–barley > tannin > terpene (P < 0.05) (Fig. 2). Treatments did not differ in total intake of foods with secondary compounds (sequence of secondary compound presentation time alfalfa–barley was fed, P = 0.32), and outcomes of preference tests did not differ across days (day effect; treatment day interaction; P > 0.10) (Fig. 2). During Preference Test 2, when lambs were offered only foods with secondary compounds, lambs in Treatments 1, 2, and 3 preferred tannin > terpene, whereas those in Treatment 4 showed similar preference for terpenes and tannins (Fig. 3). Intake of foods with secondary compounds also differed by treatments during this preference test (P = 0.006; Fig. 3). Lambs ate more food with terpenes when alfalfa–barley was fed last as opposed to first (4.88 vs. 2.63 g/kg BW;
Fig. 2. Intake of alfalfa–barley, terpene-, and tannin-containing foods offered ad libitum during Preference Test 1. Lambs were conditioned to eat alfalfa–barley before or after eating one of two possible sequences of tannin- and terpenecontaining foods. Lambs in Treatment 1 were fed three foods in the sequence alfalfa–barley ! terpene ! tannin. Lambs in Treatment 2 were fed terpene ! tannin ! alfalfa–barley. Those in Treatment 3 were fed alfalfa–barley ! tannin ! terpene. Lambs in Treatment 4 were fed tannin ! terpene ! alfalfa–barley.
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Fig. 3. Intake of terpene- and tannin-containing foods offered ad libitum during Preference Test 2. Lambs were previously conditioned to eat alfalfa–barley before or after eating one of two possible sequences of tannin- and terpene-containing foods. Lambs in Treatment 1 were fed three foods in the sequence alfalfa–barley ! terpene ! tannin. Lambs in Treatment 2 were fed terpene ! tannin ! alfalfa–barley. Those in Treatment 3 were fed alfalfa–barley ! tannin ! terpene. Lambs in Treatment 4 were fed tannin ! terpene ! alfalfa–barley.
S.E.M. = 0.47, P < 0.05). Conversely, they tended to eat less food with tannin when alfalfa– barley was fed last rather than first in the meal (5.95 vs. 7.46 g/kg BW; S.E.M. = 0.47, P < 0.15). Lambs in Treatment 3 previously conditioned with the sequence alfalfa–barley ! tannins ! terpenes ate much less terpene-containing food (2.05 vs. 5.50 g/kg BW; S.E.M. = 0.90; P < 0.05; Fig. 3) and more tannin-containing food (7.92 vs. 5.14 g/kg BW; S.E.M. = 0.90; P < 0.10; Fig. 3) than lambs in Treatment 4, previously conditioned with the sequence tannins ! terpenes ! alfalfa–barley. Total intake of foods with secondary compounds was similar among treatments (sequence of secondary compound presentation time basal diet was fed, P = 0.95), and preference tests were consistent across days (day effect; treatment day interaction; P > 0.10) (Fig. 3). 4. Discussion We determined if the temporal sequence in which foods with tannins and terpenes and a nutritious food were offered affected food intake and preference of lambs. We hypothesized that the physiological pathways whereby nutrients and secondary compounds are metabolized and the rates of metabolism influence food intake. On this basis, we predicted the sequence in which foods that vary in nutrients and secondary compounds are ingested influences food intake and preference. We found that when the alfalfa–barley food was offered in the sequence had the greatest impact on intake during conditioning and preference tests, but the sequence in which foods with tannins and terpenes were offered also affected intake and preference. 4.1. Conditioning When the nutritious alfalfa–barley was fed first, sheep did not eat much food with terpenes, and the effect was greatest for lambs in Treatment 3 fed terpenes last during conditioning. This finding is consistent with the observation that sheep learn to eat foods with terpenes best when alternative foods are restricted (Villalba et al., 2004; Shaw et al., 2006a,b), and that total intake of tannin-, terpene-, and oxalate-containing foods by sheep is greater when these foods are eaten
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prior to nutritious foods than when they are eaten after the nutritious foods (Papachristou et al., 2007). Thus, lambs are more highly motivated to consume foods with terpenes when somewhat food deprived than after eating a large meal of a nutritious food. French herders use this principle to enhance consumption of less palatable foods in grazing circuits (Meuret, 1997). Low consumption of terpene-containing food by lambs fed alfalfa–barley ! tannins ! terpenes (Treatment 3) was likely due to (1) the strong flavor of terpenes, which can deter sheep from eating foods with terpenes for several weeks (Banner et al., 2000); (2) eating alternative foods prior to terpenes in the foraging sequence (Shaw et al., 2006a,b); and (3) the lack of synergism between terpenes and tannins when food with terpenes was eaten first. Tannins are large molecules that interact with other compounds as they move slowly through the GI tract (Kumar and Singh, 1984; Min and Hart, 2003). Eating food with tannins first in a meal increases the likelihood of interaction, and possible deactivation, of food with terpenes eaten subsequently in the meal. For example, the sequential supply of tannins followed by rumen degradable protein increases the likelihood of dietary protein interacting with tannins, whereas the reverse sequence causes protein degradation and ammonia absorption before tannins can bind to proteins (Ben Salem et al., 2005). In contrast, terpenes are small non-polar molecules highly soluble in membranes; they are absorbed readily through the GI walls (Dziba et al., 2006). As opposed to sheep in Treatment 3 (alfalfa–barley ! tannins ! terpenes), sheep in Treatment 4 (tannins ! terpenes ! alfalfa–barley) began to include terpenes in their diet from the onset of conditioning, and they showed equal preference for terpenes and tannins, an effect likely linked to previous tannin consumption. Tannins and terpenes can interact synergistically to increase food intake, though no one has studied how sequence affects the outcome. Lambs (Villalba et al., 2004) and brushtail possums (Dearing and Cork, 1999) eat more when allowed to ingest tannin- and terpene-containing foods than when offered either food individually. Moreover, lambs fed 100 g of tannin-containing food and ad libitum amounts of terpene-containing food eat more terpenes and have greater preferences for food with terpenes than lambs fed tannins and terpenes ad libitum or fed tannins ad libitum and 100 g of terpene-containing food (Mote et al., 2007). These findings are consistent with landscape-level studies that show ewes with a high preference for sagebrush, a shrub high in terpenes, also consume more bitterbrush, a shrub high in tannins, compared with ewes that have a lower preference for sagebrush (Seefeldt, 2005). While further studies are required to assess how the tannin ! terpene sequence affects food consumption, all these data indicate there is a strong effect. While the synergistic effects of the sequence tannins ! terpenes may be due to tannins decreasing absorption of terpenes, lambs in Treatment 3 that also ate tannins ! terpenes never manifested this synergism, likely because they ate the alfalfa–barley prior to tannins ! terpenes. Proteins from the alfalfa–barley likely bound with tannins in the rumen (Jones and Mangan, 1977), such that tannins were less available to interact with terpenes eaten last in the sequence alfalfa–barley ! tannins ! terpenes. Foraging sequences can positively affect food intake for three reasons. First, herbivores satiate on single foods before they have necessarily met their needs for all nutrients, especially energy. Animals satiate on energy protein, micronutrients, and secondary compounds (Provenza et al., 2003). Provided intake is not limited by forage structure, satiation will reinforce the change in consumption from one food to another (Provenza, 1996). Animals fed a diet high in protein subsequently prefer foods high in energy and vice versa (Villalba and Provenza, 1999b). Second, different secondary compounds are metabolized via different pathways (Freeland and Janzen, 1974). By increasing the number of forage species consumed, particularly if their secondary
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compounds are complementary, herbivores can increase their total intake of food (Dearing and Cork, 1999; Burritt and Provenza, 2000; Villalba et al., 2004). Finally, some combinations of foods can have ameliorating and possible synergistic effects when fed in the right order and proportions. For instance, mice mix tannin and saponins in ways that nullify the effects of both compounds (Freeland et al., 1985), and lambs form preferences for medicinal supplements that ameliorate the effects of illness-inducing foods (Villalba et al., 2006). Our conditioning protocol simulated the potential contrasting sequences herbivores may face while grazing on pastures or when guided through a grazing circuit by herders (see Meuret, 1997). The protocol provided ample energy intake for animals, including a reasonable period of fast consistent with herding situations. Previous research has described 24 h as a moderate fast for sheep (Jung and Koong, 1985), and restrictions to intake of 12–24 h have been proposed as a realistic reflection of the experience of sheep grazing cool temperate pastures (Newman et al., 1994). Such restrictions in nutrient supply create different degrees of food deprivation in grazing ruminants, as well as those created by periods of vigilance at the expense of foraging effort (Laundre´ et al., 2001) or imposed by herders to reduce the risk of predation. With high stock densities commonly used in management-intensive and short-duration grazing (Savory and Butterfield, 1999; Gerrish, 2004), livestock eat foods lower in quality and higher in secondary metabolites as they deplete forage resources on a daily basis (e.g., Treatments 1 and 3). Such conditions likely challenge herbivores to learn to ‘‘mix the best with the rest’’ rather than ‘‘eat the best and leave the rest’’ (Provenza, 2003a,b) Under these conditions, some mixes of foods and sequences of eating plants are likely to be better than others at mitigating the negative effects of secondary compounds. Unfortunately, other than this study, we are not aware of research on how foraging sequences influence food intake or preference in chemically diverse pastures or rangelands. 4.2. Preference tests The objective of preference tests was to determine whether different sequences of exposure to foods during conditioning influenced subsequent preference of lambs. Preferences were conducted for a short period of time after conditioning and they were consistent. In Preference Test 1, all lambs preferred alfalfa–barley to the foods with secondary compounds. In Preference Test 2, when only foods with secondary compounds were offered, lambs in Treatments 1 (alfalfa–barley ! terpenes ! tannins), 2 (terpenes ! tannins ! alfalfa/ barley), and 3 (alfalfa–barley ! tannin ! terpene) preferred tannins to terpenes. However, lambs in Treatment 4 (tannins ! terpenes ! alfalfa/barley) ate equal amounts of terpenes and tannins, suggesting tannins affected intake of, and preference for food with terpenes. Terpene consumption was higher during conditioning for treatments fed alfalfa–barley after secondary compounds, whereas tannin consumption was higher for treatments fed alfalfa–barley before secondary compounds during conditioning. During Preference Test 2, the increased preference for food with tannins by lambs first fed alfalfa–barley was likely due to interactions between protein and tannins, experienced previously during conditioning, which attenuated the negative effects of tannins. Alfalfa and barley can induce bloat in ruminants due to high protein degradability (70–80%) (NRC, 1996). Tannins alleviate bloat by binding to proteins in the rumen (Waghorn, 1990). They also provide protein to the small intestines by binding to degradable protein in the rumen, making the protein unavailable for digestion and absorption until it reaches the more acidic abomasum (Barry et al., 2001).
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5. Conclusions Herbivores encounter an array of foods that vary in primary and secondary compounds. While the role of variety in food intake is appreciated by researchers, little is known about how the sequence of food ingestion affects food intake and preference. Our results suggest eating foods that differ in primary and secondary chemistries in different temporal arrangements creates different degrees of intake and preference for foods with secondary compounds. Thus, the sequence in which plants with different secondary compounds are consumed may affect patterns of plant use, which in turn may influence the structure of plant communities. When herbivores consume only a small suite of plant species, they can shift the balance of plant competition and reduce plant species diversity (Krueger et al., 2002; Villalba et al., 2004). In contrast, herbivores can be trained to use landscapes more evenly and to increase intake of invasive or unpalatable plant species (Provenza, 2003a,b; Provenza et al., 2003). Elucidating how sequencing affects intake of foods with nutrients and secondary compounds will enable ecosystem managers to better use grazing animals to influence the diversity of plant communities on pastures and rangelands. If people and animals learn how to use the diversity of plants in a landscape, in many areas plant diversity and wildlife habitat could be enhanced. Acknowledgements This research was supported by grants from the Utah Agricultural Experiment Station and the Initiative for the Future of Agriculture and Food Systems, USDA (Agreement No. 2001-5210311215). This paper is published with the approval of the Director, Utah Agricultural Experiment Station, and Utah State University, as paper Number 7794. We thank two anonymous reviewers for valuable comments to improve the manuscript. References Banner, R.E., Rogosic, J., Burritt, E.A., Provenza, F.D., 2000. Supplemental barley and activated charcoal increase intake of sagebrush by lambs. J. Range Manage. 53, 415–420. Barry, T.N., McNeill, D.M., McNabb, W.C., 2001. Plant secondary compounds: their impact on nutritive value and upon animal production. In: Proc. XIX Int. Grass. Conf, Sao Paulo, Brazil, pp. 445–452. Ben Salem, H., Makkar, H.P.S., Nefzaoui, A., Hassayoun, L., Abidi, S., 2005. Benefit from the association of small amounts of tannin-rich shrub foliage (Acacia cyanophylla Lindl.) with soya bean meal given as supplements to Barbarine sheep fed on oaten hay. Anim. Feed Sci. Technol. 122, 173–186. Boyle, R., McLean, S., Foley, W.J., Davies, N.W., 1999. Comparative metabolism of dietary terpene, p-cymene, in generalist and specialist folivorous marsupials. J. Chem. Ecol. 25, 2109–2127. Burritt, E.A., Provenza, F.D., 2000. Role of toxins in intake of varied diets by sheep. J. Chem. Ecol. 26, 1991–2005. Cheeke, P., Schull, L.R., 1985. Natural Toxicants in Feeds and Poisonous Plants. Avi Publishing Co., Westport, CT, pp. 64–87. Dearing, M.D., Cork, S., 1999. Role of detoxification of plant secondary compounds on diet breadth in a mammalian herbivore, Trichosurus vulpecula. J. Chem. Ecol. 25, 1205–1219. Dziba, L.E., Hall, J.O., Provenza, F.D., 2006. Feeding behavior of the lambs in relation to kinetics of 1.8-ceneole dosed intravenously or into the rumen. J. Chem. Ecol. 32, 391–408. Foley, W.J., McArthur, C., 1994. The effects and costs of allelochemicals for mammalian herbivores: an ecological perspective. In: Chivers, D.J., Langer, P. (Eds.), The Digestive System in Mammals: Food, Form and Function. Cambridge University Press, Cambridge, U.K., pp. 370–391. Foley, W.J., Iason, G.R., McArthur, C., 1999. Role of plant secondary metabolites in the nutritional ecology of mammalian herbivores: how far have we come in 25 years? In: Jung, H.G., Fahey, Jr., G.C. (Eds.), Nutritional Ecology of Herbivores. Proc. Vth Int. Symp. Nutr. Herb. Am. Soc. Anim. Sci., Illinois, pp. 130–209.
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