Feed Additives Increase Intake Of

1 Feed additives increase intake of tannin- and 2 terpene-rich plants in shrubland ecosystems 3 4 5 Jozo Rogosic1*, Zora Pilic2, Slaven Prodanovic3 and Savo Vuckovic3 6 8 1 University of Zadar, Department of Ecology, Agronomy and Aquaculture, Mihovila 9 Pavlinovića bb, 23000 Zadar, Croatia 10 11 2Department of Biology and Chemistry, Faculty of Natural Science, University of Mostar, 12 Ulica Matice hrvatske bb, 88000 Mostar, Bosnia/Herzegovina 13 14 3 University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia 15 16 17 18 19 20 21 22 23

33 34*To whom correspondence should be addressed: e-mail: [email protected]

35Tel: 385 23 302 508

36Fax: 385 23 302 509

37 38 39 40 41Abstract - Maquis and garrigues are extensive shrubland vegetation types in the

42Mediterranean region that provide important habitat for wild and domestic herbivores.

43Although the majority of these shrubs are nutritious, virtually all contain secondary

44compounds that reduce their forage value. In six trials, we examined the effect of

45polyethylene glycol (PEG), activated charcoal (Ach) and number of shrubs offered on intake

46of Mediterranean shrubs by sheep and goats. We offered 3 shrubs in trial 1, 2 shrubs in trial 2

47and 1 shrub in trial. 3. Trials were conducted with 12 sheep and 12 goats (6 PEG vs. 6 control

48and 6 Ach vs. control). In trial 1, sheep receiving PEG ate more (P=0.002) total shrubs

49(Quercus ilex, Arbutus unedo and Pistacia lentiscus) than did controls, but no such treatment

50effect was found for goats. In trial 2, supplemental PEG had a positive effect (P<0.001) on

51total shrub intake for both sheep and goats when only 2 shrubs (Arbutus unedo and Pistacia

52lentiscus) of lower palatability were offered. In trial 3, supplemental PEG had a marked

53positive effect (P<0.001) on intake of Pistacia lentiscus in both sheep and goats. PEG

54supplemented goats ate more Pistacia lentiscus (39.6 g/kg) than did PEG supplemented sheep

55(28.1 g/kg), whereas control sheep and goats ate similar amounts (12.2 and 15.3 g/kg,

56respectively). In trial 4, we initially offered 3 shrubs (Juniperus phoenicea, Helichrysum

57italicum and Juniperus oxicedrus), then 2 shrubs (Juniperus phoenicea and Helichrysum

58italicum) in trial 5, and finally 1 shrub (Juniperus phoenicea) in trial 6. Goats ate more total

59shrub biomass than did sheep (45.7 ± 1.0 g/kg BW vs. 27.2 ± 0.7 g/kg BW, respectively) in

60trial 4, and there was no difference in shrub intake (P = 0.39) between Ach-treated animals

61and controls (mean 37.5 ± 0.8 g/kg BW). Supplemental Ach had a positive effect on total

62shrub intake for both sheep and goats in all 3 trials (trial 4, P < 0.001; trial 5, P < 0.001 and

63trial 6, P < 0.03), and goats ate more shrubs than did sheep in all 3 trials (P < 0.01).

64Regardless of experiment, both species of animals showed a numerical decrease in total shrub 65intake, with or without supplemental PEG/Ach, as the number of shrub species on offer

66decreased. Our findings support the hypothesis that plant biochemical diversity plays an

67important role in diet selection, thus enabling animals to better meet their nutritional needs

68and avoid toxicity. In addition, as the number of shrubs in the diet decreased (diminution of

69biodiversity), the concentration of terpenes and tannins in diet probably increased, so

70PEG/Ach had a greater impact on shrub intake. Finally, both PEG and Ach had the same

71influence on sheep and goats throughout the experiments, even though goats always ate more

72shrubs than did sheep.

74Key words: Feed additives, Activated charcoal, Polyethylene glycol, Biodiversity, Goats,

75Sheep, Mediterranean shrubs, Secondary compounds.

77 INTRODUCTION

78Mediterranean shrubby vegetation called “maquis” and “garrigue” are widespread throughout

79the Mediterranean Basin, including the Adriatic littoral of Croatia. In Croatia and elsewhere,

80these shrublands represent traditional grazing areas and are a significant source of forage for

81sheep and goats, particularly during the dry summer.

82 Secondary compounds (e.g., phenolics, terpenes, saponins, alkaloids, glycosides,

83tannins, etc.) often limit utilization of Mediterranean shrubs and adversely affect forage

84intake. Approximately 80% of the Mediterranean shrubby species contain tannins (Rhoades,

851979), often at levels of 10% or more in the dry matter (Levin, 1976). Phenolic compounds

86(total phenols, catechins, phenolic monomers, tannins, etc.) reduce digestibility and forage

87intake of animals grazing in the Myrto-Quercetum ilicis plant community, which is a major

88vegetation type of the Mediterranean maquis (Rogosic et al., 2007). Phenolic compounds also

89limit palatability, digestibility and intake of dominant forage shrubs such as rosemary 90garrigue Erico-Rosmarinetum officinalis (Rogosic and Ivankovic, 2004).

91 Terpenoids are the largest group of plant secondary chemicals, with over 30,000

92terpenes identified structurally (Little and Croteau, 1999).Terpenes have been found to

93decrease in vitro digestibility in ruminants, suggesting antimicrobial activity, especially if the

94rumen microbial population is not adapted to the compounds (Schwartz et al., 1980). Ngugi et

95al. (1995) reported sagebrush essential oil decreased bacterial numbers in deer rumen fluid,

96but observed no evidence of microbial adaptation. Actual effects of terpenes in vivo have

97been suggested to be less because of loss during mastication and rumination (Cluff et al.,

981982), eructation of warm volatiles (Pederson and Welch, 1982), and/or absorption from the

99rumen (Freeland and Janzen, 1974). Newbold et al. (2004) reported that an essential oil

100mixture fed to sheep caused no major effects on fermentation, rumen ammonia, protozoa

101numbers, or digestion, although they did observe reduced in sacco protein degradation.

102 A number of compounds have been examined for their potential to ameliorate adverse

103effects of tannins or terpenes, including polyethylene glycol (PEG) and activated charcoal

104(Ach). PEG has a high binding affinity for tannins and been shown to increase intake of

105tannin-rich forage (Pritchard et al., 1988, Silanikove et al., 1994), probably because increased

106binding of tannins alleviates adverse postingestive consequences such as lesions in the gut

107mucosa (Reed, 1995). Activated charcoal fed to lambs also increased intake of big sagebrush

108(terpene-rich) by 30% (Banner et al., 2000).

109 The objective of the study was to determine if supplemental PEG/Ach affected intake

110of 3 tannin- and 3 terpene-rich shrubs of the Myrto-Quercetum ilicis, maquis plant community

111by sheep and goats. We also determined if the effect of supplemental PEG/Ach was

112consistent when animals were offered different combinations of shrubs or single, less

113palatable shrubs Pistacia lentiscus and Juniperus phoenicea. We hypothesized that intake of

114shrubs by both sheep and goats would increase with supplemental PEG/Ach as the number of 115shrubs increased.

116 METHODS AND MATERIALS

117Study shrubs and animals

118The experiments were conducted at an experiment station 25 km from Split in the central part

119of the Croatian Adriatic coast (lat 43052` N; long 16023` E). Three tannin- and three terpene-

120rich shrubs of the Myrto-Quercetum ilicis, maquis plant community were examined in six

121experiments, each 10 day long. The first three PEG-trials included Quercus ilex L.

122(Fagaceae), Arbutus unedo L. (Ericaceae), and Pistacia lentiscus L. (Anacardiaceae), while in

123the second three Ach-trials include shrubs include Juniperus phoenicea L. (Cupressaceae),

124Helichrysum italicum (Roth) Guss. (Asteraceae), and Juniperus oxycedrus L. (Cupressaceae).

125All 6 experiments were conducted with sheep and goats and ran consecutively.

126 The experimental sheep (n = 12, mean weight 23.08 ± 1.59 kg) were a local breed that

127is a cross between the Croatian breeds “Pramenka” and “Wunterberg”. The experimental

128goats (n = 12, mean weight 19.83 ± 1.89 kg) were a mixture of domestic goats crossed with

129Saanen and Alpine breeds. Both species of animals had an approximately equal mix of both

130sexes. All experimental animals were raised on the same farm on the island of Brac (Central

131Dalmatia) utilizing the shrubby vegetation of the Mediterranean maquis.

132 All shrubs offered to sheep and goats were hand-harvested each week in the same

133location. Leaves and one-year old twigs, 10 cm long, were clipped and placed in bags. Within

134an hour the plant material was ground to 1 cm length with a chipper, mixed for uniformity,

135placed in woven, polyethylene feed sacks, and refrigerated at 4oC. Every morning before the

136trial, bags of shrubs to be fed that day were removed from cold storage and offered

137immediately to animals.

139Experimental protocol 140Animals housed individually (1.5 x 2 m pens) in covered stalls with wire mesh sides, and

141fresh water and trace mineral salt were provided free-choice. Prior to the experiments,

142baseline intake of alfalfa pellets was determined for each animal on days 1 to 5. After

143baseline was established, all sheep and goats (n = 12) were offered all six shrubs

144simultaneously from 0900 to 1300 hr for 5 days. Shrub intake was monitored, and 12 sheep

145and 12 goats were divided into two experimental groups in each experiment (n = 6) based on

146total shrub intake. Treatment groups were unaltered during the experiments and the same 12

147sheep and 12 goats were used in all 6 experiments, but between each experiment with shrubs

148baseline was re-established using alfalfa pellets and barley at a maintenance level for 3 days.

149Experiments 1; Trials 1 – 3. The objective of the first three experiments was to assess the

150effect of PEG and energy on intake of three tannin-rich shrubs. In the first experiment, three

151shrubs (Quercus ilex, Arbutus unedo and Pistacia lentiscus), in the second experiment two

152shrubs (Arbutus unedo and Pistacia lentiscus), while in the third experiment one shrub

153Pistacia lentiscus were offered to sheep and goats. At 0800 hours each morning on days 1 to

15410 treatment sheep and goats received 200 g ground barley mixed with 25 g of PEG, whereas

155control animals did not receive ground barley or PEG. Then, all animals were offered 200 g

156of each shrub simultaneously in separate food boxes from 0900 to 1400 hours daily for 10

157days. Animals and food boxes were checked every 30 min and additional ground material was

158added as needed. Food refusals were collected and shrub consumption was calculated. At

1591500 hours animals were offered varying amounts of alfalfa pellets calculated to provide 50%

160of their maintenance energy requirements (including barley; INRA, 1989). During the

161experiment 2 and 3, we repeated the same procedure.

162Experiments 2; Trials 4 – 6. The objective of these experiments was to determine the effect

163of activated charcoal and energy on intake of three terpene-rich shrubs. In the first

164experiment, three shrubs (Juniperus phoenicea, Helichrysum italicum and Juniperus 165oxycedrus), in the second experiment two shrubs (Juniperus phoeniceae and Helichrysum

166italicum), while in the third experiment one shrub Juniperus phoenicea were offered to sheep

167and goats. During the 10-day experiment, treatment animals were given a 200 g mixed

168preload of ground barley with 20 g Ach at 0800, while control animals did not receive ground

169barley or Ach. All animals were then fed 200 g of each shrub from 0900 to 1400 hours.

170Additional shrub material was added as necessary every 30 min until 1400. Each day refused

171shrubs were collected and biomass consumption was calculated. Likewise, as in the

172experiments 1-3, at 1500 hours animals were offered varying amounts of alfalfa pellets

173calculated to provide 50% of their maintenance energy requirements (including barley; INRA,

1741989). During the experiment 2 and 3 we, repeated the same procedure.

175Statistical analysis

176The total daily consumption of all shrubs offered in each experiment was used as the

177dependent variable in the analysis. The experimental design was a completely random design.

178Animals were a random factor in the mixed model analysis (SAS 2000). The model included

179treatment (PEG/Ach vs. control), species of animal (goats vs. sheep), and the species x

180treatment interaction; individual animals were nested within treatment and species. The

181model also used days as a repeated measure with all other interactions included. All analyses

182on shrub intake were adjusted for body weight (g/kg BW).

184 RESULTS

185Experiment 1. The effect PEG on forage intake of the Mediterranean shrubs

186Trial 1. Three shrubs offered to sheep and goats

187Goats and sheep differed in total shrub consumption. Regardless of treatment, goats ate

188substantially more total shrub biomass than did sheep (60.7 vs. 45.9 ± 2.6 g/kg BW, 189respectively). There was a trend (P=0.08) towards a positive PEG effect on total shrub intake,

190with PEG-supplemented animals consuming more total shrubs than controls (56.7 vs. 50.0 ±

1912.6 g/kg BW).

192 In general, when 3 shrubs were offered to sheep and goats, either with or without

193supplemental PEG, both treatment groups and both species of animals showed a tendency for

194increasing intake from the first to the last day in all experiments. Sheep receiving PEG ate

195more (P=0.002) total shrubs than did controls (Figure 1), but no such treatment effect was

196found for goats (Figure 2). Regardless of treatment, sheep and goats ate almost the same

197amount of total shrubs (34.4 g/kg BW for sheep vs. 41.4 g/kg BW for goats). Sheep and goats

198preferred Quercus ilex (18.58 g/kg BW) and Arbutus unedo (15.95 g/kg BW) over Pistacia

199lentiscus (3.34 g/kg BW).

200Trial 2. Two shrubs offered to sheep and goats

201Supplemental PEG positively affected (P<0.001) total shrub intake when only 2 shrubs

202(Arbutus unedo and Pistacia lentiscus) of lower palatability were offered (Figure 1 and 2).

203Both sheep and goats fed supplemental PEG ate numerically more Pistacia lentiscus (17.54

204g/kg BW) compared to non-supplemented sheep or goats (7.57 g/kg BW) and especially

205compared Pistacia lentiscus in experiment 1 (3.34 g/kg BW).

206Trial 3. One shrub - Pistacia lentiscus offered to sheep and goats

207Supplemental PEG had a marked positive effect (P<0.001) on intake of Pistacia lentiscus in

208both sheep and goats (Figure 1 and 2). PEG-supplemented goats ate more Pistacia lentiscus

209(39.6 g/kg BW) than did PEG supplemented sheep (28.1 g/kg BW), whereas control sheep

210and goats ate similar amounts (12.2 and 15.3 g/kg BW, respectively).

211Experiment 2. The effect Ach on forage intake of the Mediterranean shrubs

212Trial 4. Six shrubs offered to sheep and goats 213As in experiment 1, goats again ate more shrubs than did sheep throughout experiment 4

214(P<0.01, Figure 3 and 4). Ach-treated sheep and goats ate more total shrubs than did control

215animals (P=0.002; 37.9 ± 1.4 g/kg BW versus 28.2 ± 1.3 g/kg BW). There were no species x

216treatment or treatment x day interactions (P > 0.50), but there was a species x day interaction

217as both goats and sheep increased intake of the 3 shrubs over the period (P=0.004; Figure 3

218and 4). Even though sheep and goats differed in total amounts of shrubs eaten, the rank order

219of the amount eaten for each shrub species was essentially the same for both sheep and goats.

220The mean amounts eaten of the shrubs across all treatments and animal species were:

221Juniperus oxycedrus (20.35 g/kg BW), Helichrysum italicum (18.16 g/kg BW) and Juniperus

222phoenicea (15.75 g/kg BW).

223Trial 5. Two shrubs offered to sheep and goats

224Goats had a higher total intake when offered 2 shrubs than did sheep (P=0.0003; 34.5 ± 0.9

225g/kg BW versus 24.3 ± 0.9 g/kg BW; Figure 3 and 4). Sheep and goats fed Ach ate more

226shrubs than did control animals (P=0.001; 33.9 ± 0.8 g/kg BW versus 24.8 ± 1.1 g/kg BW).

227There were no species x treatment or species x day interactions (P > 0.50), but there was a

228treatment x day interaction (P = 0.03) as charcoal-treated animals increased intake of the 2

229shrubs over the 5-day period more than the control animals (Figure 3).

230Trial 6. One shrub – Juniperus phoenicea offered to sheep and goats

231There was a treatment x day (P = 0.03) interaction as Ach-treated animals, particularly sheep,

232increased intake of Juniperus phoenicea over time compared to control animals (Figure 3 and

2334). There was also a species x day interaction (P = 0.02) as sheep increased intake over the

234period more than did goats even though sheep ate less Juniperus phoenicea biomass

235compared to goats. 236 DISCUSSION

2371. Effect of PEG on Mediterranean tannin rich shrubs intake

238PEG is a polymer that binds to tannins irreversibly over a wide range of pH, thus alleviating

239the negative effects of tannins (Landau et al., 2000). Supplemental PEG increases intake of

240tannin-containing plants by sheep and goats (Pritchard et al., 1992; Titus et al., 2000, 2001)

241and cattle (Hanningan and McNeil, 1998). Nevertheless, it is very interesting that when sheep

242and goats were offered 6 shrubs (Rogosic et al., 2007a), and when goats were offered 3

243shrubs, PEG had no beneficial effect on intake (Figure 2). In general, as the number of shrubs

244in the diet decreased, the impact of PEG on intake of shrubs increased. PEG alone increased

245consumption of the 3-shrub mix by sheep. When 3 species were offered to sheep (trial 1), and

246when 2 or 1 species offered to goats (trials 2 and 3), PEG and energy substantially increased

247intake of shrubs high in tannin.

248 Interactions among nutrients, toxins and PEG affected use of shrubs. For example, in

249trial 2 the relatively unpalatable shrub Pistacia lentiscus was not avoided when offered with

250another more palatable shrub (Arbutus unedo) even though animals were not forced to eat

251Pistacia lentiscus. Further, goats and sheep receiving PEG and energy in the 2-shrub trial ate

252much more of both shrubs than did control animals, indicating the effectiveness of

253PEG/barley in reducing the impacts of tannins from Pistacia lentiscus and Arbutus unedo

254(Villalba et al., 2002a).

255 Food selection by herbivores depends on nutritional state (Villalba et al., 2002a). In

256trials 2 and 3, the control animals did not receive supplemental feed. When ingesting toxins

257entails a metabolic cost, herbivores are likely to respond by modifying their feeding behavior

258(Foley et al., 1999). Supplemental macronutrients increase intake of foods that contain toxins

259as diverse as lithium chloride (Wang and Provenza, 1996), terpens and tannins (Rogosic et al.,

2602007), menthol (Illus and Jessop, 1996), and quebracho tannin (Villalba et al., 2002a). 261Consequently, the ability of herbivores to consume shrubs high in secondary compounds will

262depend on the interaction between the quantity and quality of nutrients available and the

263classes of toxins present in the mix of plant species (Provenza et al. 2003).

2642. Effect of Activated Charcoal on Mediterranean Terpenes Rich Shrubs Intake

265Activated charcoal adsorbs phytochemicals in shrubs and facilitates their excretion via the

266feces (Buck and Bratich, 1986). Positively charged surfaces in activated charcoal bind with

267the negatively charged molecular surface of most toxins, decreasing their bioaviability (Poage

268et al., 2000). In trial 4, when 3 shrubs were offered, sheep and goats supplemented with 200 g

269barley + 20 g Ach (treatment groups) ate more shrubs than sheep and goats (control group),

270which is not supplemented with Ach nor energy. This is similar to other results when lambs

271were supplemented with charcoal-barley mix (P = 0.71, 1.7 g charcoal/kg 0.75) and they ate

272more sagebrush than lambs supplemented only with barley (Banner et al., 2000).

273 When 3 shrubs were offered to sheep and goats, and controls were given energy as

274well, there was a positive effect from Ach on intake of 3 shrubs high in terpenes (Juniperus

275phoenicea, Helichrysum italicum and Juniperus oxicedrus; trial 4). When 2 shrubs (Juniperus

276phoenicea and Helichrysum italicum; trial 5) or 1 shrub (Juniperus phoenicea trial 6) was

277offered to sheep and goats, Ach and energy together substantially increased intake of shrubs

278high in terpenes (Figures 3 and 4). Previous work has shown that lambs that learned to eat

279foods containing a variety of toxins ate more when they could select 2 of the foods rather than

280only 1 (Villalba et al., 2004).

281 Further, goats and sheep receiving charcoal and energy in trials 4 – 6, when offered 3

282 2  1 shrub ate much more shrub than did controls, indicating the effectiveness of charcoal

283and barley in reducing the impacts of terpenes from Juniperus phoenicea, Helichrysum

284italicum and Juniperus oxicedrus. Other studies with sagebrush (Artemisia sp.) in the U.S.

285also have shown that supplemental energy impacted shrub intake when given in combination 286with activated charcoal (Banner et al., 2000; Villalba et al., 2002b).

2873. Influence of biological/biochemical diversity on shrub selection

288The vast majority of Mediterranean shrubs contain secondary compounds, thus browsing

289sheep and goats can not avoid ingesting various phytotoxins that commonly occur in those

290plants (Rogosic, et al., 2007). Most secondary compounds are ingested in amounts too low to

291cause acute symptoms of poisoning and death, and we saw no overt adverse signs of toxicity.

292This study confirmed the common view that mixed diets improve food intake (Freeland and

293Choquenot, 1990). The kinds and different amount of nutrients and toxins in the

294Mediterranean shrubs fed to sheep and goats probably influenced toxin satiation, which is not

295fixed but dynamically determined by the physiological state of animal relative to the quality

296and quantity of nutrients and toxins available (Villalba et al., 2002a). In essence, toxins cause

297herbivores to eat a variety of plants to meet their needs for nutrients because the pathways of

298detoxification are saturable (Foley et al., 1999). Thus, as animals satiate on particular toxins

299they must find alternative food sources containing complementary nutrients and toxins

300(Villalba al. al., 2002b). The satiety hypothesis and ensuing nutrient-nutrient, nutrient-toxin,

301and toxin-toxin interactions help to explain the dynamics of these interactions and the benefits

302of varied diets (Provenza et al., 2003).

303 As the number of shrubs decreased from trial 1 to trial 3 and from trial 4 to trial 6, the

304concentration of tannins and terpenes in shrub biomass offered to animals increase, so the

305total shrub intake decreased numerically for both sheep and goats. In other words, these

306results clearly showed that combinations of more shrubs offered to animals promoted greater

307intake. These results are consistent with the satiety hypothesis (Provenza et al., 2003) that

308interactions among flavors, nutrients, and toxins (i.e., secondary compounds) lead to

309enhanced intakes, even of lower preference shrub such as Juniperus phoenicea and Pistacia

310lentiscus. 311 Depending on prevailing conditions, sheep and goats browsing on Mediterranean

312shrubby vegetation may be able to mix their diets to provide sufficient energy and protein

313while reducing toxin loads. The shrubs offered in these experiments varied in amounts of

314macronutrients (Rogosic et al., 2006), and also in kinds and amounts of secondary compounds

315such as terpenes (high terpenes: Juniperus phoenicea, Helichrysum italicum and Juniperus

316oxycedrus), tannins (high tannins: Arbutus unedo, Quercus ilex and Pistacia lentiscus).

317 Our results suggest that interaction between macronutrients, PEG (in trial 1 - 3) and/or

318Ach (in trial 4 - 6), flavor and toxins influenced how sheep and goats mixed their diets and

319utilized shrubs, and that supplemental macronutrients enhanced intake of food that contain

320terpenes and tannins. Likewise, different concentrations of nutrients in plant species may have

321different effects on selection of food by sheep and goats depending on the classes and

322concentration of toxins in maquis plant community.

324 CONCLUSIONS

325Shrubby vegetation of the Mediterranean maquis dominates rangelands throughout the

326Mediterranean Basin, including the Adriatic littoral of Croatia. Sheep and goats are an

327environmentally and economically sound alternative for using the forage potential of

328Mediterranean shrubs. Increasing use of these shrubs by livestock would likely enhance the

329production of grasses and forbs and create a more diverse mix of plants. Grazing by livestock

330also reduces the likelihood and the impacts of fires, common in these regions.

331 Most Mediterranean shrubs contain large quantities of secondary compounds that limit

332intake and cause animals to eat a variety of foods. In this study, as the number of shrub

333species in the diet decrease (trials 1 - 3 and trails 4 - 6), so too did intake. Both PEG and Ach

334supplement positively influenced shrub intake when offered 3  2 1 shrubs to sheep and

335goats. Likewise PEG and Ach had the same influence on sheep and goats throughout the 336experiments, and it had the most influence on both sheep and goats when shrub availability

337was reduced.

338 Our findings suggest that biochemical diversity plays a very important role in

339herbivore’s diet selection, enabling animals to better meets their nutritional needs and avoid

340toxicity.

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Sheep 50 3 shrubs 2 shrubs 1 shrub Trial 1 Trial 2 Trial 3 a 40 a

) Quercus ilex . b

w Arbutus unedo . 30 b a

Pistacia lentiscus g k / g (

e 20 k a t

n b I 10

0 p p p u p p p u u o u u u ro ro r o ro ro g g -g r g g - - L g - - L G - L G O E O G O E R P R E R P T T P T N N N O O O C C C 462 463Figure 1. Total shrub intake by sheep offered three (Quercus ilex, Arbutus unedo, and

464Pistacia lentiscus), two (Arbutus unedo, and Pistacia lentiscus), or one (Pistacia lentiscus)

465shrub species. Different letters indicate that charcoal-treated animals and controls differed

466(Trial. 1, P= 0.002; Trial 2, P 0.001; Trial 3, P 0,001) within an experiment.

Goats 60 3 shrubs 2 shrubs 1 shrub Trial 2 Trial 3 Trial 1 a 50 a )

. a a Quercus ilex w 40 .

b Arbutus unedo

g Pistacia lentiscus k / 30 g

0 p u p p p p u u u u p ro o o o o u g r r r r o - g -g -g -g r G - L -g E L G G L P O E O E R P R P O T T R N N T O N O C O 468 C C

470Figure 2. Total shrub intake by goats offered three (Quercus ilex, Arbutus unedo, and Pistacia

471lentiscus), two (Arbutus unedo, and Pistacia lentiscus), or one (Pistacia lentiscus) shrub

472species. Different letters indicate that charcoal-treated animals and controls differed (Trial 1,

473P= 0.002; Trial 2, P 0.001; Trial 3, P 0.001) within an experiment.

475 482 481 480 479 478 477 476 experiment. experiment. controls(Trial1,P differed and animals ( one italicum, bysheep( intake offered 3.Totalshrub three Figure

S Intake (g/kg b.w.) h 15 20 25 30 35 10 Juniperus phoenicea e

e 0 5 p C and S h h a e r e c p o

Juniperus oxycedrus a C l o shrubs3 n 1 Trial tr a o l

S h ) shrub species. Different letters indicate that charcoal-treated charcoal-treated that indicate letters species. Different ) shrub e e

p b C S h h a r e c e

), two( o p a C l

n shrubs2 tr Trial2  o

0.01; Trial 2, P= 0.001; Trial3,P=an Trial2,P= 0.02)within 0.001; 0.01; l Juniperus phoenicea Juniperus phoenicea

S h e b e p C

Juniperus phoenicea, Helichrysum Helichrysum Juniperus phoenicea, S h h a e r e c p o a C l a o n tr

o 1shrub l Trial3 and and Juniperusoxycedrus Helichrysumitalicum Juniperusphoenicea b Helichrysum italicumHelichrysum ), or 491 490 489 488 487 486 485 484 483 experiment. experiment. controls(Trial1,P differed and animals ( one italicum, bygoats( intake offered 4.Totalshrub three Figure Intake (g/kg b.w.) Juniperus phoenicea 10 20 30 40 50 0

and G o a t

Juniperus oxycedrus C h a r c Trial 1

o a a G l o a t

C 3 shrubs

) shrub species. Different letters indicate that charcoal-treated charcoal-treated that indicate letters species. Different ) shrub o n tr G o b o l a t C h ), two( a r c o a l a

 G 2shrubs 0.01; Trial 2, P= 0.001; Trial3,P=an Trial2,P= 0.02)within 0.001; 0.01; Juniperus phoenicea Juniperus phoenicea o Trial2 a t C o n tr G o l b o a Juniperus phoenicea, Helichrysum Helichrysum Juniperus phoenicea, t C h a r c o a l a G o

and and a 1shrub

t Trial3 C o

Helichrysum italicumHelichrysum n tr o b

l Juniperus oxycedrus Helichrysumitalicum Juniperus phoenicea ), or