Consumption of Pistacia lentiscus foliage alleviates coccidiosis in young goats

Veterinary Parasitology 186 (2012) 165–169

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Consumption of Pistacia lentiscus foliage alleviates coccidiosis in young goats A. Markovics a , I. Cohen b , H. Muklada b , T.A. Glasser c , L. Dvash b , E.D. Ungar b , H. Azaizeh d , S.Y. Landau b,∗ a

Department of Parasitology, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel Department of Agronomy and Natural Resources, Institute of Plant Sciences, Agricultural Research Organization, the Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel c The Ramat Hanadiv Nature Park, Zikhron Yaakov, Israel d Institute of Applied Research (Affiliated with University of Haifa), The Galilee Society, Shefa-Amr 20200, Israel b

a r t i c l e

i n f o

Article history: Received 26 September 2011 Received in revised form 27 November 2011 Accepted 29 November 2011 Keywords: Eimeria Caprine Tannin Browse Coccidiosis Mediterranean

a b s t r a c t Coccidiosis near weaning is a major cause of diarrhea, ill-thrift, and impaired performance in small ruminants. A recent survey showed that in villages of the Samaria Hills, Israel, shepherds treat young, weaned goat kids afflicted with diarrhea by cutting and feeding them the foliage of Pistacia lentiscus L. (lentisk) or by tethering them close to lentisk bushes which they browse. The aim of the present study was to assess whether lentisk leaves do indeed have anti-coccidial value, and, if positive, to ascertain the role of tannins in this effect. We monitored for 24 (Experiment 1) and 30 (Experiment 2) days the effect of lentisk feeding on the development of naturally occurring coccidiosis in weaned kids artificially infected with parasitic nematodes. In Experiment 1, kids were infected with nematodes and fed lentisk foliage (PIS) or cereal hay (HAY). Coccidiosis developed at the early stage of the nematode infection, when dietary treatments were initiated. Kids in the PIS group had a lower (P < 0.02) concentration of oocysts per gram feces (opg). In Experiment 2, aimed at verifying if tannins are the active component in lentisk foliage, coccidiosis occurred at the peak of the nematode infection, before experimental diets were initiated. Dietary treatments were: cereal hay (HAY), or lentisk foliage consumed without (PIS) or with (PISPEG) a 20-g daily supplement of polyethylene glycol (PEG; MW 4000), a molecule that impairs tannin-bonding with proteins. Goats fed the PIS diet had lower fecal opg counts than counterparts of the HAY (P < 0.001) and PISPEG (P < 0.002) treatments. Fecal opg counts for the HAY and PISPEG treatments did not differ, suggesting that the anti-coccidial moiety in lentisk was indeed tannins. Our results strongly suggest that: (i) in agreement with the ethno-veterinary anecdotal evidence, exposure of young, weaned goat kids to lentisk foliage alleviates coccidiosis; and (ii) this positive effect is associated with tannins. As coccidiosis is a major affliction of kids, providing them with tannin-rich browse near weaning could be an environmentally friendly way of improving their welfare and health status, in particular under bio-organic farm management. © 2011 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. Tel.: +972 3 9683492; fax: +972 3 9669642. E-mail address: [email protected] (S.Y. Landau). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.11.072

Most extensively managed Mediterranean goat flocks are exposed to Eimeria infections (e.g., De la Fuente and Alunda, 1992; Sayin et al., 1986). Even though all animals host Eimeria protozoa, it is mainly young kids near weaning

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that suffer from coccidiosis, resulting in ill-thrift, decreased feed intake, and mortality (Gregory and Norton, 1986). As kids are weaned at the end of spring, a period favorable to nematode infections, coccidiosis and helminthiosis superimpose in multispecies etiologies characterized by prolonged diarrhea (De la Fuente et al., 1993). A recent survey (Landau et al., 2010a) showed that in villages of the Samaria Hills, Israel, shepherds use foliage of lentisk (Pistacia lentiscus L.; Family: Anacardiaceae; common names: lentisk, mastic tree, sarees (Arabic)) to treat goats afflicted with diarrhea, either by cutting and feeding or by tethering the goats close to lentisk bushes. Lentisk is native of coastal and hilly areas throughout the Mediterranean region. In the Middle-East, lentisk infusions are used in humans to treat jaundice (Said et al., 2002), diarrhea and stomach ache (Chevallier, 1996). Landau et al. (2010b) demonstrated the anthelmintic value of lentisk foliage in goats. Lentisk leaf chemistry is characterized by an exceptionally high concentration of polyethyleneglycol-binding tannins (Landau et al., 2004), including hydrolizable tannins (Romani et al., 2002), and a variety of terpenes (Barazani et al., 2003). Nevertheless, the plant is not toxic, even when fed to goats as the sole source of dietary roughage (Silanikove et al., 1996a). Aqueous extracts of leaves from Azadirachta indica decreased oocyst excretion in Eimeria-infected poultry as efficaciously as nicarbazin (Biu et al., 2006). Ripe A. indica and Melia azedarach fruits showed anti-coccidial value in poultry (Ali Tipu et al., 2002) and goats (Madibela and Kelemogile, 2008). Although aqueous extracts of foliage of the Meliacea family contain tannins (Madibela and Kelemogile, 2008), there is no prior evidence that tannins constitute the anti-coccidial moiety. The effects of lentisk and tannins on coccidiosis that we report on here came to light serendipitously while examining their effects on nematode infection. Although not their original purpose, the two experiments described below provide evidence of the anti-coccidial value of lentisk leaves in goats, and of its attribution to tannins. 2. Materials and methods Two experiments were carried out at the Ramat Hanadiv Nature Park, at the south of the Mount Carmel ridge, Israel. Welfare regulations of the Israel Council on Animal Care Guidelines (ICACG, 1994) were followed throughout the experiments. 2.1. Experiment 1 Experiment 1 was carried out with 26 local female kids aged between 6 and 7 months and weighing initially 27.8 ± 1.8 kg (average ± SEM). On 5 August, kids were allotted to two experimental diets consisting of 400 g (fresh weight) of a commercial concentrate (Ambar Feed Mills, Hadera, Israel) containing 16% crude protein (CP) together with an ad libitum allowance of either lentisk foliage (PIS; n = 13) or cereal hay (HAY, n = 13). On 16 August, fecal oocyst counts (oocysts per gram feces; opg) – performed using a modified McMaster technique with a detection level of 100 oocysts per gram (opg) – did not differ between

groups (750 ± 500 and 840 ± 570 opg for the HAY and PIS treatments, respectively). Nine goats in the PIS treatment were penned individually from 16 August to 16 September in order to quantify tannin intake. The remaining PIS goats and the HAY goats were group-fed in separate corrals. All goats were infected with 8000 and 5000 L3 mixed-species nematode larvae – Teladorsagia cirumcinta, Trichostrongylus colubriformis and Chabertia ovina – on 19 August and 26 August, respectively. Fecal samples were collected rectally from each animal on 16 and 19 August and then four times at weekly intervals until 18 September. As nematode fecal egg count (FEC) values were 0 for all kids until 16 September, and reached only 200 eggs per gram (epg) at the end of experiment, on 18 September, it can be inferred that the start of coccidial infection occurred before, and developed in the very early stage of nematode infection. 2.2. Experiment 2 Experiment 2 studied the effect of lentisk tannins on strongyle-infected young local goats (Landau et al., 2010b). In brief, 28 weaned, young female goats were assigned to the experiment on 5 October and fed 600 g of concentrate (16% CP) and wheat hay ad libitum. They were infected with doses of 7500 and 2500 L3 mixed larvae encompassing 3 species – Teladorsagia cirumcinta, Trichostrongylus colubriformis and Chabertia ovina – on 1 and 8 September, respectively. The experiment was designed to offer tanninrich browse once FEC reaches a minimal level, therefore, we collected feces weekly to monitor the development of infection. No Eimeria oocysts were found in the feces incubated to manufacture the infecting doses or in the infecting doses. On 5 October, while monitoring the development of the strongyle infection, the mean count was 2680 ± 548 opg. At that time, the nematode mean FEC was still very low (66 epg). The build-up of coccidiosis was confirmed on 13 October, with a mean count of 2843 ± 2007 opg. At that stage, the strongyle mean FEC was 250 ± 89 epg. On 22 October we initiated dietary treatments. At that stage, goats were 215 ± 1.6 days old and weighed 24.7 ± 1.1 kg. The animals were penned individually and allotted to one of three diets which they received for 30 days. Body weight was balanced between diets. Treatments were: a control diet of hay and concentrate (C); a diet of lentisk foliage comprising twigs and leaves freshly collected in the nature park (PIS); and a diet of lentisk foliage plus 20 g d−1 of Polyethyleneglycol (PEG 4000; PISPEG). Strongyle FEC peaked with a mean of 1042 ± 250 epg on 5 November and decreased thereafter to 534 ± 250 epg on 18 November, when dietary treatments were discontinued. Thus we monitored the coccidiosis episode for 24 days at the climax of a strongyle infection. Fecal samples were taken at days 11, 13, 16, 21, 24, after initiation of dietary treatments. Rations were formulated to meet all nutrient requirements with a daily intake of 0.025 × BW (body weight). The daily intake of PEG-binding tannins was planned to be 1.5 g kg−1 BW, i.e., more than needed to bond with all the tannins in the lentisk diet treatment and to counteract their effects on nutrient intake and digestibility in young goats (Silanikove et al., 1996b). This dosage ensured that if

A. Markovics et al. / Veterinary Parasitology 186 (2012) 165–169 Table 1 The contents of ash, crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) and poly-ethylenebinding-tannins (PEG-b-T), and the in vitro dry matter digestibility (IVDMD), as percentage of dry matter, of the feeds used in experiments 1 and 2. Lentisk foliage Experiment 1 Ash CP NDF ADF ADL PEG-b-T IVDMD

5.3 ± 0.1 8.5 ± 0.1 38.7 ± 0.4 28.2 ± 0.4 16.0 ± 0.1 21.5 ± 0.4 31.5 ± 0.5

Hay Experiment 2 5.0 ± 0.3 8.3 ± 0.6 35.8 ± 2.4 26.1 ± 1.7 14.9 ± 1.0 21.0 ± 0.3 32.1 ± 0.5

6.1 10.1 64.2 36.8 5.8 0 49.6

lentisk foliage had anti-coccidial activity, this activity was neutralized by PEG, then it could be inferred that tannins are the anti-coccidial moiety (see rationale in Landau et al., 2010b). Lentisk foliage was collected daily in the park, leaves were manually stripped from stems and fed within one hour after collection. 2.3. Chemical analyses of diets Samples of hay, concentrate, and lentisk foliage were ground to pass a 1-mm sieve. The in vitro digestibility of dry matter (IVDMD) was evaluated according to Tilley and Terry (1963). Crude protein (CP) was assayed by using an automated Kjeldahl method (AOAC method 976.05; AOAC, 1990), and neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were assayed according to Goering and Van Soest (1970). The content of PEG-binding tannins (PEG-b-T) was determined by near infrared spectrometry without extraction, according to Landau et al. (2004). Ash content was established by ashing at 450  C for 5 h. The same hay and concentrate were used in Experiments 1 and 2. A sample of lentisk foliage as fed was taken 10 and 15 times throughout Experiments 1 and 2, respectively. Chemical composition is shown in Table 1.

18000

tanint

167 HAY

PIS

80

16000

70

14000

60

12000

50

10000

40

8000

30

6000 4000

20

2000

10

0

15-Aug

25-Aug

4-Sep

14-Sep

0

Fig. 1. Fecal oocyst counts and tannin intake in Experiment 1: kids received hay (HAY, circles; n = 13) or lentisk (P. lentiscus;PIS, squares; n = 13) foliage 5 August to 18 September. Strongyle infection was on 18 August. Lines indicate fecal oocyst counts in both treatments (mean ± SE). The intake of PEG-binding tannins (tanint, g d−1 ) in the PIS group is indicated by columns (mean ± SE). X-axis: date Y-axis: Oocyst count (opg).

3. Results 3.1. Experiment 1 Intake of PEG-binding tannins from lentisk stabilized in the range 52–60 g d−1 , i.e., approximately 2 g kg−1 BW d−1 . Fecal opg counts for the two treatments were similar in the early phase of the experiment but then diverged greatly (Fig. 1). In the analysis of fecal oocyst counts, the effect of days was significant (P < 0.05) but the interaction day × diet and the covariate effects were not. While fecal opg values peaked at approximately 13,000 in the HAY group, lentiskfed kids had opg values of 2600. Overall, opg values were lower for PIS than for HAY (P < 0.02). 3.2. Experiment 2 The intake of PEG-binding tannins from P. lentiscus was 29.3 ± 4.3 and 26.3 ± 2.3 g d−1 for goats in the PIS and PISPEG groups, respectively (Fig. 2). At the peak of coccidian infection, fecal opg values were 131,000, 56,000, and 143,000 for the HAY, PIS, and PISPEG treatments,

2.4. Statistical analyses Fecal counts were (square-root +0.5) transformed in order to normalize the data and stabilize variances. In order to test the effect of diets on opg, we used the MIXED procedure of SAS version 9.1 (SAS, 2002). Terms in the model were diet, kid within diet as a random effect, day of sampling as the repeated measure, and interaction between diet and day of sampling. The covariance structure used was AR(1). In Experiment 1, we verified that the management of goats as a group or in cages did not affect fecal opg counts and the management factor was not included in statistical analyses. The average opg counts of the two measurements before dietary treatments were initiated served as covariate for both experiments. We separated means on sampling days by contrast t-tests using the Bonferroni correction for multiple testing.

Fig. 2. Oocyst counts in Experiment 2: kids received hay (HAY, circles; n = 10) or lentisk (P. lentiscus) foliage with (PISPEG, triangles; n = 9) or without (PIS, squares; n = 9) a supplement of poly(ethylene)-glycol (20 g d−1 , MW 4000). Arrow indicates start of experimental diets (22 October): Means ± SE. X-axis: date. Y-axis: Oocyst count (opg).

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respectively, i.e., much higher than in Experiment 1. The effect of day of measurement on opg was significant (P < 0.0001) and the treatment × day interaction tended to be significant (P < 0.07), but the effect of covariate (opg counts before experimental diets were given) was not (P = 0.38). PIS-fed kids had lower opg counts than counterparts fed hay or lentisk plus PEG (P < 0.002), and the two latter treatments did not differ between them (P = 0.92). 4. Discussion Although designed to address a different, albeit often related, clinical condition in young goats, results of both experiments present strong evidence that consumption of P. lentiscus foliage alleviates naturally occurring coccidiosis infection. This is consistent with the ethno-veterinary practice of tethering diarrheic young goats near lentisk shrubs (Landau et al., 2010a). Madibela and Kelemogile (2008) demonstrated that the oocyst count of goats feeding on ripe M. azedarach fruit remained stable at approximately 6400 opg for one month, while that of control animals soared to 33,000 opg (P < 0.001). Because the fruit pulp contains 4% of condensed tannins on a dry matter (DM) basis, these authors attributed this effect to tannins. However, M. azedarach fruit also contain a wide array of limonoids that have anti-protozoan properties (Roy and Saraf, 2006), as shown in vivo with apes (Huffman and Seifu, 1989). Therefore, the specificity of tannins against coccidian infection had to be established. This was achieved in Experiment 2 of the present study: when tannin-binding with protein was impaired by PEG, P. lentiscus did not alleviate the development of coccidiosis (Fig. 2). To the best of our knowledge, this is the first published evidence demonstrating the specific anti-coccidial value of tannins in vivo. Previous studies showed that dietary tannins decrease protozoa counts in the ruminal fluid of small ruminants in a specific way that is reversed by PEG (Yanez Ruiz et al., 2004). In an in vitro study with goats (Hur et al., 2005), feeding 40 g condensed tannins (CT) DM/day from fresh pine needles or oak leaves in combination with lucerne chaff acted rapidly as a coccidiostat, as demonstrated by a sharp decrease in oocyst production. OPG was reduced to 7% (pine) and 15% (oak) of pre-treatment levels within ten days. An in vitro study with avian coccidian parasites showed that an aqueous extract of pine bark impaired oocyst sporulation (Molan et al., 2009). This suggests that the mechanism by which tannins alleviate coccidiosis could be decreased infection rate mediated by impaired sporulation of oocysts. What would be the dietary tannin contents needed to control coccidiosis? Some of the goats did not consume all their P. lentiscus allowance in the PIS group, which provides an opportunity to study the relationship between fecal opg and the intake of tannins around the peak of infection. Goats in the PISPEG group were withdrawn from this analysis. A power function best described the relationship between tannin intake and opg (Fig. 3) but tannins accounted for only 33% of the variation in opg at the peak of infection. In the study of Madibela and Kelemogile (2008), where the opg values peaked at 35,000 in the controls,

500000 450000 400000 350000 300000 250000 200000 y = 83787e-0.04x R² = 0.322

150000 100000 50000 0 0

10

20

30

40

50

60

Fig. 3. Mean fecal oocyst count (opg, Y-axis) of goats in the PIS (n = 9) and HAY (n = 9) groups as a function of the daily intake of PEG-binding tannins (g d−1 ) at the peak of coccidian infection in Experiment 2 (2 November). Tannin intake explained 32% of opg variation. X-axis: Intake of PEG-binding tannins (g d−1 ). Y-axis: Oocyst count (opg).

an amount of tannins from M. azedarach fruits as small as 7.3 g d−1 controlled naturally occurring coccidiosis in goats weighing approximately 20 kg, i.e., 0.36 g kg−1 BW d−1 . In Experiment 1 of the present study, tannin intake at the peak of coccidian infection was 55 g d−1 , or approximately 2 g kg−1 BW d−1 , and opg values decreased from 14,000 to less than 4000. In Experiment 2, opg values peaked at 140,000 for HAY goats, compared with 60,000 in the PIS goats, indicating that the tannin intake of 27 g d−1 , or 1 g kg−1 BW d−1 , was not sufficient to control the coccidiosis outburst. Comparison of opg values in Experiments 1 and 2 suggests that a dose of 2 g of lentisk tannins kg−1 BW d−1 should control coccidiosis. We previously showed that a daily intake of 1 g of lentisk tannins kg−1 BW achieved almost total suppression of fecal strongyle egg excretion following mixed-species infection (Landau et al., 2010b). It seems, therefore, that a higher dose of tannins is needed to control coccidiosis than to control helminthiosis. The requirement for tannins as anti-coccidial agent is a function of infection severity, and dose response studies are needed. Finally, individual resistance to coccidiosis is probable, where opg values vary from 0 to more than 450,000 opg in control goats (Fig. 3), suggesting that response to infection varies greatly among animals. Also, opg values were much higher in Experiment 2, where coccidian infection occurred at the peak of helminth infection, which suggests that the immune status in Experiment 2 was more challenged than in Experiment 1. Tannins decrease protein availability (Silanikove et al., 1996b; Landau et al., 2010b), which in turn impairs immunity to parasites (Kyriazakis and Houdijk, 2006). So there is a trade-off between a negative role of tannins on protein metabolism, and a positive, direct action on parasites. The optimal dosage should maximize anti-coccidian value while minimizing impairment of host immunity. This trade-off has still to be studied for coccidiosis. 5. Conclusions Consumption of lentisk (Pistacia lentiscus L.) foliage after natural coccidian infection decreased fecal excretion

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of oocysts. This anti-coccidial effect can be attributed to tannins. As consumers have negative perceptions of chemical coccidiostats, dietary tannins could be an environmentally-friendly alternative, in particular under bio-organic management of livestock, where coccidiostats are banned. Acknowledgements We thank the Ministry of Science of Israel for partial funding of this study in the framework of Regional Research and Development programs. We are indebted to Dr Hillary Voet for professional assistance in statistical analyses, and to staff of the Ramat Hanadiv Nature Park – in particular, Mr Hugo Jan Trago – for allowing us to carry out this study with their herd. ARO publication no. 111/2011. References Ali Tipu, M., Pasha, T.N., Ali, Z., 2002. Comparative efficiency of salinomycin sodium and neem fruit (Azadirachta indica) as feed additive anti-coccidials in broilers. Intl. J. Poultry Sci. 1, 91–93. AOAC, 1990. Official Methods of Analysis, 15th ed. Assoc. Off. Anal. Chem., Arlington, VA. Barazani, O., Dudai, N., Golan-Goldhirsh, A., 2003. Comparison of Mediterranean Pistacia lentiscus genotypes by random amplified polymorphic DNA, chemical, and morphological analyses. J. Chem. Ecol. 29, 1939–1952. Biu, A.A., Yusuf, S.D., Rabo, J.S., 2006. Use of Neem (Azidirachta indica) aqueous extract as a treatment for poultry coccidiosis in Borno State, Nigeria. Af. Sci. 7, 147–153. Chevallier, A., 1996. The Encyclopedia of Medicinal Plants. Dorling Kindersley, London. De la Fuente, C., Alunda, J.M., 1992. A quantitative study of Eimeria infections of goats from Central Spain. Vet. Parasitol. 41, 7–15. De la Fuente, C., Cuquerella, M., Carrera, L., Alunda, J.M., 1993. Effect of subclinical coccidiosis in kids on subsequent trichostrongylid infection after weaning. Vet. Parasitol. 45, 177–183. Goering, H.K., Van Soest, P.J., 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agriculture Handbook No. 379. ARS-USDA, Washington, DC. Gregory, M.W., Norton, C.C., 1986. Caprine coccidiosis. Goat Vet. Soc. J. 7, 32–34. Huffman, M.A., Seifu, M., 1989. Observations on the illness and consumption of a possibly medicinal plant Vernonia amygdalina by a wild chimpanzee in the Mahale Mountains National Park, Tanzania. Primates 30, 51–63.

169

Hur, S.N., Molan, A.L., Cha, J.O., 2005. Effects of feeding condensed tannin-containing plants on natural coccidian infection in goats. Asian-Australasian J. Anim. Sci. 18, 1262–1266. ICACG, 1994. Israel Council on Animal Care Guidelines: Legislation on Animal Welfare (defending animal rights). Paragraph 14. Knesset Law Pub, Jerusalem, Israel (in Hebrew). Kyriazakis, I., Houdijk, J., 2006. Immunonutrition: nutritional control of parasites. Small Rumin. Res. 62, 79–82. Landau, S., Dvash, L., Decandia, M., Cabiddu, A., Shapiro, F., Molle, G., Silanikove, N., 2004. Determination of poly(ethylene glycol)-binding to browse foliage, as an assay of tannin, by near-infrared reflectance spectroscopy. J. Agric. Food Chem. 52, 638–642. Landau, S.Y., Azaizeh, H., Muklada, H., Abu-Rabia, A., Abu-Siam, S., 2010a. Ethno-veterinary use of plants in Israel. Report to Nekudat Hen, Keren HaNadiv, the Rothschild Foundation (Jerusalem). Landau, S., Azaizeh, H., Muklada, H., Glasser, T., Ungar, E.D., Baram, H., Abbas, N., Markovics, A., 2010b. Anthelmintic activity of Pistacia lentiscus foliage in two Middle Eastern breeds of goats differing in their propensity to consume tannin-rich browse. Vet. Parasitol. 173, 280–286. Madibela, O.R., Kelemogile, K.M., 2008. Exposure of Melia azadirachta fruits to Eimeria lowers oocyst output in yearling Tswana goats. Small Rumin. Res. 76, 207–210. Molan, A.L., Liu, Z., De, S., 2009. Effect of pine bark (Pinus radiata) extracts on sporulation of coccidian oocysts. Folia Parasitol. 56, 1–5. Romani, A., Pinelli, P., Galardi, C., Mulinacci, N., Tattini, A., 2002. Identification and quantification of galloyl derivatives, flavonoid glycosides and anthocyanins in leaves of Pistacia lentiscus. Phytochem. Anal. 13, 79–86. Roy, A., Saray, S., 2006. Limonoids: overview of significant bio-active triterpenes distributed in plant kingdom. Biol. Pharm. Bull. 29, 191–201. Said, O., Khalil, K., Fulder, S., Azaizeh, H., 2002. Ethnopharmacological survey of medicinal herbs in Israel, the Golan Heights and the West Bank region. J. Ethnopharmacol. 83, 251–265. Sayin, F., Kahyaoglu, T., Cakmak, A., 1986. Incidence of Eimeiria species found in sheep and goats from Aegean Sea Coast of Turkey. Ankara Univ. Vet. Fak. Derg. 33, 90–96 (in Turkish). SAS, 2002. SAS/STAT guide. Version 9, 1. SAS Institute, Cary, NC, p. 3. Silanikove, N., Gilboa, N., Nir, I., Perevolotsky, A., Nitsan, Z., 1996a. Goats fed tannin-containing leaves do not exhibit toxic syndrome. Small Rumin. Res. 21, 195–201. Silanikove, N., Gilboa, N., Nir, I., Perevolotsky, A., Nitsan, Z., 1996b. Effect of a daily supplementation of polyethylene glycol on intake and digestion of tannin-containing leaves (Quercus calliprinos, Pistacia lentiscus and Ceratonia siliqua) by goats. J. Agric. Food Chem. 44, 199–205. Tilley, J.M.A., Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Brit. Grassl. Soc. 18, 104–111. Yanez Ruiz, D.R., Moumen, A., Martin Garcia, A.I., Molina Alcaide, E., 2004. Ruminal fermentation and degradation patterns, protozoa population, and urinary purine derivatives excretion in goats and wethers fed diets based on two-stage olive cake: effect of PEG supply. J. Anim. Sci. 82, 2023–2032.