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Ruminal fermentation and degradation patterns, protozoa population and urinary purine derivatives excretion in goats and wethers fed diets based on olive leaves¹

Unidad de Nutrición Animal, Estación Experimental del Zaidín (CSIC), Armilla (Granada), Spain

	Abstract

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Olives leaves, accrued during the processing of olive harvests for oil extraction, are poor in N, rich in crude fat and ADF (1.19, 8.03 and 28.2 g/100 g of DM, respectively), and relatively low in condensed tannins (11.1 mg/g of DM). Three experiments were conducted in a 2 x 3 (two animal species: goats vs. wethers; and three experimental diets: olive leaves without or with polyethylene glycol supply and olive leaves supplemented with barley and faba beans) factorial design to evaluate ruminal degradation and passage kinetics (Exp. 1), fermentation pattern and protozoa population (Exp. 2), and urinary purine derivatives excretion (Exp. 3). Polyethylene glycol was supplied to evaluate the effects of condensed tannins contained in olive leaves. Ruminal degradability of CP was low in both goats and wethers, although goats showed higher (P < 0.05) values than wethers. Supplementation of olive leaves with barley and faba beans increased (P < 0.001) ruminal degradability of DM and CP. Both goats and wethers fed olive leaves showed similarly low particulate fractional passage rates (0.021 and 0.023/h, respectively). Ingestion of olive leaves promoted lowNH₃-N and VFA concentrations, which reflect poor microbial activity. These concentrations, especially that of VFA, increased when barley and faba beans were added. Ingestion of olive leaves affected ruminal protozoa: Entodiniomorphida showed low concentrations and Holotricha completely disappeared. When animals received a diet based on olive leaves, barley, and faba beans, Holotricha appeared in the ruminal liquor and Entodiniomorphida increased (P < 0.001). In goats and wethers fed olive leaves alone, urinary allantoin excretion was very low (163 and 164 µmol/kg BW^0.75 in goats and wethers, respectively), and moderate values (352 and 389 µmol/kg BW^0.75 in goats and wethers, respectively) were observed when a diet of olive leaves, barley, and faba beans was fed. The polyethylene glycol supply did not have an effect in goats or in wethers, indicating the lack of an effect of condensed tannins in olive leaves. Ingestion of olive leaves promotes a low microbial activity, although its supplementation with readily degraded carbohydrates and protein improves microbial activity and, as a consequence, increases its ruminal degradation. In general, for most of the measured variables, there were no animal species x diet interactions. Thus, goats and wethers had similar ruminal activities when fed diets based on olive leaves.

Key Words: Goats • Olive Leaves • PEG • Protozoa • Purine Derivatives • Sheep

	Introduction

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Olive culture is economically and socially important in the Mediterranean area, Spain being the number one producer (4,945,000 t/yr; MAPYA, 2003). Cleaning olives during oil extraction generates important amounts (200,000 t/yr) of small branches and olive leaves (OL). This by-product is traditionally used in Mediterranean countries as an alternative source of nutrients for small ruminants during periods of scarce feed supplies. Different studies carried out with OL reflected a low nutritive value (Alibés et al., 1982; Delgado Pertíñez, 1994; Martín García et al., 2003), especially for protein, and these studies suggested higher suitability of supplementary proteins than nonprotein nitrogen for improving use of OL nutrients (Martín García, 2001). Nevertheless, no information is available concerning in vivo ruminal environment promoted by OL fermentation, and this aspect would be of importance to learn the true potential of OL as an alternative forage. It has been accepted that goats utilize OL nutrients more efficiently than sheep, but there are no comparative works that support this idea. Previous studies conducted in vitro (Martín García et al., 2003; Molina Alcaide et al., 2003) showed that use of OL nutrients could be limited by phenolic compounds, such as condensed tannins (CT), which can form complexes with dietary proteins, carbohydrates, and microbial enzymes, thereby decreasing protein availability (Mangan, 1988). Those effects can vary depending on animal species (Makkar, 2003) and need further study.

The objective of the present work was the assessment of ruminal fermentation and degradation characteristics, protozoa counts, and urinary purine derivative (PD) excretion in goats and wethers fed OL supplemented or not with barley grain and faba beans. The effect of supplying polyethylene glycol (PEG), a compound that forms complexes with tannins (Silanikove et al., 2001), on those variables was studied to evaluate the potential effect of tannins.

	Material and Methods

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Feeds

The OL and small branches, previously separated from the olives by forced ventilation, were collected daily in the oil mill industry. After collection, OL were dried at room temperature for 1 wk, and then chopped (10 cm length) before being delivered to the animals.

Animals and Diets

Three consecutive experiments were conducted in adult, dry, nonpregnant Granadina goats and Segureña wethers. In Exp. 1, ruminal degradation profiles and passage rates of particles were determined. In Exp. 2, fermentation patterns and protozoa counting in the ruminal liquor were also determined. In both experiments, three ruminally cannulated goats (43 ± 2.1 kg BW) and three ruminally cannulated wethers (69 ± 4.3 kg BW) were used. In Exp. 3, the urinary purine derivatives and creatinine excretion were determined in six noncannulated goats (46 ± 3.1 kg BW) and six noncannulated wethers (72 ± 5.1 kg BW). The experiments were carried out by following Spanish Research Council guidelines (Approval No. 123/03) concerning the use of animals in research, which is in compliance with the European Directive 86/609. In the three consecutive experiments, animals were fed three experimental diets (Table 1): 1) OL ad libitum (OL diet), 2) OL ad libitum plus 20 or 30 g/d of PEG (molecular weight 4,000; Fluka 81240, Sigma-Aldrich, Madrid, Spain) dissolved in 1 L of drinking water for goats and wethers, respectively (diet OLP), and 3) OL ad libitum supplemented with barley and faba beans (diet OLSUP). Level of supplementation with either barley or faba beans is detailed in Table 1. To achieve the total consumption of PEG, 1 L of PEG solution was offered every morning. Once this volume of water was consumed, additional drinking water was provided ad libitum. Diets were offered once daily at 0900. Each diet included a 20-d adaptation period. A mineral-vitamin mixture was also supplied to the animals (20 and 30 g/d for goats and wethers, respectively), which was formulated (as-fed basis) with 277 g of NaCl, 270 g of ash from "two-stage dried olive cake" combustion, 250 g of (PO₄)₂H₄Ca, 200 g of MgSO₄, 8.5 mg of CoO, 4 mg of Se, 2.5 mg of I, and 83,500 and 16,700 IU of vitamins A and D, respectively, per kilogram.

Experiment 1

Ruminal Degradation Profiles. Samples of dried OL were mill ground to pass a 2-mm screen, and aliquots of approximately 2.5 g were weighed in nylon bags (7 x 10 cm and 46-µm pore), which were immediately placed in the rumen of cannulated animals fed each experimental diet (Table 1) and incubated during 0, 4, 8, 16, 24, 48 and 72 h. Two bags per animal and incubation time were used. After incubation, bags were washed in a washing machine for 20 min, stomached (vigorous mechanical pummeling between two metal plates; IUL S. A. Instruments, Barcelona, Spain) for 5 min (Michalet Doreau and Ould Bach, 1992) to detach ruminal microbial cells, and then dried at 60°C for 48 h. Residual DM was analyzed for N content. Degradation profiles were calculated by the nonlinear model described by Ørskov and McDonald (1979). Effective degradability (ED) in the rumen was calculated as ED = a + [(b x c)/(c + k)], where a is the water soluble fraction, b the potentially degradable (insoluble) fraction, c is the degradation rate of b, and k is the fractional passage rate out of the rumen, which was assumed to be 0.031 and 0.025/h in goats and wethers, respectively (García et al., 1995).

Fractional Passage Rate. Chromium-mordanted OL fiber (Udén et al., 1980) was placed directly into the rumen of each animal (25 g of DM) immediately before feeding. Feces were individually collected by rectal grab sampling at 6, 9, 12, 24, 28, 32, 48, 53, 72, and 79 h after the dosage of mordanted material and stored at –20°C until analysis for chromium sesquioxide (Cr₂O₃; Aguilera et al., 1988). The Cr₂O₃ concentration in feces samples was determined by spectrophotometry (372 nm) in distilled water solutions after ashing and fusing the samples with an alkaline fusion mixture (10 parts of Na₂CO₃, 10 parts of K₂CO₃, and four parts of KNO₃) and by using a standard curve. The digesta fractional passage rate (k) was calculated as the regression slope of chromium concentration logarithm in feces with time after dosing (Grovum and Williams, 1977), according to the equation log_e Y = log_e A – kt, where log_e Y and log_e A are the natural logarithms of chromium concentration in feces at zero time and after a given time, respectively, and t is the time after marker administration.

Experiment 2

Ruminal contents were collected from ruminally cannulated goats and wethers with a manual vacuum, placing the hose in different parts of the rumen at 0, 2, 4, and 6 h after feeding. Collection was carried out over 2 d. Approximately 100 mL of ruminal contents was collected. The contents were strained through two layers of cheesecloth, and pH was immediately measured using a pH meter (model 691, Metrohm, Herisau, Switzerland). A 5-mL aliquot was combined with 1 mL of 0.2 N HCl for ammonia N (NH₃-N) analysis. Additionally, 3 mL were combined with 0.5 mL of 1% HgCl (vol/vol) and 0.5 mL of 25% metaphosphoric acid (wt/vol) for total and individual VFA analyses. Both subsamples were frozen at –20°C before analyses.

Ruminal samples were prepared for protozoa counting by following the procedure described by Dehority (1984). Ciliate protozoa were counted using a Neubauer counting cell. Twenty aliquots per sample were counted. In every sample, Entodiniomorphida and Holotricha protozoa were separately recorded.

Experiment 3

Six animals of each species maintained in individual metabolism crates (124 x 50 x 95 cm) were used. A bucket (5 L) containing 100 mL (for wethers) and 50 mL (for goats) of 10% H₂SO₄ (vol/vol) to keep the final pH below 3 was placed under the crate for urine collection. Urine was collected daily for 5 d before feeding, weighed, and a subsample of 100 mL was stored at –20°C for purine derivative and creatinine concentration analyses.

Laboratory Analyses

Samples of feeds and daily refusals were individually taken during 7 d, thawed, and mixed before analysis. They were then mill ground (1-mm screen) and analyzed for DM, OM, crude fat, and total N, according to the AOAC (1984) methods. The GE was determined in an adiabatic calorimeter, and NDF, ADF, and ADL were analyzed by the sequential procedure of van Soest and Masson (1991), using the Ankom^200/220 fiber analyzer (Ankom, 2000). Neutral detergent fiber was assayed with sodium sulfite and without -amylase. Both NDF and ADF were expressed without residual ash.

Free, protein-bound, and fiber-bound condensed tannins were determined in feed samples using the procedure proposed by Pérez Maldonado and Norton (1996). Condensed tannins from quebracho powder (Roy Wilson Dickson Ltd., Mold, U.K.) were used as standard.

Individual and total VFA were determined by gas chromatography (Isac et al., 1994). Ammonia-N concentration was determined following the Weatherburn (1967) technique.

Urinary purine derivatives (xanthine, hypoxanthine, uric acid, and allantoin) and creatinine were determined following the procedure described by Balcells et al. (1992) using HPLC analysis, which consisted of a multisolvent delivery system (Waters model 710 B, Milford, MA), an injector (WISP model 710 B), a multiwave-length detector (model 481 Lambda-Max, Waters, set to 205 nm) and, double 4.0 mm x 250 mm S5 ODS 2 analytical columns (Waters Sphericorb). Purine derivatives and creatinine were quantified by peak integration using Waters HPLC systems software Millenium³².

Statistical Analyses

Data obtained in each of the three experiments were analyzed by the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). The model included diet, animal species, and their interaction as fixed effects. In each of the three experiments, three periods were considered and animals were randomly fed one of the three experimental diets in a crossover design. Because there was no interaction between main effects and sampling time, ruminal fermentation data are presented as averaged across 0 to 6 h after feeding. Protozoa population counts were transformed (log₁₀) and statistically analyzed for each sampling time separately, as there was a significant interaction with sampling time. If a value of P < 0.05 was detected, differences among means and variable interactions were tested with the Bonferroni t-test.

	Results and Discussion

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Feed Composition

The ingredient and chemical compositions of the experimental diets are shown in Table 1. Previous studies have reported high variability in OL chemical composition, especially for DM, OM, and crude fat, depending on factors such as origin (Molina Alcaide et al., 2003), storage, and drying systems (Gómez Cabrera et al., 1992; Delgado Pertíñez et al., 2000). Despite this variability, OL are generally rich in cell walls, poor in N, and relatively rich in crude fat. Our results are in the range of previously reported studies. Olive leaves supplemented with barley and faba beans resulted in an increase in N, a slightly decreased NDF content, and a strong decrease in crude fat.

A low concentration of condensed tannins was detected in OL. The methodology used in the present work, which determines three fractions of condensed tannins (free and bound to protein and fiber; Pérez Maldonado and Norton, 1996), has not been applied previously to OL. Even so, total condensed tannins concentration (11.1 mg/g of DM) was similar to values observed by others using different methodologies (Fegeros et al., 1995: 10.7 mg/g of DM; Martín García et al., 2003: 8.30 mg/g DM).

Ruminal Degradation Profiles

In situ ruminal DM and CP degradability of OL was low in both goats and wethers, and increased (P < 0.001) when OL were supplemented with barley and beans (Table 2). Low ruminal degradability of CP and DM could be due to high OL fat content and to incomplete removal of ruminal microbes from the undegraded residues in the nylon bags by washing and stomaching procedures (Martín-Oruúe et al., 1998; Hvelplund and Weisbjerg, 2000). That underestimation is especially important for protein due to the high N content of microbes (Varvikko and Lindberg, 1985). Samples incubated in the rumen of goats showed increased potentially degradable (insoluble) fractions and effective degradability values for DM and CP than in wethers. These results corroborate those found in previous studies (García et al., 1995; Martín García et al., 2003), indicating goats have a better capacity for degrading poor-quality feedstuffs. However degradation rate values of CP were higher in wethers. This could be caused by a mathematical artifact due to the low potentially degradable (insoluble) fraction values found in this animal species. Polyethylene glycol (OLP diet) supply did not affect OL ruminal degradation compared with OL incubated in animals fed OL diet, either in goats or wethers. The lack of PEG effect may be due to the low tannin content of OL.

Table 2 also shows the fractional passage rate and mean retention time of particles in the gastrointestinal tract of goats and wethers fed the experimental diets. There were no differences (P = 0.471) between goats and wethers, which is in contrast with previous results obtained with animals grazing pastures from semiarid lands (García et al., 1995).

Although only limited information from comparative studies is available concerning particulate passage rate in goats and sheep, results are contradictory due to methodological differences (type and dose of the marker), nature of the diet, and feeding conditions (Watson and Norton, 1982; Katoh et al., 1988; Domingue et al., 1991). In agreement with our results, Aitchison et al. (1986a, b) observed that the addition of a moderate amount of concentrate to diets based on perennial ryegrass (Lolium perenne) hay either slightly increased digesta fractional passage rate or did not affect it at all.

Ruminal Fermentation Pattern

Table 3 shows averaged values (0 to 6 h after feeding) of pH and concentrations of NH₃-N and VFA in the ruminal liquor of goats and wethers fed the experimental diets. Averaged across both species, pH values were decreased (P < 0.001) when OL were supplemented with barley and beans (7.18 and 6.78, average values for OL and OLSUP diets, respectively), whereas the addition of PEG had no effect on this variable. Ruminal liquor obtained from goats showed lower pH values (P < 0.01) compared with ruminal liquor taken from wethers.

Olive leaves supplemented with barley and faba beans (diet OLSUP) increased (P < 0.05) NH₃-N ruminal concentration in goats, promoting values close to those considered to be optimal for microbial activity in animals fed lignocellulosic materials, whereas no response was observed in wethers. This different response to supplementation could be a function of a more efficient N recycling in goats than in wethers (Domingue et al., 1991; Tisserand et al., 1991).

Total VFA concentration in the ruminal liquor of both animal species fed OL was low (27.9 mM). This value is in the range, or even lower than those found in sheep and goats fed cereal straw (Antoniou and Hadjipanayiotou, 1985; Fondevilla et al., 1994) or low-quality grass (Molina Alcaide et al., 1997). Low total VFA, isobutyric, and isovaleric concentrations indicate low protein degradability, which affects fiber degradation, as those values were lower than the proposed as optimal for fibrolytic microorganism activity (Hume, 1970). A stable and low fermentative activity from 0 to 6 h after feeding was also observed, which could, in part, explain the observed long OL particulate retention time (47.7 and 43.5 h in goats and wethers, respectively) compared with those determined in the same animal species fed alfalfa hay (29.4 and 31.3 h, respectively; Molina Alcaide et al., 2000).

The PEG supply only increased (P <0.01) isobutyrate and butyrate concentrations in both animal species, indicating no effect of the CT contained in OL on carbohydrate fermentation.

Olive leaves supplemented with barley grain and faba beans increased (P < 0.001) total VFA concentration compared with OL fed alone in both animal species, and reached values characteristic of medium-quality diet fermentation (Ørskov and Ryle, 1998). An increase in acetate:propionate ratio was observed in goats as well (3.74 to 4.82), supporting the idea that an adequate supplementation may lead to a higher degradation of OL structural carbohydrates (Wiseman and Cole, 1988; Reddy et al., 1989).

Ruminal Protozoa Population

Holotricha protozoa completely disappeared in the ruminal liquor of both animal species fed OL. These microorganisms have considerable capacity for taking up soluble compounds (Jouany, 1996), decreasing when fed diets rich in cellulolytic materials (Jouany, 1989), which is the case with OL. It has been previously reported (Williams, 1989) that diets high in lipids are toxic for ruminal protozoa, as they have limited ability to metabolize them. Decreased protozoa number due to high dietary fat has been reported in several studies (Clemens et al., 1974; Sutton et al., 1983, Tesfa, 1993). Ivan et al. (2001) observed that Holotricha were the most susceptible to toxic effects of oils, especially with oils rich in unsaturated fatty acids, which agrees with our results. Fat present in OL comes from the break up of olives during their separation from leaves and, as a consequence, this olive oil is rich in unsaturated fatty acids (Uceda and Hermoso, 1997). When animals received supplemented OL, Holotricha (data not shown) appeared in the collected ruminal liquor (20.3, 66.0, 81.9, 110.6, and 12.9, 37.3, 59.5, 45.4 x 10³ cells/mL, in goats and wethers at 0, 2, 4, and 6 h, respectively, after feeding).

Entodiniomorphida protozoa counts in ruminal liquor of goats and wethers fed OL (Table 4) were low and constant from 0 to 6 h after feeding. When animals received the OLSUP diet, Entodiniomorphida increased (P < 0.001) up to values close to those reported in animals fed good-quality diets (Ivan et al., 2001; Hindrichsen et al., 2002). Higher values (P < 0.05) of Entodiniomorphida protozoa were found in ruminal samples collected at 0, 2, and 6 h after feeding in goats vs. wethers fed OLSUP. The same species differences were observed by Santra et al. (1998) in goats and sheep fed diets with a similar roughage:concentrate ratio (65:35) as in the OLSUP diet.

Mean urinary daily PD and creatinine excretion of goats and wethers is shown in Table 5. Allantoin was the main PD detected in the urine of goats and wethers, with values ranging from 155 to 352 and from 142 to 389 µmol/kg BW^0.75, respectively. Urinary allantoin excretion in goats and sheep fed OL (164 µmol/kg BW^0.75, respectively) was in the same range as values reported for sheep fed barley straw (88.5 to 215 µmol/kg BW^0.75; Laurent and Vignon, 1979) or barley straw treated with 50 g of NaOH/kg DM (170 µmol/kg BW^0.75; Balcells et al., 1993b). These results suggest that, in terms of promoted microbial activity, OL and cereal straw have similar qualities. The values found in animals fed OL and those estimated as endogenous excretion coming from tissue turnover in goats (202 µmol/kg BW^0.75; Belenguer et al., 2002) and sheep (190.2 µmol/kg BW^0.75; Balcells et al., 1991) were similar, indicating low microbial activities in the rumen of both animal species when fed OL. This idea is supported by the low NH₃-N concentrations observed in animals fed OL. Polyethylene glycol supply did not increase allantoin excretion, which indicates that CT in OL did not affect microbial protein synthesis in the rumen. In both animal species, OL supplemented with barley grain and faba beans increased urinary allantoin excretion to values that were in the range reported for sheep fed medium-quality diets (Chen et al., 1992; Balcells et al., 1993a).

When animals were fed the OLSUP diet, total PD (allantoin, uric acid, xanthine, and hypoxanthine) urinary excretion was 424 and 491 µmol/kg BW^0.75 for goats and wethers, respectively. These values are in the range of values reported for sheep fed medium-quality diets using the same methodology (Balcells et al., 1993a; Ben Salem et al., 1999; 2000), and confirm that OL supplemented with barley grain and faba beans promotes adequate microbial activity in the rumen.

	Implications

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The characteristics of ruminal degradation and fermentation indicate that olive leaves are poor forage, similar to cereal straws. Ingestion of olive leaves promotes low microbial protein synthesis and strongly affects the ruminal protozoa population in goats and wethers. Supplementation of olive leaves with readily degraded carbohydrates and protein leads to adequate microbial activity and, as a consequence, increases ruminal degradation of olive leaves. This result implies that olive leaves can be used as an alternative forage in the Mediterranean area when adequately supplemented. Condensed tannins present in olive leaves did not affect nutrient use in the rumen; however, antinutrients other than tannins might be involved in the low N availability. Goats and wethers had similar ruminal activities when fed diets based on olive leaves.

	Footnotes

 
¹ This research was supported by "Consejería de Agricultura y Pesca" of Junta de Andalucía (Project CAO 01-003). D. R. Yáñez Ruiz gratefully acknowledges support from Fundación Ramón Areces.

² Correspondence: Camino del Jueves, s/n 18100 (phone: +34-958-572757; fax: +34-958-572753; e-mail: molina{at}eez.csic.es).

Received for publication July 31, 2003. Accepted for publication June 28, 2004.

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