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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 supply¹

Unidad de Nutrición Animal, Estación Experimental del Zaidín (CSIC), Camino del Jueves, sn, 18100 Armilla, Granada, Spain

	Abstract

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Three experiments were conducted in Granadina goats and Segureña wethers fed at maintenance level to evaluate the effect of including a mixture of barley and a new by-product derived from olive oil extraction (two-stage dried olive cake) on 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 the animals to evaluate the effects of tannins contained in the by-product. The experimental diets were as follows: alfalfa hay and alfalfa hay plus a concentrate, formulated with two-stage dried olive cake, barley, and a mineral-vitamin mixture either with or without the addition of polyethylene glycol to the drinking water. The inclusion of two-stage dried olive cake in the diet resulted in an increase of condensed tannins. Ruminal VFA concentration in goats and wethers increased (P < 0.05) and ammonia N (NH₃-N) concentration decreased (P < 0.05). The inclusion of two-stage dried olive cake decreased (P < 0.001) urinary allantoin excretion only in wethers. Ruminal degradation profiles and fractional passage rates were similar in goats and wethers. The polyethylene glycol supply increased (P < 0.001) DM and N degradation rates in both animal species but did not modify the fractional passage rate. Ruminal fermentation patterns were also similar in goats and wethers and were affected by polyethylene glycol supply. In general, Entodiniomorphida and Holotricha protozoa counts were higher (P < 0.05) in the rumen of goats than of wethers. Protozoa count in wethers responded more to polyethylene glycol supply than in goats. The present work presents the first data obtained from a comparative study with sheep and goats concerning urinary excretion of purine derivatives. The excretion was similar in both animal species when fed alfalfa hay; however, polyethylene glycol affected only urinary allantoin excretion in wethers. Results suggest a greater sensitivity of wethers than of goats to two-stage olive cake condensed tannins.

Key Words: Goats • Polyethylene Glycol • Protozoa • Purine Derivatives • Sheep • Two-Stage Olive Cake

	Introduction

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The olive oil industry has important economical and social roles in the Mediterranean region. Since 1995, a new system is being applied in olive oil extraction that generates large amounts (3,417.2 t/yr in 1996 to 2000) of a new by-product, called two-stage olive cake, which includes the remainders of pulp, stones, skin, and vegetable waters (Hermoso et al., 1995). This by-product is often extracted and dried, and the stones are partially removed (two-stage dried olive cake, TSDOC). The three-stage olive cake (by-product obtained from the traditional "three-stage" extraction system, previously used) has been used for nutrition studies in nonproducing (Hadjipanayiotou, 1998) and pregnant ewes (Aguilera et al., 1992) and cows (O’Donovan, 1983). It has also been used as part of supplements for grazing animals (Ben Salem and Nefzaoui, 2003). However, little information exists about nutrient utilization of the TSDOC in ruminants. Martín García et al. (2003) showed that nutrient utilization from TSDOC could be limited by condensed tannins (CT), which decrease protein availability and microbial protein production (Mangan, 1988). The effects can vary depending on tannin type, concentration, and animal species (Makkar, 2003). Polyethylene glycol (PEG) has been successfully used for nutritional evaluation of CT-rich foods (Silanikove et al., 2001). On the other hand, a better ability for adapting to diets rich in lignocellulosic materials and tannins has been reported in goats compared with sheep (Silanikove et al., 1994, 1996), although little comparative information in both animal species exists, especially concerning ruminal activity.

Our objective was to evaluate ruminal degradation and passage kinetics, fermentation patterns, protozoa population and urinary purine derivatives (PD) excretion in goats and wethers fed diets based on TSDOC. The potential effect of CT contained in this by-product was addressed by supplying PEG to the animals.

	Materials and Methods

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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 determined. In both experiments, three ruminally cannulated goats (43 ± 2.1 kg live weight [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 the established guidelines of the Spanish Research Council (approval No. 123/03) concerning the use of animals in research, which are in compliance with European Directive 86/609. Throughout the three experiments, animals were fed at the maintenance level (Aguilera et al., 1986; Prieto et al., 1990) the following three experimental diets (Table 1): first, alfalfa hay plus a commercial mineral-vitamin supplement (AH) and second, alfalfa hay and a concentrate formulated with barley, two-stage olive cake (TSDOC), and a mineral and vitamin mixture (AHCO). The TSDOC is a solid by-product obtained from the desiccation, extraction, and partial pitting of the crude "two stage" olive cake, produced after extracting the olive oil by centrifugation of the whole olives. The third experimental diet (AHCOP) was the AHCO diet plus 20 and 30 g/d, respectively, for goats and wethers, of polyethylene glycol (PEG, molecular weight 4,000; Fluka 81240), dissolved in 1 L of drinking water. To ascertain the total consumption of PEG, 1 L of the PEG solution was offered every morning. Once this volume of water was consumed, additional drinking water was provided ad libitum. Foods and polyethylene glycol solution were offered once in the morning (0900). Every experiment included a 20-d adaptation period to each diet. The mineral-vitamin mixture used with AH diet was formulated with 300 g of sodium chloride and 700 g of a commercial vitamin-mineral (Ovifort; NANTA, S.A., Madrid, Spain), which had the following composition (per kilogram): Ca, 155 mg/g; P, 97 mg/g; Mg, 97 mg/g; Se, 6 µg/g; Co, 14 µg/g; Fe, 2.7 mg/g; Zn, 3.9 mg/g; Mn, 3 mg/g; S, 28 mg/g; retinol, 120 mg/kg; cholecalciferol, 2 mg/kg; and -tocopherol, 150 mg/kg. The mineral-vitamin mixture included in diets AHCO and AHCOP was formulated (per kilogram) with NaCl, 277 g; ash from TSDOC combustion, 270 g; (PO₄)₂H₄Ca, 250 g; MgSO₄, 200 g; I, 25 mg; CoO, 8.5 mg; Se, 4 mg; IO, 2.5 mg; and 83,500 and 16,700 IU of vitamins A and D. In both cases, the mineral requirements of the animals were met (ARC, 1980).

Experiment 1

Ruminal Degradation Profiles. Samples of alfalfa hay and concentrate were mill-ground to pass a 2-mm screen, and aliquots of approximately 2 g were placed in nylon bags (7 x 10 cm and a 46-µm pore; Sefar Maissa, S.A., Barcelona, Spain). Bags were incubated during 0, 4, 8, 16, 24, 48, and 72 h in the rumen of cannulated animals fed one of the experimental diets containing the ingredient to be tested. Two bags per animal and incubation time were used. After incubation, bags were washed in a washing machine during 20 min, then stomached (residual material was subjected to vigorous mechanical pummeling between two metal plates, [IUL Instruments GmbH]) for 5 min (Michalet Doreau and Ould Bach, 1992) to remove the bacterial population from bag residues and, lastly, dried at 60°C. Aliquots of the residual DM were used for N analyses. The degradation profiles were calculated by the nonlinear model described by Ørskov and McDonald (1979). The effective degradability (ED) in the rumen was calculated, using the nonlinear regression (NLIN) procedure of SAS (SAS Inst. Inc., Cary, NC), as ED = a + [(b x c)/(c + k)], where a is the water-soluble fraction, b the potentially degradable (insoluble) fraction, c the rate of degradation of b, and k the passage rate of the digesta 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. Twenty-five grams of chromium-mordanted fiber (Udén et al., 1980) of the corresponding experimental diet was directly placed in the rumen of every animal (three goats and three wethers) 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 dosing the mordanted material and stored at –20°C until analyzed 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 samples 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, respectively, at zero time and after a given time, and t is the time after marker administration.

Experiment 2

Ruminal content samples were drawn from three goats and three wethers fitted with ruminal cannulas at 0, 2, 4, and 6 h after feeding during two nonconsecutive days. Approximately 100 mL of ruminal content was strained through two layers of cheesecloth and pH measured immediately. A subsample of 5 mL was combined with 1 mL of HCl 0.2 N for NH₃-N analysis. Additionally, 3 mL of rumen liquor was combined with 0.5 (vol/vol) mL HgCl 1% and 0.5 mL 25% metaphosphoric acid (wt/vol) for VFA analyses. Both subsamples were frozen at –20°C until laboratory analyses.

Ruminal samples were prepared for protozoa counting by following the procedure described by Dehority (1984). Strained ruminal liquor was preserved by adding an equal volume (5 mL) of 18.5% (vol/vol) formaldehyde. Then, two drops of Brilliant green dye were added to 1 mL of the sample, mixed, and allowed to stand overnight. After staining, 9 mL of 30% glycerol solution was added, giving a 1:20 dilution of the original ruminal liquid. Ciliate protozoa were counted using a Neubauer counting cell. Twenty aliquots per sample were counted. In every sample, the numbers of Entodiniomorphida and Holotricha were separately recorded.

Experiment 3

After 20 d of adaptation to the experimental diets, six goats and six wethers maintained in individual metabolism crates, were used for determining the urinary purine derivatives and creatinine excretion. A bucket (5 L), containing 100 mL (for wethers) and 50 mL (for goats) of 10% (vol/vol) sulfuric acid, to keep the final pH below 3, was placed under the crate for urine collection. Urine was collected daily for 5 d and weighed, and a subsample of 100 mL was stored at –20°C for purine derivatives and creatinine concentrations analyses.

Laboratory Analyses

Samples of offered feed were individually taken for 7 d and mixed before analysis to determine chemical composition. Samples were mill-ground (1-mm screen) and analyzed for DM, OM, crude fat and N, according to the AOAC (1984) methods; GE was determined in an adiabatic calorimeter; NDF, ADF, and ADL analyses were performed by the sequential procedure of Van Soest and Masson (1991), using the Ankom 200/220 fiber analyzer (Ankom, 2000). The NDF was assayed with sodium sulfite and without -amylase, and both NDF and ADF were expressed without residual ash.

Free (FCT), protein-bound (P-CT), and fiber-bound (F-CT) 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 in aliquots of strained ruminal liquor by gas chromatography (Isac et al., 1994). The NH₃-N concentration was determined following the Weatherburn (1967) technique.

Urinary PD (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 (model 710 B; Waters, Milford, MA), an injector (model 710 B; WISP), a multiwavelength detector (model 481; Lambda-Max, Waters; set to 205 nm) and a double 4.0-mm x 250-mm S5 ODS 2 analytical column (Waters Sphericorb). Purine derivatives and creatinine were quantified by peak integration using the Waters HPLC systems software Millenium 32.

Statistical Analysis

Data obtained in each of the three experiments were analyzed by the GLM procedure of SAS. In each experiment, three periods were considered, and in each period one animal of each species in Exp. 1 and 2 and two of each species in Exp. 3 were randomly fed one of the three experimental diets in a crossover design. If a value of P < 0.05 was detected, differences among means and variable interactions were tested with a Bonferroni t-test. In Exp. 1, main effects and interaction were separately analyzed for each ingredient (AH and CO). Protozoa population counts were transformed (log₁₀) before statistical analysis.

	Results and Discussion

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The inclusion of three-stage olive cake in the diet of grazing animals allowed farmers to decrease the daily cost of gain of lambs by 38 and 18%, respectively (El Hag et al., 2002; Ben Salem and Nefzaoui, 2003). However, little information exists about nutrient availability and utilization of the TSDOC, a new by-product, which is more abundant in Spain and Italy, where the new extraction technology is extensively applied. Because it is abundant in semiarid areas, which have a scarcity of natural pastures and conventional animal foods, its use in ruminant feeding could contribute to the development of Mediterranean livestock production systems, thus replacing, to some extent, more expensive feeds (mainly, cereal grains).

However, the use of TSDOC in ruminant feeding could have detrimental effects on ruminal activity owing to the presence of CT (Martín García et al., 2003). There is a need to define the optimum concentration and type of CT in various sources in order to decrease dietary protein degradation in the rumen and increase postruminal absorption of nonammonia N and, at the same time, obtain high VFA production. It also seems that CT may be digested differently by different ruminants. Although some materials have low CT concentrations (<5 g/100 g of DM), tannins could still have an important effect. In those cases, PEG can help to reveal this effect (Silanikove et al., 2001).

Feed Composition

The chemical composition of diets formulated with AH and TSDOC was not very different from that of AH, although a decrease in N and an increase in total condensed tannins were observed (Table 1). The information about TSDOC chemical composition is scarce, but in general it is similar to that of olive cake obtained from the previously used three-stage technology (Molina Alcaide et al., 2003). However, two-stage olive cake includes vegetable waters, obtained separately in the three-stage technology, which are rich in polyphenols (Hermoso et al., 1995; Ramos Cormenzana et al., 1996). As a consequence, a higher content of tannins could be expected in TSDOC in comparison with three-stage olive cake. In fact, we found 4.73 g/100 g of DM condensed tannins in TSDOC and Nefzaoui (1985) found 1.36 g/100 of DM in the three-stage olive cake.

Ruminal Degradation Profiles

In general, degradation profiles of both alfalfa hay and concentrate were similar in goats and wethers (Table 2). Higher values, although not significant, of ED and degradation rates of CP were observed in goats fed the AHCOP diet compared with wethers. These results corroborate those found in previous studies (Isac et al., 1994; Molina Alcaide et al., 2000) concerning the lack of significant differences between goats and sheep in DM and CP degradation rates and effective degradability of medium- to good-quality feedstuffs. The AH had lesser (P < 0.01) a-values and greater (P < 0.01) b-values of both DM or CP when samples were incubated in the rumen of both animal species fed the AH diet compared with the AHCO diet; however, DM and CP effective degradability of AH were not affected.

Some authors stated that low concentrations of CT (1 to 5 g/100 g of DM) have beneficial nutritional effects attributed to the formation of stable CT-protein complexes in the rumen, which decreases microbial degradation of dietary proteins, increasing their availability after the subsequent dissociation of the complexes in the lower tract (Jones and Mangan, 1977). Our ruminal degradability studies did not show such an effect in goats and wethers fed diets containing TSDOC.

Fractional Passage Rate

In our trials, no significant differences in the fractional rate of digesta passage in goats and wethers (Table 2) were found. Previous comparative studies of particulate matter kinetics carried out in our laboratory with unrestrained animals demonstrated similar outflow rates of stained particles in goats and sheep (Isac et al., 1994; Molina Alcaide et al., 2000). Other authors reported slower (Ndosa, 1980; Domingue et al., 1991) or faster (Huston et al., 1986; Katoh et al., 1988) digesta passage rates in goats than in sheep.

Silanikove et al. (2001) studied the effect of condensed tannins on digesta passage rate along the gastrointestinal tract in goats fed carob leaves with and without PEG addition. They observed that the overall effect resulted in a delay of particulate matter passage throughout the gastrointestinal tract, owing to the interaction of tannins with digestive enzymes and the epithelial lining the intestine. However, they did not report an effect of PEG treatment on ruminal particulate matter retention time. Content of condensed tannins in carob leaves used in that study was low (0.7 g/100 g of DM), although they showed high PEG-binding capacity, equivalent to that of species with higher tannin content (3.5 to 25.4 g/100 g of DM; Silanikove et al., 1996). Reports concerning CT effects on these aspects are few and inconsistent (Waghorn et al., 1994, Barry and McNabb, 1999), reflecting an important variability, which seems to depend not only on the CT amount supplied to the animals, but also on the type of tannins, with different microbial and endogenous digestive enzyme binding capacities (Makkar, 2003).

Ruminal Fermentation Pattern

Table 3 shows the average values of pH and concentrations of NH₃-N and VFA at different sampling times in the ruminal liquor of goats and wethers fed the experimental diets. Values of pH were in the range to allow an optimal cellulolytic activity, except for diets AHCO and AHCOP in wethers, with values below 6.2. Goats showed a better buffering activity than sheep as stated by Silanikove (2000). The PEG supply seemed to stimulate carbohydrate fermentation in wethers because it promoted a pH decrease (P < 0.05) between the AHCO and AHCOP diets.

Many authors suggested that concentrations of CT below 50 g/kg of DM in the diet do not have important effects on ruminal fermentation (Barry and McNabb, 1999; Salawu et al., 1999). Our results concerning PEG effect on pH and NH₃-N and VFA concentrations were similar in the ruminal liquor obtained from goats or wethers and, in the case of pH, it could indicate that negative effects of tannins on degradative enzymes may have been lower with PEG. Besides concentration of total tannins, both the distribution of the different fractions (free or bound) and their nature would also influence the animal response to tannins and, consequently, to PEG supply. Bound CT presumably caused less effect on ruminal activity than free CT that may directly interact with microbial enzymes (Pérez Maldonado and Norton, 1996). Regarding CT nature, a diet containing 1.8% of condensed tannins from Lotus pedunculatus caused a significant decrease in ruminal NH₃-N and BCVFA concentrations, whereas addition of PEG to a diet containing the same level of condensed tannins (1.8%) from Lotus corniculatus had lesser effects (Waghorn and Shelton, 1997). The effect of PEG also depends on the level of dietary protein, and the higher the protein content in the diet, the lesser the effect of PEG (Makkar and Becker, 1996). In the current study, alfalfa hay provided N in excess, which could partly explain the slight effect found on ruminal fermentation, especially on NH₃-N concentrations.

Ruminal Protozoa Population

The total protozoa (Entodiniomorphida and Holotricha) present in the rumen of goats and wethers fed alfalfa hay (AH) ranged from 504 to 1,035 x 10³ cells/mL and from 274 to 946 x 10³ cells/mL (Table 4), respectively, corresponding to the highest values of ruminal liquor samples taken just before feeding. Entodiniomorphida protozoa accounted for around 90% of total protozoa. This finding was in agreement with values observed by other authors (Santra et al., 1998; Hindrichsen et al., 2002) using similar sampling and counting methodologies. However, the variability that the literature reflects, owing to factors such as type of diet, animal species, individuals, and sampling methodology, suggests that we must analyze data with caution for comparative purposes. The progressive decrease of ciliate protozoa in the ruminal liquor of wethers and goats from 0 to 6 h after feeding could be ascribed to sequestration of Entodiniomorphida (Kamra et al., 1991) but also to the dilution effect of saliva influx and passage rate. When animals were fed the AHCO diet, no significant effect on Entodiniomorphida protozoa was observed except for a decrease (P < 0.05) in wethers at 0 h and in goats at 2 h after feeding. Goats showed higher Entodiniomorphida counts at 2, 4, and 6 h than wethers. The same interspecies differences were observed by Santra et al. (1998) in animals fed diets with a similar roughage:concentrate ratio. These authors associated decreased protozoa in sheep with lower NDF and ADF digestibility than in goats. In our laboratory, we observed the same effect (unpublished data). The PEG treatment (AHCOP diet) significantly increased Entodiniomorphida protozoa in wethers independently of the sampling time, resulting in even higher counts than those obtained in animals fed AH diet, due to combined positive effects of starch presence as well as removal of the deleterious effect of CT. The PEG significantly increased (P < 0.05) Holotricha protozoa in goats at 0 and 6 h and in wethers at 0 and 2 h. Makkar et al. (1995) observed a significant decrease in both types of protozoa with the addition of 0.2 mg of quebracho powder per milliliter of ruminal liquor in a RUSITEC device. Although tannin effects on ruminal protozoa counts are variable in assays carried out in vivo (Makkar, 2003), some evidence exists for lower protozoal number in the presence of tannins. Wang et al. (1994) also found lower protozoa numbers in the rumen of sheep fed diets containing tannins compared with those receiving an intraruminal infusion of PEG. In our work, this effect was detected only in the rumen of wethers, which could partly explain the major sensibility of this animal species to diets containing tannins compared with goats (Silanikove, 2000). The significant effect of PEG supply on NH₃-N, BCVFA, and total VFA concentrations could partly be explained by the increased concentrations of protozoa in the rumen, given that Entodiniomorphida and Holotricha ruminal protozoa, respectively, take part in the degradation of lowly soluble particulate dietary proteins and in taking up soluble compounds from the medium, respectively (Jouany, 1996).

Mean values for daily excretion of PD and creatinine in the urine of goats and wethers are shown in Table 5. Allantoin was the main PD detected in the urine of goats and wethers, and its excretion ranged from 435 to 487 and from 301 to 460 µmol/kg BW^0.75, respectively. In the case of wethers, an increase (P < 0.001) of allantoin excretion was promoted by PEG supply. The same effect was observed by Ben Salem et al. (2000) in sheep fed Acacia cyanophylla (20 to 50 g/kg of DM CT) supplemented with PEG. No data have been published concerning comparative urinary excretion of PD in sheep and goats. No changes, owing to the consumed diet, in urinary excretion of allantoin precursors (uric acid, hypoxanthine, and xanthine) were observed either in wethers or in goats.

Urinary PD excretion is used to predict or estimate ruminal microbial protein synthesis in ruminants. The principle is that duodenal purine bases, as a microbial marker, are efficiently absorbed at the small intestine and the majority of their metabolites excreted via the kidney, with a specific urinary recovery in each animal species (Balcells et al., 1991; Belenguer et al., 2002).

The urinary PD excretion values obtained with goats and wethers are similar to those observed in sheep fed similar quality diets (Chen et al., 1992; Hindrichsen et al., 2002). The addition of PEG only had a significant effect (P < 0.05) in wethers. It may be speculated that this fact is due to a better adaptation capacity of goats vs. sheep to tannin-rich diets as reported by different authors (Narjisse et al., 1995; Silanikove, 2000). Ben Salem et al. (1999, 2000) observed increased urinary PD excretions in sheep fed Acacia cyanophila (3.2 CT g/kg of DM) when adding PEG (19 to 27 mg PEG/kg BW^0.75) in feed blocks. However, Pérez Maldonado and Norton (1996) did not find a significant difference in microbial protein outflow between Border Leicester x Merino sheep and cashmere goats fed Desmodium intortum (3.35 CT g/kg of DM) using ³⁵S as a microbial marker. Various factors—such as techniques used for condensed tannins analysis, structure of tannins, and the ability of ruminal microbes to perform efficiently in the presence of tannins—could explain those different effects. The present work provides the first data obtained from a comparative study of goats vs. sheep concerning the urinary excretion of PD and suggests a higher sensitivity of sheep to diets containing tannins from TSDOC compared with goats.

	Implications

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Ruminal fermentation characteristics and urinary purine derivatives excretion were similar in goats and wethers fed alfalfa hay; however, when goats and wethers received diets containing two-stage olive cake they behaved differently. As reflected from polyethylene glycol supply, condensed tannins had some effects on ruminal degradation, fermentation activities, and the protozoa population in both animal species. Wethers were more affected than goats by two-stage olive cake consumption, especially in terms of protozoa counts and urinary allantoin excretion. These effects were reversible by the addition of polyethylene glycol. Our results pointed out the suitability of a potential use of two-stage olive cake in the practical feeding of small ruminants. However, ruminal activity in goats seems to be more adapted than in wethers to diets containing two-stage olive cake, which indicates a greater suitability for feeding this by-product to goats than to wethers, but this must be confirmed in practical assays.

	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—phone: +34 958 572757; fax +34 958 572753; e-mail: molina{at}eez.csic.es.

Received for publication June 23, 2003. Accepted for publication March 16, 2004.

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Top Abstract Introduction Materials and Methods Results and Discussion Implications Literature Cited

 


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