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Effect of different drying procedures on the nutritive value of olive (Olea europaea var. europaea) leaves for ruminants
Available online at www.sciencedirect.com
Animal Feed Science and Technology 142 (2008) 317–329
Effect of different drying procedures on the nutritive value of olive (Olea europaea var. europaea) leaves for ruminants A.I. Mart´ın-Garc´ıa, E. Molina-Alcaide ∗ Estaci´on Experimental del Zaid´ın (CSIC), Profesor Albareda 1, 18008 Granada, Spain Received 19 February 2007; received in revised form 16 July 2007; accepted 4 September 2007
Abstract Fresh, freeze-, air- and oven-dried at 60 ◦ C and 100 ◦ C olive leaves (OL) were studied in order to determine the effect of different drying procedures on OL chemical composition, in vitro digestibility, ruminal degradability, and intestinal digestibility. The drying procedure affected all the parameters measured except for gross energy (GE; P=0.194). Protein-bound condensed tannins (CT) decreased (P=0.001) with freeze-, air- and 60 ◦ C drying (from 1.25 up to 0.82 g/kg dry matter, DM). Total CT were only decreased (P=0.001) by drying at 60 ◦ C (from 10.0 to 6.24 g/kg DM). The in vitro crude protein (CP) digestibility increased (P<0.001) with drying except for oven-drying at 100 ◦ C up to 58%. Values for CP digestibility found in freeze- and air-dried OL were not different (P>0.05). No differences (P>0.05) were observed between CP digestibility in air- and oven-dried at 60 ◦ C OL. Effective degradability of DM and CP increased from 0.53 to 0.62 (P=0.005) and from 0.46 to 0.64 (P=0.002), respectively after treatment. The apparent
Abbreviations: a, rapidly degradable fraction; ADF, acid detergent fibre; ADIN, acid detergent insoluble nitrogen; ADL, sulphuric acid lignin; ANOVA, analysis of variance; b, potentially degradable fraction; c, rate of degradation of the potentially degradable fraction; CP, crude protein; CT, condensed tannins; DM, dry matter; ED, effective degradability; GE, gross energy; LW, live weight; NA, not applicable; NDF, neutral detergent fibre; OL, olive leaves; OM, organic matter; PD, potential degradability; SDS, sodium dodecyl sulphate; S.E.M., standard error of the mean; t, tonnes. ∗ Corresponding author. Tel.: +34 958572757; fax: +34 958572753. E-mail address: [email protected] (E. Molina-Alcaide). 0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2007.09.005
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intestinal digestibility of undegraded CP in the rumen was only affected (P=0.046) by ovendrying, which increased it from 0.33 to 0.39. As air-drying did not have detrimental effects on the OL nutritive value it could be an appropriate, simple and low-cost procedure for olive-leaves preservation. © 2007 Elsevier B.V. All rights reserved. Keywords: Olive leaves; Chemical composition; Drying; In vitro digestibility; Degradability; Intestinal digestibility
1. Introduction Olive leaves (OL) account for around 3–5% of the total biomass processed in the olive oil mill (Delgado Pert´ın˜ ez et al., 1998). Its nutrient availability is similar to that of cereal straws (Mart´ın Garc´ıa et al., 2003) and it has been commonly used for livestock in Mediterranean countries. It is of especial importance to optimise the use of by-products such as OL to be used in animal feeding under dry conditions. The seasonal and high OL production (3,600,000 t of olives in Spain during 2005; MAPA, 2006) requires adequate preservation. As the preservation procedure may lead to variations in OL nutritive value (Delgado Pert´ın˜ ez et al., 1998), it is important to investigate suitable procedures not only to preserve OL but also to improve its nutritive value. Ensiling OL appears to be impractical (Parellada et al., 1982) due to the physical structure, high dry matter (DM) content, low density and lack of fermentable sugars. Practical storage procedures of plant materials such as wilting or air-drying involve water removal and may have different effects (Mupangwa et al., 2003) depending on plant type. High temperatures may diminish volatile substances, such as short-chain fatty acids and alcohols (McDonald et al., 1991) or organic matter (Acosta and Kothmams, 1978) and induce chemical and enzymatic changes of cell-wall components, proteins, carbohydrates (L´opez et al., 1995) and secondary compounds such as tannins (Ahn et al., 1989). Of especial importance might be the formation of Maillard products during sun-drying process (Dzowela et al., 1995). These factors might affect ruminal degradation (Dakowski et al., 1996), apparent digestibility (Palmer and Schlink, 1992) or nutrient availability at the intestine (Ahn et al., 1997). Available information on drying effect on OL nutritive value is scarce and often contradictory. High temperatures have been reported to decrease OL intake and nutritive value (Maymone et al., 1950; Delgado Pert´ın˜ ez et al., 1998, 2000), although Nefzaoui (1987) observed no change in OL nutritive value with drying. On the other hand, no work is available on the effect of drying on OL amino acid composition and protein ruminal degradation or intestinal digestibility, which are the main factors determining the protein value of feedstuffs for ruminants. The aim of the present work was to evaluate the effect of different drying procedures on the chemical composition, in vitro digestibility, ruminal degradability, and intestinal digestibility of olive leaves in order to state the most adequate and practical procedure to preserve this by-product without affecting or even improving its nutritive value.
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2. Materials and methods 2.1. Olive leaves Olive leaves from olive cleaning were obtained in January 2001 in an oil mill (Romeroliva, Granada, Spain). Different samples of OL were studied: fresh, freeze-dried, air-dried for 7 days in the shade at room temperature, and dried in a forced-air oven at 60 ◦ C for 48 h and 100 ◦ C overnight. After oven-drying, the OL were equilibrated at room temperature for 48 h. Both fresh and dried OL were crushed and stored at 4 ◦ C until chemically analysed. 2.2. Animals Granadina goats (39.4 ± 2.62 kg LW), fitted with permanent ruminal cannulas, were used. The animals were housed in individual pens and had free access to water and were fed two equal meals at 08:00 and 16:00 h at maintenance level (Prieto et al., 1990), a goodquality alfalfa hay and a mineral–vitamin mixture (20 g/d which was formulated with 277 g NaCl, 270 g of ash from two-stage dried olive cake combustion, 250 g (PO4 )2 H4 Ca, 200 g MgSO4 , 8.5 mg CoO, 4 mg Se, 2.5 mg I, and 83,500 and 16,700 IU vitamins A and D, respectively, per kilogram). All management and experimental procedures were carried out in strict accordance to the Spanish guidelines (Act No. 1201/2005 of 10 October 2005) for experimental animal protection and by trained personnel. Fistulated goats remained in good health and welfare throughout the experimental and inter-experimental periods. Ruminal content for in vitro digestibility determination was manually extracted through the cannula using a rubber hose and applying a small hand vacuum. 2.3. In vitro digestibility This parameter was determined according to Tilley and Terry (1963) by using the Daisy II incubator (Ankom® Technology, New York, NY, Ankom, 2007) as described by Mart´ın Garc´ıa et al. (2004). The rumen inoculum was individually withdrawn from three ruminally fistulated goats 2 h after morning feeding, transferred into pre-warmed (39 ◦ C) thermo bottles and squeezed through four layers of cheesecloth under anaerobic conditions. A pooled sample was prepared by combining equal squeezed volumes from each animal. The inoculum was a mixture (4:1 v/v) of rumen liquor and artificial saliva (McDougall, 1948). Samples (0.5 ± 0.05 g) were weighted in filter bags (Ankom® Technology, # F57) and three samples per treatment were incubated in the incubator. Two incubation runs were carried out. Every incubation run included two blanks and three standard samples for which the apparent in vivo DM digestibility was known. The in vitro DM and CP digestibility were calculated as DM and CP disappearance, respectively, per unit of original DM and CP. Residual DM and CP were corrected for those in the blanks. 2.4. Rumen degradability It was measured in situ by using the nylon-bag technique described by Madsen and Hvelplund (1994). Approximately 3 g of samples, ground to pass through a 2 mm mesh,
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were weighed in nylon bags (10 cm × 7 cm, 46 m of pore size). Five incubation runs were carried out with two ruminally fistulated goats in each run. One bag per treatment and time of incubation was simultaneously incubated in the rumen of each goat for 0, 4, 8, 16, 24, 48 and 72 h. At the end of the incubation period, the nylon bags were rinsed under cold running tap water to remove large particles of food, and then stored at −20 ◦ C. After completion of all incubations, the bags were washed with cold water in a washing machine for 20 min, and the residue was subjected to vigorous mechanical pummelling between two metal plates (Masticator, IUL Instruments GmbH, K¨onigswinter, Germany) for 5 min (Michalet-Doreau and Ould-Bah, 1992) to detach bacteria associated with food particles and, finally, dried for 48 h at 60 ◦ C. The profiles of DM and CP degradation were calculated according to Ørskov and McDonald (1979). The effective degradability (ED) was calculated as ED = a + [(b × c)/(c + k)], where a is the soluble fraction, b the potentially degradable insoluble fraction, c the degradation rate of b and k the fractional outflow rate with a value of 0.021 per h, previously determined (Y´an˜ ez Ruiz et al., 2004) in Granadina goats fed OL at maintenance level. 2.5. Intestinal digestibility The intestinal digestibility of dietary and ruminally undegraded protein was determined by using the procedure of Calsamiglia and Stern (1995). Three Granadina goats, fitted with permanent ruminal cannula were used. Eight nylon bags (10 cm × 7 cm, 46 m of pore size) with approximately 3 g of sample per bag per treatment were successively incubated in the rumen of one of three cannulated goats in order to obtain a minimum residual N of 60 mg per treatment. After rumen preincubation, the bags were washed and dried at 60 ◦ C for 48 h. Pooled residues containing 15 mg of N were incubated for 1 h in 10 ml of a 1 N HCl-pepsin solution. After incubation, pH was neutralised with 5 ml of 1 N NaOH and, 13.5 ml of a pH 7.8 phosphate buffer containing 37.5 mg of pancreatin were added to the solution and incubated at 38 ◦ C. After a 24 h incubation period, 3 ml of 1000 g/l trichloroacetic acid (Panreac, 131067) solution were added to precipitate the undigested proteins and N content in supernatant was measured. Dietary and ruminally undegraded CP apparent digestibility were calculated as described by Mart´ın Garc´ıa et al. (2004). The true intestinal digestibility of rumen-undegraded CP was calculated by using the equation of Hvelplund and Madsen (1990) [(1 − CP effective degradability) − (1 − apparent intestinal digestibility of dietary CP)]/(1 − CP effective degradability). An estimation of ruminally degradable and intestinally digestible dietary protein was also obtained from 1 − ADIN as suggested Krishnamoorthy et al. (1982). 2.6. Chemical analyses All samples were ground through a 1 mm sieve before analysis and, DM content was determined by drying to a constant weight in a forced-air oven at 103 ± 1 ◦ C using the AOAC (1984) method number 7.008. The organic matter (OM) was calculated from the ash content, determined by ashing in a muffle furnace for 3 h at 550 ◦ C following the AOAC (1984) method number 7.010. The ether extract was determined by extraction with petroleum ether (boiling point: 40–60 ◦ C) following the AOAC (1984) method number
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7.045. Gross energy (GE) was determined in an adiabatic bomb calorimeter (Gallenkamp & Co. Ltd., London) according to the methodology described by Prieto et al. (1990). Neutral detergent fibre (NDFom), acid detergent fibre (ADFom) and sulphuric acid lignin (ADLsa) analyses were performed by the sequential procedure of Van Soest et al. (1991) using the Ankom 200 Fiber Analyzer (Ankom® Technology, New York, NY) (Ankom, 2007). The NDF was assayed with sodium sulphite and without alpha amylase. Both NDF and ADF were expressed without residual ash. Total nitrogen was determined by Kjeldahl analysis according to the method recommended by the AOAC (1984), number 2.055. Acid detergent insoluble N (ADIN) was determined by Kjeldahl analysis of ADFom residues. Free, protein- and fibre-bound condensed tannins (CT) were sequentially extracted following the procedure described by Terril et al. (1992) and modified by P´erez Maldonado and Norton (1996). The free-CT fraction was extracted using aqueous acetone (70%), diethyl ether and ethyl acetate and partitioned into the aqueous phase to exclude pigments and small phenolic compounds. Subsequent extraction with boiling sodium dodecyl sulphate (SDS), triethanolamine and 2-mercaptoethanol yielded protein-bound tannins. The residue from this SDS extraction was washed with methanol and butanol to yield fibre-bound tannins. Analyses of the free, protein-bound and fibre-bound condensed tannins were conducted according to the butanol–HCl method of Porter et al. (1986) with the modifications proposed by Terril et al. (1992). Condensed tannins from quebracho powder (Roy Wilson Dickson Ltd., Mold, U.K.) were used as standard. Total amount of CT was calculated by adding the amounts of free, protein- and fibre-bound CT in the sample. The amino acid N content was determined by high-performance liquid chromatography (HPLC) using the Waters® Pico-Tag method, which involves precolumn derivatization with phenylisothiocyanate. Protein hydrolysis was performed in 6 N HCl using sealed and evacuated tubes at 110 ◦ C for 24 h (Fern´andez F´ıgares et al., 1997). Cysteine and methionine were determined as cysteic acid and methionine sulphone, respectively, obtained by oxidation with performic acid before 6 N HCl hydrolysis. 2.7. Statistical analysis The effect of drying on OL chemical composition, in vitro digestibility, ruminal degradability profiles and apparent intestinal digestibility was tested by ANOVA using the Minitab Statistical Software Package (Minitab, New York, NY). Significant differences between means for each treatment were assessed by Tukey or Bonferroni t-tests for paired or unequal groups, respectively. 3. Results 3.1. Chemical composition The drying procedure affected all the parameters measured (Table 1) except for GE and total CT (P=0.194). The OM content decreased (P=0.001) when OL was freeze-
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Olive leaves
Fresh
Freeze-dried
Air-dried
Dried 60 ◦ C
Dried 100 ◦ C
P-value
S.E.M.
DM (g/kg fresh matter) Organic matter Ether extract Gross energy (MJ/kg DM) Neutral detergent fibre Acid detergent fibre Sulphuric acid lignin Total nitrogen Acid detergent insoluble nitrogen (g/g total nitrogen) Condensed tannins Free Fibre-bound Protein-bound Total
694d
622a
630b
641c
870c 97.6c 20.0 399bc 267b 158ab 11.4b 0.509a
863b 87.4b 19.7 358a 245a 152a 11.0ab 0.556ab
865bc 82.2ab 19.9 415c 279b 166b 11.1ab 0.486a
861b 78.1a 19.9 398bc 270b 163ab 11.3b 0.595b
696d 847a 95.2c 19.6 376ab 249a 155a 10.6a 0.676c
0.000 0.001 0.001 0.194 0.001 0.001 0.027 0.036 0.009
0.85 0.51 0.48 0.59 1.67 0.92 1.03 0.06 0.001
4.02b 4.73bc 1.25b 10.0c
2.25a 6.72c 0.95a 9.92c
2.50a 5.72bc 1.40b 9.57bc
0.004 0.001 0.001 0.001
0.07 0.14 0.02 0.12
2.95ab 4.13ab 0.82a 7.90ab
a–d: In a row means without a common letter differ (P<0.05); S.E.M.: standard error of the mean.
2.80a 2.53a 0.91a 6.24a
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Table 1 Chemical composition (g/kg DM) of fresh and dried olive leaves
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Table 2 Amino acid composition (g amino acid-N/kg Total N) of fresh and dried olive leaves Fresh
Freeze-dried
Air-dried
Dried 60 ◦ C
Dried 100 ◦ C
Aspartic acid Glutamic acid Serine Glycine Histydine Arginine Threorine Alanine Proline Tyrosine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Lysine Essential Non-essential
27.5 35.1 44.5 79.6 25.4 162 46.8 73.8 84.2 32.3 74.8 5.32 1.60 58.8 104. 51.8 19.1 547 379
32.3 47.4 40.6 65.6 27.8 125 40.5 47.4 68.0 29.0 47.1 7.40 2.91 35.3 60.9 31.3 21.0 396 333
44.3 53.4 39.8 63.6 28.1 122 39.0 49.9 74.2 28.3 51.6 4.82 2.30 40.9 71.2 36.7 44.4 438 356
31.2 46.3 36.0 49.9 20.3 165 40.0 51.1 99.3 35.1 52.7 3.62 2.01 39.9 71.7 38.2 47.4 478 351
33.2 48.1 44.0 68.9 16.3 105 41.4 52.7 94.8 30.0 56.4 5.02 1.81 44.4 74.5 40.4 18.1 401 374
Totala
926
729
794
829
774
a
Without tryptophane.
dried and dried at 60 and 100 ◦ C. A trend to decrease with some drying procedures was also found for GE. Freeze-drying decreased the NDFom content (P=0.001) and ADFom decreased (P=0.001) both after freeze-drying and 100 ◦ C oven-drying. Total N was slightly affected (P=0.036) by drying in the oven at 100 ◦ C. The lowest ADIN values corresponded to air-dried and fresh samples but rose (P=0.009) with drying at 60 and 100 ◦ C. Free CT decreased (P=0.004) after freeze-drying and 60 and 100 ◦ C oven-drying. Fibrebound CT decreased (P=0.001) only after drying at 60 ◦ C. Protein-bound CT decreased (P=0.001) after freeze-, air- and 60 ◦ C drying. Total CT were only decreased (P=0.001) by drying at 60 ◦ C. Olive leaves were rich in arginine, leucine, proline, glycine, valine and alanine and low in cysteine, methionine and lysine (Table 2). All the treatments tended to diminish the total amino acid content. 3.2. In vitro digestibility Drying OL improved (P=0.005) DM digestibility with the exception of 60 ◦ C ovendrying (Table 3). No differences were found (P>0.05) when comparing the different drying procedures. The CP digestibility increased (P<0.001) with drying except for oven-drying at 100 ◦ C. Values for CP digestibility found in freeze and air dried OL were not different (P>0.05). No differences (P>0.05) were observed between CP digestibility in air- and ovendried OL at 60 ◦ C.
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Table 3 In vitro digestibility of fresh and dried olive leaves Dry matter
Crude protein
Fresh Freeze-dried Air-dried Oven-dried at 60 ◦ C Oven-dried at 100 ◦ C
0.53a 0.60b 0.59b 0.58ab 0.59b
0.36a 0.57c 0.53bc 0.48b 0.37a
P-value S.E.M.
0.005 0.0053
0.000 0.0047
a–c: In a column the means without a common letter differ (P<0.05); S.E.M.: standard error of the mean.
3.3. Rumen degradability The DM and CP ruminal degradation profiles of fresh and dried OL are shown in Table 4. Significant differences among treatments were found for rapidly degradable fraction (a) of DM (P=0.006) and CP (P=0.003). Both values increased with all drying procedures except at 60 ◦ C. The potentially degradable fraction (b), degradation rate (c) and potential degradability (PD) were not affected (P=0.505, 0.161, and 0.473, respectively) by any treatment. However, effective degradability was affected (P=0.005 and 0.002, respectively for DM and CP) by the treatment except for oven-drying at 60 ◦ C in the case of DM. Table 4 Ruminal degradation profiles of fresh and dried olive leaves a
b
c (h−1 )
PD
EDa
Dry matter Fresh Freeze-dried Air-dried Oven-dried at 60 ◦ C Oven-dried at 100 ◦ C
0.22a 0.32c 0.29bc 0.24ab 0.29bc
0.52 0.49 0.48 0.51 0.48
0.032 0.033 0.040 0.045 0.045
0.73 0.80 0.77 0.75 0.78
0.53a 0.61b 0.60b 0.59ab 0.62b
P-value S.E.M.
0.006 0.005
0.628 0.01
0.161 0.002
0.452 0.01
0.005 0.004
Crude protein Fresh Freeze-dried Air-dried Oven-dried at 60 ◦ C Oven-dried at 100 ◦ C
0.12a 0.21b 0.20b 0.11a 0.20b
0.70 0.71 0.73 0.77 0.68
0.020 0.026 0.030 0.033 0.038
0.82 0.92 0.92 0.88 0.88
0.46a 0.60b 0.62b 0.57b 0.64b
P-value S.E.M.
0.003 0.005
0.505 0.002
0.161 0.002
0.473 0.002
0.002 0.007
a–c: In a column means without a common letter differ (P<0.05); PD: potential degradability (a + b). a ED: effective degradability, calculated as a + [(b × c)/(c + k)], were k = 0.021 h−1 (Y´ an˜ ez Ruiz et al., 2004); S.E.M.: standard error of the mean.
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Table 5 Apparent and true intestinal digestibility of dietary and ruminally undegraded protein of fresh and dried olive leaves Apparent intestinal digestibility
True intestinal digestibility of undegraded crude proteina
Estim. N dig.b
Dietary CP
Ruminally undegraded CP
Fresh Freeze-dried Air-dried Oven-dried 60 ◦ C Oven-dried 100 ◦ C
0.56a 0.71b 0.73b 0.74b 0.74b
0.33a 0.35abc 0.36abc 0.38bc 0.39c
0.19 0.28 0.29 0.40 0.28
0.49 0.44 0.51 0.41 0.32
P-value S.E.M.
0.011 0.01
0.046 0.007
NA
NA
a–c: In a column means without a common letter differ (P<0.05); S.E.M.: standard error of the mean; NA: not applicable. a True intestinal digestibility of undegraded crude protein = [(1 − CP effective degradability) − (1 − apparent intestinal digestibility of dietary CP)]/(1 − CP effective degradability) (Hvelplund and Madsen, 1990). b Ruminally degradable and intestinally digestible dietary protein (Krishnamoorthy et al., 1982). Estimated, assuming zero digestibility of acid detergent insoluble N, as 1 − ADIN.
3.4. Apparent intestinal digestibility Values of estimated intestinal digestibility for fresh and dried OL are shown in Table 5. The apparent dietary CP digestibility increased (P=0.011) with drying and no differences were observed when values for OL dried with different procedures were compared. The apparent intestinal digestibility of undegraded CP in the rumen was only affected (P=0.046) by oven-drying at 60 and 100 ◦ C without differences (P>0.05) between different drying treatments. The calculated true intestinal digestibility of undegraded CP trends to increase (from 0.19 to 0.40) only by drying OL at 60 ◦ C. The degradable digestible dietary CP in the rumen and in the intestine (1 − ADIN) tended to decrease with drying treatments with the exception of air-drying. 4. Discussion One of the main concerns when low-density and seasonal by-products are considered as sources of nutrients for livestock is their preservation. Procedures have to be simple and low cost and, additionally, they must not affect the nutritive value of the by-product to be preserved or even improve it. These effects need to be known. On the other hand, for comparative purposes, it is necessary to take into account the ideal initial status of the source, simulating the conditions of fresh consumption. Although freeze-drying is an expensive technique we choose it as a reference method because it stops enzymatic activity of plants (Jones, 1981). Deinum and Maassen (1994) considered that it is possible to obtain results close to the ideal state mainly because ice crystals do not break down membranes and cell walls.
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4.1. Chemical composition The lack of drying effect on GE olive leaves agreed with Alomar et al. (1999) observations on pasture silages. The unclear response of drying on ADL might be due to the formation of analytical artefacts derived from the Maillard products (Van Soest, 1994). In contrast to our results, Delgado Pert´ın˜ ez et al. (1998) found decreased ADL after OL freeze-, air- or 60 ◦ C oven drying. Maillard products may be as well responsible for the increased ADIN promoted by drying OL at 60 and 100 ◦ C. The slight effect of drying on total N found on OL agrees with results observed in different plant leaves (Stewart et al., 2000; Parissi et al., 2004). The results of the present work appear to confirm the reported presence of tannins in OL (Fegeros et al., 1995; Mart´ın Garc´ıa et al., 2003; Y´an˜ ez Ruiz et al., 2004) and disagree with Delgado Pert´ın˜ ez et al. (1998), who found no tannins. Most of the CT in OL appeared to be free and fibre-bound, coinciding with observations of Y´an˜ ez Ruiz et al. (2004). The information available on the effect of drying plant leaves on CT is contradictory. Our results agree with those of Norton and Ahn (1997), who observed lower free and protein-bound CT as a result of drying tree leaves. Stewart et al. (2000) observed a reduction of fibre-bound CT and an increase in protein-bound CT when leaves were freezeor air-dried. Makkar (2003) reported that the effect of drying on the CT of plant leaves might depend on the moisture content. He reported decreased tannins only after heating or lyophilizing tree leaves with high moisture contents. The results underline the importance of determining tannins in the stage in which the plants are going to be offered to the animals, in order to establish a close association between CT concentration and type and their nutritive effects. Information on OL amino acid composition is scarce. Our values are similar to those registered by Mart´ın Garc´ıa et al. (2003) but higher than those reported by Y´an˜ ez Ruiz et al. (2004). Dakowski et al. (1996) reported a decrease in rapeseed meal individual amino acids due to heating, especially arginine, cystine and lysine, the latter being the most sensitive to heating. This was not the case in OL where cysteine and lysine tended to increase with airor 60 ◦ C oven-drying. More research is need in order to verify these results and to elucidate the reasons. 4.2. In vitro digestibility Information available on OL in vitro digestibility is very scarce. Delgado Pert´ın˜ ez et al. (1991) reported values for DM close to those found in OL air- and oven-dried at 65 ◦ C in the present study. Concerning CP, our results were higher than those reported in vivo (0.13 and 0.24, respectively for OL air- and 65 ◦ C dried) by Delgado Pert´ın˜ ez et al. (1998). The positive effect of freeze-drying on in vitro OL digestibility was also observed by Stewart et al. (2000) with Callandria calothyrsus leaves. Similar in vitro digestibility was found in freeze and air-dried OL. The last procedure is easier to use in practical conditions. The increased CP digestibility after freeze-, air- and, oven-dried at 60 ◦ C may be due, in some degree, to the effect of these treatments on protein-bound CT. Since total CT was only affected by oven-drying at 60 ◦ C, it seems of importance to determine the different CT fractions in order to explain their effects on nutrient availability.
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4.3. Rumen degradability The OL solubility (0 h of incubation) is close to its rapidly degradable fraction (a), in agreement with others (Mart´ın Garc´ıa et al., 2003; Y´an˜ ez Ruiz et al., 2004). Our results seem to confirm the observations made in vitro and agree with Makkar (2003), who reported marginally increases in DM degradability of some tree leaves after drying. Parissi et al. (2004) found also different effects of freeze- and 60 ◦ C oven-drying on gas production in different tree leaves, depending on their ADL content. On the other hand, Palmer and Schlink (1992), studying fresh leaves of Calliandra calothyrsus, found higher DM degradability in fresh than in freeze-, air- or 65 ◦ C over-dried material. The improved CP effective degradability of OL promoted by some of the treatments may be due, in some degree, to the decreased amount of protein-bound CT as discussed for in vitro digestibility. However, this effect was not found in vivo by using polyethylene glycol as tannins binding compound (Y´an˜ ez Ruiz et al., 2004). 4.4. Intestinal digestibility The protein value of feeds for ruminants depends largely on the amount of undegraded protein in the rumen that reach the duodenum and on its intestinal digestion. Increased intestinal digestibility of dietary protein due to heating has been described (Vanhatalo and Aronen, 1991). This may be a consequence of the decreased ruminal degradation (Walz and Stem, 1989). The results obtained in the present work indicate a trend to decrease CP potentially degradable fraction (b) after OL drying, especially oven-dried at 100 ◦ C. This treatment promotes the highest (0.39) value of apparent intestinal digestibility of undegraded protein in the rumen.
5. Conclusions Freeze-dried olive leaves does not appear to represent fresh material as major differences were found in chemical composition, in vitro digestibility, effective rumen degradability and apparent intestinal digestibility of CP. Air-drying in the shade could be an appropriate, simple and low-cost procedure for olive-leaf preservation, as no detrimental effects on the by-product nutritive value were detected. Although further in vivo research is needed, it appears that drying olive leaves at high temperatures is not justified from a nutritive standpoint. This is of practical importance as olive leaf production is seasonal but should be used throughout the year.
Acknowledgments This research was supported by Junta de Andaluc´ıa (Consejer´ıa de Agricultura y Pesca, Project CAO01-003). Thanks go to L. del Boz and M.C. S´anchez for technical assistance and Romeroliva company for providing olive leaves.
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References Acosta, R.A., Kothmams, M.M., 1978. Chemical composition of oesophageal-fistula forage samples as influenced by drying method and salivary leaching. J. Anim. Sci. 47, 691–698. Ahn, J.H., Elliott, R., Norton, B.W., 1997. Oven drying improves the nutritional value of Calliandra calothyrsus and Gliricidia sepium as supplements for sheep given low-quality straw. J. Sci. Food Agric. 75, 503–510. Ahn, J.H., Robertson, B.M., Elliott, R., Gutteridge, R.C., Ford, C.W., 1989. Quality of tropical browse legumes: tannin content and protein degradation. Anim. Feed Sci. Technol. 27, 147–156. Alomar, D., Fuchslocher, R., Stockebrand, S., 1999. Effects of oven- or freeze-drying on chemical composition and NIR spectra of pasture silage. Anim. Feed Sci. Technol. 80, 309–319. Ankom, 2007. Procedures for automated fibre and in vitro analysis. Accessed at http://www.ankom.com. AOAC, 1984. Official Methods of Analysis of the Association of Official Analytical Chemist International. In: Williams, S. (Ed.), 14th AOAC International. Arlintong, Virginia, USA. Calsamiglia, S., Stern, M.D., 1995. A three-step in vitro procedure for estimating intestinal digestion of protein in ruminants. J. Anim. Sci. 73, 1459–2146. Dakowski, P., Weisbjerg, M.R., Hvelplund, T., 1996. The effect of temperature during processing of rapeseed meal on amino acid degradation in the rumen and digestion in the intestine. Anim. Feed Sci. Technol. 58, 213–226. Deinum, B., Maassen, A., 1994. Effects of drying temperature on chemical composition and in vitro digestibility of forages. Anim. Feed Sci. Technol. 46, 75–86. Delgado Pert´ın˜ ez, M., Chesson, A., Gordon, J.P., Garrido, A., G´omez Cabrera, A., 1998. Effect of different drying systems for the conservation of olive leaves on their nutritive value for ruminants. Ann. Zootech. 47, 141– 150. Delgado Pert´ın˜ ez, M., Garrido, A., Guerrero, J.E., Ortiz, V., 1991. Effect of different drying on the nutritive value of the olive leaf. Options M´editerran´eennes 16, 157–159. Delgado Pert´ın˜ ez, M., G´omez Cabrera, A., Garrido, A., 2000. Predicting the nutritive value of the olive leaf (Olea europaea): digestibility and chemical composition and in vitro studies. Anim. Feed Sci. Technol. 87, 187–201. Dzowela, B.H., Hove, L., Mafongoya, P.L., 1995. Effect of drying method on the chemical composition and in vitro digestibility of multi-purpose tree and shrub fooders. Trop. Grassl. 29, 263–269. Fegeros, K., Zervas, G., Apsokardos, F., Vastardis, J., Apostolaki, E., 1995. Nutritive evaluation of ammonia treated olive tree leaves for lactating sheep. Small Rumin. Res. 17, 9–15. Fern´andez F´ıgares, I., Prieto, C., Nieto, R., Aguilera, J.F., 1997. Free amino acid concentrations in plasma, muscle and liver as indirect measures of protein adequacy in growing chickens. Anim. Sci. 64, 529–539. Hvelplund, T., Madsen, J., 1990. A study of the quantitative nitrogen metabolisnm in the gastro-intestinal tract, and the resultant new protein evaluation system for ruminants. The AAT-PBV system. Thesis. Jones, D.I.H., 1981. Chemical composition and nutritive value. In: Hodgson, J., Baker, R.D., Davies, A., Laidlaw, A.S., Leaver, J.D. (Eds.), Sward Measurement Handbook. British Grassland Society, Hurley, Maidenhead, Berkshire, pp. 243–265. Krishnamoorthy, U., Sniffen, C.J., Van Soest, P.J., 1982. Nitrogen fractionation in ruminant feedstuffs for feed evaluation. In: Proceedings of Cornell Nutrition Conference, pp. 59–102. L´opez, S., Hovell, F.D.D., Manyuchi, B., Smart, I., 1995. Comparison of sample preparation methods for the determination of the rumen degradation characteristics of fresh and ensiled forages by the nylon bag technique. Anim. Sci. 60, 439–450. Madsen, J., Hvelplund, T., 1994. Prediction of in situ protein degradability in the rumen: results of a European ring test. Livest. Prod. Sci. 39, 201–212. Makkar, H.P.S., 2003. Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds. Small. Rumin. Res. 49, 241–256. MAPA, 2006. Anuario estad´ıstico del Ministerio de Agricultura, Pesca y Alimentaci´on (Statistical Yearbook of the Agriculture and Fishing Spanish Ministry). Accessed at: http://www.mapa.es/. Mart´ın Garc´ıa, A.I., Moumen, A., Y´an˜ ez Ruiz, D.R., Molina Alcaide, E., 2003. Chemical composition and nutrients availability for goats and sheep of two-stage olive cake and olive leaves. Anim. Feed Sci. Technol. 107, 61–74. Mart´ın Garc´ıa, A.I., Y´an˜ ez Ruiz, D.R., Moumen, A., Molina Alcaide, E., 2004. Effect of polyethylene-glycol on the chemical composition and nutrient availability of olive (Olea europaea var. europaea) by-products. Anim. Feed Sci. Technol. 114, 159–177.
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Maymone, B., Sblendoiro, A., Ceci Ginestrelli, D., 1950. Ricerche sulla composizione chimica, sulla digeribilit`a e sul valore nutritivo delle foglie di olivo (Olea Europea L.) verdi, essicate, insilate. Ann. Ist. Sper. Zootechnol. 4, 1–19. McDonald, P., Henderson, A.R., Heron, S.J.E., 1991. The Biochemistry of Silage, second ed. Chalcombe Publications, Marlow, pp. 340. McDougall, E.I., 1948. Studies on ruminant saliva 1. The composition and output of sheep’s saliva. Biochem. J. 43, 99–109. Michalet-Doreau, B., Ould-Bah, M., 1992. In vitro and in sacco methods for the estimation of dietary nitrogen degradability in the rumen: a review. Anim. Feed Sci. Technol. 40, 57–86. Mupangwa, J.F., Ngongoni, N.T., Hamudikuwanda, H., 2003. Effects of stage of maturity and method of drying on in situ nitrogen degradability of fresh herbage of Cassia rotundifolia, Lablab purpureus and Macroptilium atropurpureum. Livestock Research for Rural Development 15 (5). Retrieved January 25, 2007, from http://www.cipav.org.co/lrrd/lrrd15/5/mupa155.htm. Nefzaoui, A., 1987. Valorisation des sous-produits de l’olivier dans l’agriculture et l’industrie 1. Les grignons. Agr. Horticult. 1, 26–48. Norton, B.W., Ahn, J.H., 1997. A comparison of fresh and dried Calliandra calothyrsus supplements for sheep given a basal diet of barley straw. J. Agric. Sci. 129, 485–494. Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 92, 499–503. Palmer, B., Schlink, A.C., 1992. The effect of drying on the intake and rate of digestion of the legume Calliandra calothyrsus. Trop. Grassl. 26, 89–93. Parellada, G., G´omez Cabrera, A., Oca˜na, F., Garrido, A., 1982. Utilizaci´on del ram´on de olivo en la alimentaci´on animal: efectos de diversos tratamientos f´ısicos y de la forma de conservaci´on. Avances en Alimentaci´on y Mejora Animal 15, 15–19. Parissi, Z.M., Khazaal, K., Nastis, A.S., Tsiouvaras, C.N., 2004. Assessment of the effect of drying methods on the chemical composition and in vitro gas production of two woody species. Options M´editerran´eennes 59, 141–145. P´erez Maldonado, R.A., Norton, B.W., 1996. Digestion of 14 C labelled condensed tannins from Desmodium intortum in sheep and goats. Br. J. Nutr. 76, 501–513. Prieto, C., Aguilera, J.F., Lara, L., Fonoll´a, J., 1990. Protein and energy requirements for maintenance of indigenous Granadina goats. Br. J. Nutr. 63, 155–163. Porter, L.J., Hrstich, L.N., Chan, B.G., 1986. The conversion of proanthocyanidins and prodelphinidins in cyanidin and delphinidin. Phytochemistry 25, 223–230. Stewart, J.L., Mould, F., Mueller-Harvey, I., 2000. The effect of drying treatment on the fodder quality and tannin content of two provenances of Calliandra calothyrsus Meissner. J. Sci. Food Agric. 80, 1461–1468. Terril, T.H., Douglas, G.B., Foote, A.G., Purchas, R.W., Wilson, G.F., Barry, T.N., 1992. Effect of condensed tannins upon body growth, wool growth and rumen metabolism in sheep grazing sulla (Hedysarum coronarium) and perennial pasture. J. Agric. Sci. 119, 265–273. Tilley, J.M.A., Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Soc. 18, 104–111. Vanhatalo, A.O., Aronen, I.P., 1991. The ruminal and intestinal degradation estimates of either untreated or physically treated rapeseed meals measured by nylon bag techniques. In: GCIRC 8th Int. Rapeseed Congress, 9–11 July, Saskatoon, Sask., Canada, pp. 424–429. Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant. O&B Books, Corvallis (OR), USA, pp. 174–176. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Walz, D.M., Stem, M.D., 1989. Evaluation of various methods for protecting soya-bean protein from degradation by rumen bacteria. Anim. Feed Sci. Technol. 25, 111–122. Y´an˜ ez Ruiz, D.R., Mart´ın Garc´ıa, A.I., Moumen, A., Molina Alcaide, E., 2004. Ruminal fermentation and degradation patterns, protozoa population and urinary purine derivates excretion in goats and wethers fed diets based on olive leaves. J. Anim. Sci. 82, 3006–3014.
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Effect of different drying procedures on the nutritive value of olive (Olea europaea var. europaea) leaves for ruminants A.I. Mart´ın-Garc´ıa, E. Molina-Alcaide ∗ Estaci´on Experimental del Zaid´ın (CSIC), Profesor Albareda 1, 18008 Granada, Spain Received 19 February 2007; received in revised form 16 July 2007; accepted 4 September 2007
Abstract Fresh, freeze-, air- and oven-dried at 60 ◦ C and 100 ◦ C olive leaves (OL) were studied in order to determine the effect of different drying procedures on OL chemical composition, in vitro digestibility, ruminal degradability, and intestinal digestibility. The drying procedure affected all the parameters measured except for gross energy (GE; P=0.194). Protein-bound condensed tannins (CT) decreased (P=0.001) with freeze-, air- and 60 ◦ C drying (from 1.25 up to 0.82 g/kg dry matter, DM). Total CT were only decreased (P=0.001) by drying at 60 ◦ C (from 10.0 to 6.24 g/kg DM). The in vitro crude protein (CP) digestibility increased (P<0.001) with drying except for oven-drying at 100 ◦ C up to 58%. Values for CP digestibility found in freeze- and air-dried OL were not different (P>0.05). No differences (P>0.05) were observed between CP digestibility in air- and oven-dried at 60 ◦ C OL. Effective degradability of DM and CP increased from 0.53 to 0.62 (P=0.005) and from 0.46 to 0.64 (P=0.002), respectively after treatment. The apparent
Abbreviations: a, rapidly degradable fraction; ADF, acid detergent fibre; ADIN, acid detergent insoluble nitrogen; ADL, sulphuric acid lignin; ANOVA, analysis of variance; b, potentially degradable fraction; c, rate of degradation of the potentially degradable fraction; CP, crude protein; CT, condensed tannins; DM, dry matter; ED, effective degradability; GE, gross energy; LW, live weight; NA, not applicable; NDF, neutral detergent fibre; OL, olive leaves; OM, organic matter; PD, potential degradability; SDS, sodium dodecyl sulphate; S.E.M., standard error of the mean; t, tonnes. ∗ Corresponding author. Tel.: +34 958572757; fax: +34 958572753. E-mail address: [email protected] (E. Molina-Alcaide). 0377-8401/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2007.09.005
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intestinal digestibility of undegraded CP in the rumen was only affected (P=0.046) by ovendrying, which increased it from 0.33 to 0.39. As air-drying did not have detrimental effects on the OL nutritive value it could be an appropriate, simple and low-cost procedure for olive-leaves preservation. © 2007 Elsevier B.V. All rights reserved. Keywords: Olive leaves; Chemical composition; Drying; In vitro digestibility; Degradability; Intestinal digestibility
1. Introduction Olive leaves (OL) account for around 3–5% of the total biomass processed in the olive oil mill (Delgado Pert´ın˜ ez et al., 1998). Its nutrient availability is similar to that of cereal straws (Mart´ın Garc´ıa et al., 2003) and it has been commonly used for livestock in Mediterranean countries. It is of especial importance to optimise the use of by-products such as OL to be used in animal feeding under dry conditions. The seasonal and high OL production (3,600,000 t of olives in Spain during 2005; MAPA, 2006) requires adequate preservation. As the preservation procedure may lead to variations in OL nutritive value (Delgado Pert´ın˜ ez et al., 1998), it is important to investigate suitable procedures not only to preserve OL but also to improve its nutritive value. Ensiling OL appears to be impractical (Parellada et al., 1982) due to the physical structure, high dry matter (DM) content, low density and lack of fermentable sugars. Practical storage procedures of plant materials such as wilting or air-drying involve water removal and may have different effects (Mupangwa et al., 2003) depending on plant type. High temperatures may diminish volatile substances, such as short-chain fatty acids and alcohols (McDonald et al., 1991) or organic matter (Acosta and Kothmams, 1978) and induce chemical and enzymatic changes of cell-wall components, proteins, carbohydrates (L´opez et al., 1995) and secondary compounds such as tannins (Ahn et al., 1989). Of especial importance might be the formation of Maillard products during sun-drying process (Dzowela et al., 1995). These factors might affect ruminal degradation (Dakowski et al., 1996), apparent digestibility (Palmer and Schlink, 1992) or nutrient availability at the intestine (Ahn et al., 1997). Available information on drying effect on OL nutritive value is scarce and often contradictory. High temperatures have been reported to decrease OL intake and nutritive value (Maymone et al., 1950; Delgado Pert´ın˜ ez et al., 1998, 2000), although Nefzaoui (1987) observed no change in OL nutritive value with drying. On the other hand, no work is available on the effect of drying on OL amino acid composition and protein ruminal degradation or intestinal digestibility, which are the main factors determining the protein value of feedstuffs for ruminants. The aim of the present work was to evaluate the effect of different drying procedures on the chemical composition, in vitro digestibility, ruminal degradability, and intestinal digestibility of olive leaves in order to state the most adequate and practical procedure to preserve this by-product without affecting or even improving its nutritive value.
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2. Materials and methods 2.1. Olive leaves Olive leaves from olive cleaning were obtained in January 2001 in an oil mill (Romeroliva, Granada, Spain). Different samples of OL were studied: fresh, freeze-dried, air-dried for 7 days in the shade at room temperature, and dried in a forced-air oven at 60 ◦ C for 48 h and 100 ◦ C overnight. After oven-drying, the OL were equilibrated at room temperature for 48 h. Both fresh and dried OL were crushed and stored at 4 ◦ C until chemically analysed. 2.2. Animals Granadina goats (39.4 ± 2.62 kg LW), fitted with permanent ruminal cannulas, were used. The animals were housed in individual pens and had free access to water and were fed two equal meals at 08:00 and 16:00 h at maintenance level (Prieto et al., 1990), a goodquality alfalfa hay and a mineral–vitamin mixture (20 g/d which was formulated with 277 g NaCl, 270 g of ash from two-stage dried olive cake combustion, 250 g (PO4 )2 H4 Ca, 200 g MgSO4 , 8.5 mg CoO, 4 mg Se, 2.5 mg I, and 83,500 and 16,700 IU vitamins A and D, respectively, per kilogram). All management and experimental procedures were carried out in strict accordance to the Spanish guidelines (Act No. 1201/2005 of 10 October 2005) for experimental animal protection and by trained personnel. Fistulated goats remained in good health and welfare throughout the experimental and inter-experimental periods. Ruminal content for in vitro digestibility determination was manually extracted through the cannula using a rubber hose and applying a small hand vacuum. 2.3. In vitro digestibility This parameter was determined according to Tilley and Terry (1963) by using the Daisy II incubator (Ankom® Technology, New York, NY, Ankom, 2007) as described by Mart´ın Garc´ıa et al. (2004). The rumen inoculum was individually withdrawn from three ruminally fistulated goats 2 h after morning feeding, transferred into pre-warmed (39 ◦ C) thermo bottles and squeezed through four layers of cheesecloth under anaerobic conditions. A pooled sample was prepared by combining equal squeezed volumes from each animal. The inoculum was a mixture (4:1 v/v) of rumen liquor and artificial saliva (McDougall, 1948). Samples (0.5 ± 0.05 g) were weighted in filter bags (Ankom® Technology, # F57) and three samples per treatment were incubated in the incubator. Two incubation runs were carried out. Every incubation run included two blanks and three standard samples for which the apparent in vivo DM digestibility was known. The in vitro DM and CP digestibility were calculated as DM and CP disappearance, respectively, per unit of original DM and CP. Residual DM and CP were corrected for those in the blanks. 2.4. Rumen degradability It was measured in situ by using the nylon-bag technique described by Madsen and Hvelplund (1994). Approximately 3 g of samples, ground to pass through a 2 mm mesh,
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were weighed in nylon bags (10 cm × 7 cm, 46 m of pore size). Five incubation runs were carried out with two ruminally fistulated goats in each run. One bag per treatment and time of incubation was simultaneously incubated in the rumen of each goat for 0, 4, 8, 16, 24, 48 and 72 h. At the end of the incubation period, the nylon bags were rinsed under cold running tap water to remove large particles of food, and then stored at −20 ◦ C. After completion of all incubations, the bags were washed with cold water in a washing machine for 20 min, and the residue was subjected to vigorous mechanical pummelling between two metal plates (Masticator, IUL Instruments GmbH, K¨onigswinter, Germany) for 5 min (Michalet-Doreau and Ould-Bah, 1992) to detach bacteria associated with food particles and, finally, dried for 48 h at 60 ◦ C. The profiles of DM and CP degradation were calculated according to Ørskov and McDonald (1979). The effective degradability (ED) was calculated as ED = a + [(b × c)/(c + k)], where a is the soluble fraction, b the potentially degradable insoluble fraction, c the degradation rate of b and k the fractional outflow rate with a value of 0.021 per h, previously determined (Y´an˜ ez Ruiz et al., 2004) in Granadina goats fed OL at maintenance level. 2.5. Intestinal digestibility The intestinal digestibility of dietary and ruminally undegraded protein was determined by using the procedure of Calsamiglia and Stern (1995). Three Granadina goats, fitted with permanent ruminal cannula were used. Eight nylon bags (10 cm × 7 cm, 46 m of pore size) with approximately 3 g of sample per bag per treatment were successively incubated in the rumen of one of three cannulated goats in order to obtain a minimum residual N of 60 mg per treatment. After rumen preincubation, the bags were washed and dried at 60 ◦ C for 48 h. Pooled residues containing 15 mg of N were incubated for 1 h in 10 ml of a 1 N HCl-pepsin solution. After incubation, pH was neutralised with 5 ml of 1 N NaOH and, 13.5 ml of a pH 7.8 phosphate buffer containing 37.5 mg of pancreatin were added to the solution and incubated at 38 ◦ C. After a 24 h incubation period, 3 ml of 1000 g/l trichloroacetic acid (Panreac, 131067) solution were added to precipitate the undigested proteins and N content in supernatant was measured. Dietary and ruminally undegraded CP apparent digestibility were calculated as described by Mart´ın Garc´ıa et al. (2004). The true intestinal digestibility of rumen-undegraded CP was calculated by using the equation of Hvelplund and Madsen (1990) [(1 − CP effective degradability) − (1 − apparent intestinal digestibility of dietary CP)]/(1 − CP effective degradability). An estimation of ruminally degradable and intestinally digestible dietary protein was also obtained from 1 − ADIN as suggested Krishnamoorthy et al. (1982). 2.6. Chemical analyses All samples were ground through a 1 mm sieve before analysis and, DM content was determined by drying to a constant weight in a forced-air oven at 103 ± 1 ◦ C using the AOAC (1984) method number 7.008. The organic matter (OM) was calculated from the ash content, determined by ashing in a muffle furnace for 3 h at 550 ◦ C following the AOAC (1984) method number 7.010. The ether extract was determined by extraction with petroleum ether (boiling point: 40–60 ◦ C) following the AOAC (1984) method number
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7.045. Gross energy (GE) was determined in an adiabatic bomb calorimeter (Gallenkamp & Co. Ltd., London) according to the methodology described by Prieto et al. (1990). Neutral detergent fibre (NDFom), acid detergent fibre (ADFom) and sulphuric acid lignin (ADLsa) analyses were performed by the sequential procedure of Van Soest et al. (1991) using the Ankom 200 Fiber Analyzer (Ankom® Technology, New York, NY) (Ankom, 2007). The NDF was assayed with sodium sulphite and without alpha amylase. Both NDF and ADF were expressed without residual ash. Total nitrogen was determined by Kjeldahl analysis according to the method recommended by the AOAC (1984), number 2.055. Acid detergent insoluble N (ADIN) was determined by Kjeldahl analysis of ADFom residues. Free, protein- and fibre-bound condensed tannins (CT) were sequentially extracted following the procedure described by Terril et al. (1992) and modified by P´erez Maldonado and Norton (1996). The free-CT fraction was extracted using aqueous acetone (70%), diethyl ether and ethyl acetate and partitioned into the aqueous phase to exclude pigments and small phenolic compounds. Subsequent extraction with boiling sodium dodecyl sulphate (SDS), triethanolamine and 2-mercaptoethanol yielded protein-bound tannins. The residue from this SDS extraction was washed with methanol and butanol to yield fibre-bound tannins. Analyses of the free, protein-bound and fibre-bound condensed tannins were conducted according to the butanol–HCl method of Porter et al. (1986) with the modifications proposed by Terril et al. (1992). Condensed tannins from quebracho powder (Roy Wilson Dickson Ltd., Mold, U.K.) were used as standard. Total amount of CT was calculated by adding the amounts of free, protein- and fibre-bound CT in the sample. The amino acid N content was determined by high-performance liquid chromatography (HPLC) using the Waters® Pico-Tag method, which involves precolumn derivatization with phenylisothiocyanate. Protein hydrolysis was performed in 6 N HCl using sealed and evacuated tubes at 110 ◦ C for 24 h (Fern´andez F´ıgares et al., 1997). Cysteine and methionine were determined as cysteic acid and methionine sulphone, respectively, obtained by oxidation with performic acid before 6 N HCl hydrolysis. 2.7. Statistical analysis The effect of drying on OL chemical composition, in vitro digestibility, ruminal degradability profiles and apparent intestinal digestibility was tested by ANOVA using the Minitab Statistical Software Package (Minitab, New York, NY). Significant differences between means for each treatment were assessed by Tukey or Bonferroni t-tests for paired or unequal groups, respectively. 3. Results 3.1. Chemical composition The drying procedure affected all the parameters measured (Table 1) except for GE and total CT (P=0.194). The OM content decreased (P=0.001) when OL was freeze-
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Olive leaves
Fresh
Freeze-dried
Air-dried
Dried 60 ◦ C
Dried 100 ◦ C
P-value
S.E.M.
DM (g/kg fresh matter) Organic matter Ether extract Gross energy (MJ/kg DM) Neutral detergent fibre Acid detergent fibre Sulphuric acid lignin Total nitrogen Acid detergent insoluble nitrogen (g/g total nitrogen) Condensed tannins Free Fibre-bound Protein-bound Total
694d
622a
630b
641c
870c 97.6c 20.0 399bc 267b 158ab 11.4b 0.509a
863b 87.4b 19.7 358a 245a 152a 11.0ab 0.556ab
865bc 82.2ab 19.9 415c 279b 166b 11.1ab 0.486a
861b 78.1a 19.9 398bc 270b 163ab 11.3b 0.595b
696d 847a 95.2c 19.6 376ab 249a 155a 10.6a 0.676c
0.000 0.001 0.001 0.194 0.001 0.001 0.027 0.036 0.009
0.85 0.51 0.48 0.59 1.67 0.92 1.03 0.06 0.001
4.02b 4.73bc 1.25b 10.0c
2.25a 6.72c 0.95a 9.92c
2.50a 5.72bc 1.40b 9.57bc
0.004 0.001 0.001 0.001
0.07 0.14 0.02 0.12
2.95ab 4.13ab 0.82a 7.90ab
a–d: In a row means without a common letter differ (P<0.05); S.E.M.: standard error of the mean.
2.80a 2.53a 0.91a 6.24a
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Table 1 Chemical composition (g/kg DM) of fresh and dried olive leaves
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Table 2 Amino acid composition (g amino acid-N/kg Total N) of fresh and dried olive leaves Fresh
Freeze-dried
Air-dried
Dried 60 ◦ C
Dried 100 ◦ C
Aspartic acid Glutamic acid Serine Glycine Histydine Arginine Threorine Alanine Proline Tyrosine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Lysine Essential Non-essential
27.5 35.1 44.5 79.6 25.4 162 46.8 73.8 84.2 32.3 74.8 5.32 1.60 58.8 104. 51.8 19.1 547 379
32.3 47.4 40.6 65.6 27.8 125 40.5 47.4 68.0 29.0 47.1 7.40 2.91 35.3 60.9 31.3 21.0 396 333
44.3 53.4 39.8 63.6 28.1 122 39.0 49.9 74.2 28.3 51.6 4.82 2.30 40.9 71.2 36.7 44.4 438 356
31.2 46.3 36.0 49.9 20.3 165 40.0 51.1 99.3 35.1 52.7 3.62 2.01 39.9 71.7 38.2 47.4 478 351
33.2 48.1 44.0 68.9 16.3 105 41.4 52.7 94.8 30.0 56.4 5.02 1.81 44.4 74.5 40.4 18.1 401 374
Totala
926
729
794
829
774
a
Without tryptophane.
dried and dried at 60 and 100 ◦ C. A trend to decrease with some drying procedures was also found for GE. Freeze-drying decreased the NDFom content (P=0.001) and ADFom decreased (P=0.001) both after freeze-drying and 100 ◦ C oven-drying. Total N was slightly affected (P=0.036) by drying in the oven at 100 ◦ C. The lowest ADIN values corresponded to air-dried and fresh samples but rose (P=0.009) with drying at 60 and 100 ◦ C. Free CT decreased (P=0.004) after freeze-drying and 60 and 100 ◦ C oven-drying. Fibrebound CT decreased (P=0.001) only after drying at 60 ◦ C. Protein-bound CT decreased (P=0.001) after freeze-, air- and 60 ◦ C drying. Total CT were only decreased (P=0.001) by drying at 60 ◦ C. Olive leaves were rich in arginine, leucine, proline, glycine, valine and alanine and low in cysteine, methionine and lysine (Table 2). All the treatments tended to diminish the total amino acid content. 3.2. In vitro digestibility Drying OL improved (P=0.005) DM digestibility with the exception of 60 ◦ C ovendrying (Table 3). No differences were found (P>0.05) when comparing the different drying procedures. The CP digestibility increased (P<0.001) with drying except for oven-drying at 100 ◦ C. Values for CP digestibility found in freeze and air dried OL were not different (P>0.05). No differences (P>0.05) were observed between CP digestibility in air- and ovendried OL at 60 ◦ C.
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Table 3 In vitro digestibility of fresh and dried olive leaves Dry matter
Crude protein
Fresh Freeze-dried Air-dried Oven-dried at 60 ◦ C Oven-dried at 100 ◦ C
0.53a 0.60b 0.59b 0.58ab 0.59b
0.36a 0.57c 0.53bc 0.48b 0.37a
P-value S.E.M.
0.005 0.0053
0.000 0.0047
a–c: In a column the means without a common letter differ (P<0.05); S.E.M.: standard error of the mean.
3.3. Rumen degradability The DM and CP ruminal degradation profiles of fresh and dried OL are shown in Table 4. Significant differences among treatments were found for rapidly degradable fraction (a) of DM (P=0.006) and CP (P=0.003). Both values increased with all drying procedures except at 60 ◦ C. The potentially degradable fraction (b), degradation rate (c) and potential degradability (PD) were not affected (P=0.505, 0.161, and 0.473, respectively) by any treatment. However, effective degradability was affected (P=0.005 and 0.002, respectively for DM and CP) by the treatment except for oven-drying at 60 ◦ C in the case of DM. Table 4 Ruminal degradation profiles of fresh and dried olive leaves a
b
c (h−1 )
PD
EDa
Dry matter Fresh Freeze-dried Air-dried Oven-dried at 60 ◦ C Oven-dried at 100 ◦ C
0.22a 0.32c 0.29bc 0.24ab 0.29bc
0.52 0.49 0.48 0.51 0.48
0.032 0.033 0.040 0.045 0.045
0.73 0.80 0.77 0.75 0.78
0.53a 0.61b 0.60b 0.59ab 0.62b
P-value S.E.M.
0.006 0.005
0.628 0.01
0.161 0.002
0.452 0.01
0.005 0.004
Crude protein Fresh Freeze-dried Air-dried Oven-dried at 60 ◦ C Oven-dried at 100 ◦ C
0.12a 0.21b 0.20b 0.11a 0.20b
0.70 0.71 0.73 0.77 0.68
0.020 0.026 0.030 0.033 0.038
0.82 0.92 0.92 0.88 0.88
0.46a 0.60b 0.62b 0.57b 0.64b
P-value S.E.M.
0.003 0.005
0.505 0.002
0.161 0.002
0.473 0.002
0.002 0.007
a–c: In a column means without a common letter differ (P<0.05); PD: potential degradability (a + b). a ED: effective degradability, calculated as a + [(b × c)/(c + k)], were k = 0.021 h−1 (Y´ an˜ ez Ruiz et al., 2004); S.E.M.: standard error of the mean.
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Table 5 Apparent and true intestinal digestibility of dietary and ruminally undegraded protein of fresh and dried olive leaves Apparent intestinal digestibility
True intestinal digestibility of undegraded crude proteina
Estim. N dig.b
Dietary CP
Ruminally undegraded CP
Fresh Freeze-dried Air-dried Oven-dried 60 ◦ C Oven-dried 100 ◦ C
0.56a 0.71b 0.73b 0.74b 0.74b
0.33a 0.35abc 0.36abc 0.38bc 0.39c
0.19 0.28 0.29 0.40 0.28
0.49 0.44 0.51 0.41 0.32
P-value S.E.M.
0.011 0.01
0.046 0.007
NA
NA
a–c: In a column means without a common letter differ (P<0.05); S.E.M.: standard error of the mean; NA: not applicable. a True intestinal digestibility of undegraded crude protein = [(1 − CP effective degradability) − (1 − apparent intestinal digestibility of dietary CP)]/(1 − CP effective degradability) (Hvelplund and Madsen, 1990). b Ruminally degradable and intestinally digestible dietary protein (Krishnamoorthy et al., 1982). Estimated, assuming zero digestibility of acid detergent insoluble N, as 1 − ADIN.
3.4. Apparent intestinal digestibility Values of estimated intestinal digestibility for fresh and dried OL are shown in Table 5. The apparent dietary CP digestibility increased (P=0.011) with drying and no differences were observed when values for OL dried with different procedures were compared. The apparent intestinal digestibility of undegraded CP in the rumen was only affected (P=0.046) by oven-drying at 60 and 100 ◦ C without differences (P>0.05) between different drying treatments. The calculated true intestinal digestibility of undegraded CP trends to increase (from 0.19 to 0.40) only by drying OL at 60 ◦ C. The degradable digestible dietary CP in the rumen and in the intestine (1 − ADIN) tended to decrease with drying treatments with the exception of air-drying. 4. Discussion One of the main concerns when low-density and seasonal by-products are considered as sources of nutrients for livestock is their preservation. Procedures have to be simple and low cost and, additionally, they must not affect the nutritive value of the by-product to be preserved or even improve it. These effects need to be known. On the other hand, for comparative purposes, it is necessary to take into account the ideal initial status of the source, simulating the conditions of fresh consumption. Although freeze-drying is an expensive technique we choose it as a reference method because it stops enzymatic activity of plants (Jones, 1981). Deinum and Maassen (1994) considered that it is possible to obtain results close to the ideal state mainly because ice crystals do not break down membranes and cell walls.
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4.1. Chemical composition The lack of drying effect on GE olive leaves agreed with Alomar et al. (1999) observations on pasture silages. The unclear response of drying on ADL might be due to the formation of analytical artefacts derived from the Maillard products (Van Soest, 1994). In contrast to our results, Delgado Pert´ın˜ ez et al. (1998) found decreased ADL after OL freeze-, air- or 60 ◦ C oven drying. Maillard products may be as well responsible for the increased ADIN promoted by drying OL at 60 and 100 ◦ C. The slight effect of drying on total N found on OL agrees with results observed in different plant leaves (Stewart et al., 2000; Parissi et al., 2004). The results of the present work appear to confirm the reported presence of tannins in OL (Fegeros et al., 1995; Mart´ın Garc´ıa et al., 2003; Y´an˜ ez Ruiz et al., 2004) and disagree with Delgado Pert´ın˜ ez et al. (1998), who found no tannins. Most of the CT in OL appeared to be free and fibre-bound, coinciding with observations of Y´an˜ ez Ruiz et al. (2004). The information available on the effect of drying plant leaves on CT is contradictory. Our results agree with those of Norton and Ahn (1997), who observed lower free and protein-bound CT as a result of drying tree leaves. Stewart et al. (2000) observed a reduction of fibre-bound CT and an increase in protein-bound CT when leaves were freezeor air-dried. Makkar (2003) reported that the effect of drying on the CT of plant leaves might depend on the moisture content. He reported decreased tannins only after heating or lyophilizing tree leaves with high moisture contents. The results underline the importance of determining tannins in the stage in which the plants are going to be offered to the animals, in order to establish a close association between CT concentration and type and their nutritive effects. Information on OL amino acid composition is scarce. Our values are similar to those registered by Mart´ın Garc´ıa et al. (2003) but higher than those reported by Y´an˜ ez Ruiz et al. (2004). Dakowski et al. (1996) reported a decrease in rapeseed meal individual amino acids due to heating, especially arginine, cystine and lysine, the latter being the most sensitive to heating. This was not the case in OL where cysteine and lysine tended to increase with airor 60 ◦ C oven-drying. More research is need in order to verify these results and to elucidate the reasons. 4.2. In vitro digestibility Information available on OL in vitro digestibility is very scarce. Delgado Pert´ın˜ ez et al. (1991) reported values for DM close to those found in OL air- and oven-dried at 65 ◦ C in the present study. Concerning CP, our results were higher than those reported in vivo (0.13 and 0.24, respectively for OL air- and 65 ◦ C dried) by Delgado Pert´ın˜ ez et al. (1998). The positive effect of freeze-drying on in vitro OL digestibility was also observed by Stewart et al. (2000) with Callandria calothyrsus leaves. Similar in vitro digestibility was found in freeze and air-dried OL. The last procedure is easier to use in practical conditions. The increased CP digestibility after freeze-, air- and, oven-dried at 60 ◦ C may be due, in some degree, to the effect of these treatments on protein-bound CT. Since total CT was only affected by oven-drying at 60 ◦ C, it seems of importance to determine the different CT fractions in order to explain their effects on nutrient availability.
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4.3. Rumen degradability The OL solubility (0 h of incubation) is close to its rapidly degradable fraction (a), in agreement with others (Mart´ın Garc´ıa et al., 2003; Y´an˜ ez Ruiz et al., 2004). Our results seem to confirm the observations made in vitro and agree with Makkar (2003), who reported marginally increases in DM degradability of some tree leaves after drying. Parissi et al. (2004) found also different effects of freeze- and 60 ◦ C oven-drying on gas production in different tree leaves, depending on their ADL content. On the other hand, Palmer and Schlink (1992), studying fresh leaves of Calliandra calothyrsus, found higher DM degradability in fresh than in freeze-, air- or 65 ◦ C over-dried material. The improved CP effective degradability of OL promoted by some of the treatments may be due, in some degree, to the decreased amount of protein-bound CT as discussed for in vitro digestibility. However, this effect was not found in vivo by using polyethylene glycol as tannins binding compound (Y´an˜ ez Ruiz et al., 2004). 4.4. Intestinal digestibility The protein value of feeds for ruminants depends largely on the amount of undegraded protein in the rumen that reach the duodenum and on its intestinal digestion. Increased intestinal digestibility of dietary protein due to heating has been described (Vanhatalo and Aronen, 1991). This may be a consequence of the decreased ruminal degradation (Walz and Stem, 1989). The results obtained in the present work indicate a trend to decrease CP potentially degradable fraction (b) after OL drying, especially oven-dried at 100 ◦ C. This treatment promotes the highest (0.39) value of apparent intestinal digestibility of undegraded protein in the rumen.
5. Conclusions Freeze-dried olive leaves does not appear to represent fresh material as major differences were found in chemical composition, in vitro digestibility, effective rumen degradability and apparent intestinal digestibility of CP. Air-drying in the shade could be an appropriate, simple and low-cost procedure for olive-leaf preservation, as no detrimental effects on the by-product nutritive value were detected. Although further in vivo research is needed, it appears that drying olive leaves at high temperatures is not justified from a nutritive standpoint. This is of practical importance as olive leaf production is seasonal but should be used throughout the year.
Acknowledgments This research was supported by Junta de Andaluc´ıa (Consejer´ıa de Agricultura y Pesca, Project CAO01-003). Thanks go to L. del Boz and M.C. S´anchez for technical assistance and Romeroliva company for providing olive leaves.
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