Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol87.Issue3-4.Oct2000:
Animal Feed Science and Technology
87 (2000) 187±201
Predicting the nutritive value of the olive leaf
(Olea europaea): digestibility and chemical
composition and in vitro studies
Manuel Delgado-PertõÂnÄeza,*,
Augusto GoÂmez-Cabrerab, Ana Garridob
a
Dpto. Ciencias Agroforestales, Universidad de Sevilla, Ctra. Utrera Km. 1, 41013 Sevilla, Spain
b
Dpto. ProduccioÂn Animal, Universidad de CoÂrdoba, Apdo. 3048, 14080 CoÂrdoba, Spain
Received 17 November 1998; received in revised form 11 August 1999; accepted 10 August 2000
Abstract
Eight tests of olive leaf digestibility were carried out with sheep. The olive leaves were collected using
different procedures (four samples of leaves from dried branches and another four of leaves removed
from chopped branches and dried) and stored for varying times. Marked differences in OMD were
observed between the two types of procedures and between different times of storage. The greatest loss
of nutritive value was in the chopped samples. In the leaves from dried branches, with storage periods
longer than 9 months, the digestible organic matter content ranged between 431 and 448 g kgÿ1 OM,
while in the chopped samples, the value ranged between 360 g kgÿ1 OM (in a sample stored for only 1
month) and 177 g kgÿ1 OM (in a sample with obvious signs of fermentation).
The sample set was used together with another, evaluated prior to this work, to study the prediction of
in vivo digestibility (OMD, DMD and CPD) from chemical and biological (dry matter disappearance
after in vitro incubation with rumen liquor±pepsin or pepsin±cellulase solutions) parameters. In the case
of OMD, the best predictions were obtained with the pepsin±cellulase method (r 0:91, RSD 4:6)
and NDF (r ÿ0:91, RSD 4:6). # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Olive leaves; Nutritive value; Sheep; In vitro studies; Predicting in vivo digestibility
1. Introduction
In Mediterranean grassland ruminant production systems, the nutritive input from
natural grazing is much reduced. In Syria, the supply was estimated as between 14 and
*
Corresponding autor. Tel.: 34-423-3669; fax: 34-423-2644.
E-mail address: [email protected] (M. Delgado-PertõÂnÄez).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 9 5 - 4
188
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
35%, depending on the characteristics of the year (Treacher et al., 1993). Various
management strategies are used to complement the supply. They include long-range
(trashumance) and short-range ¯ock migration, but above all, the use of crop residues, in
particular those of cereals (Flamant, 1992).
Apart from cereals, an important crop in the Mediterranean basin is the olive, with the
region producing 98% of the world total (approximately 11 million tons) (FAO, 1995).
The use of by-products of this crop (leaf and olive pressings) has been part of the farming
tradition of the region (Sansoucy et al., 1985). However, the specialization in production
that has led to monocultivation of the olive in wide areas has also meant the absence of
animals that could directly consume the plentiful prunings. The prunings must therefore
be moved away from where they are produced, involving operations to increase their
density to make their transport and storage easier (Delgado-PertõÂnÄez et al., 1994).
Various reviews of work on the use of the olive leaf (Sansoucy et al., 1985; Nefzaoui
and Zidani, 1987) demonstrate a wide range in its nutritive value. It is therefore
essential to identify the factors responsible for such diversity and to obtain equations
for the determination of nutritive value, so that each batch can be assigned a correct
value.
Previous work (GoÂmez-Cabrera et al., 1992) has shown a minor effect of variety,
season and year in the nutritive value, but a greater importance of conservation by
drying the leaves, mainly when they were not attached to the branches. The main aim
of this work was to predict the nutritive value of the olive leaf from various chemical and
biological parameters. As there is no standard system of collection and manipulation,
emphasis has been placed on obtaining very different materials. Therefore we
included leaves dried on the branches or obtained after chopping the branches, to
con®rm our previous results (GoÂmez-Cabrera et al., 1992) and sometimes by increasing
storage time.
2. Materials and methods
2.1. Effects of different drying methods on nutritive value of olive leaf
2.1.1. Collection and storage of olive leaves
Mature branches were collected during routine pruning of Picual and Hojiblanca
cultivars of olive trees (Olea europaea L.).
A set of eight samples of olive leaves were evaluated (Table 1).
2.1.1.1. Dried branches
2.1.1.1.1. Leaves from air-dried branches. Leaves dried on pruned branches in the air
and under cover for 3 months. The branches were then crushed with a tractor and the
leaf was coarsely separated by hand and stored for 21 months (sample B24) and 39
months (sample B42) piled on the ground. Prior to the digestibility tests, the leaf was
mechanically separated from residual wood (of which some 15±20% remained at the end
of the process).
Treatment
Fresh leaves
Dried branches (oven-dried)
Item
a
FL
a,b
ODa/ODb
a
Short description of treatment
Storage time
Leaves from the branches
2±5 days
Branches oven dried at 60±658C
16 h
Dried branches (air-dried)
BOut3
B3a/B3ba,b
B24
B42
Branches air-dried outdoors
Branches air-dried under cover
Leaves from B3b were separated and stored for another 21 months
Leaves from B3b were separated and stored for another 39 months
3
3
3
3
Dried branches (bales)
BB9
BB10
Branches baled 7 days after pruning and stored under cover
Branches baled 17 days after pruning and stored under cover
7 days 9 months
17 days 9 months
Chopped branches
BCa
BC2a
BC24
BC42
BC1
BC12-F
Branches chopped and leaves stored in piles outside, without rain
Material BC stored in evacuated airtight plastic bags for a further month
Branches chopped and leaves stored in piles under cover
Branches chopped and leaves stored in piles under cover
Branches chopped and leaves stored in piles outside, without rain
Branches chopped and wetted leaves stored in piles inside (slight fermentation)
5±10 days
5±10 days 1 month
24 months
42 months
10±15 days
12 months
a
b
Material evaluated previously by our team (GoÂmez-Cabrera et al., 1992 and unpublished data).
Two samples obtained in 2 years.
months
months
21 months
39 months
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 1
Olive leaves obtained after different methods of drying
189
190
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
2.1.1.1.2. Leaves from baled branches. Branches of less than 3 cm in diameter at the base
were baled into high-pressure bales (0:6 m 0:4 m 0:3 m) using a LERDA machine.
Some samples were baled 7 days (sample BB9) or 17 days (sample BB10) after pruning
and stored under cover for 9 months. The leaves were then separated from the branches by
hand. Both samples were wetted by rain before they were baled and placed under cover
with sample BB9 receiving somewhat more rain.
2.1.1.2. Partially wetted chopped branches. Pruned olive branches were chopped while
fresh with a DORCH chopper (Kapinka) and stored in piles under cover for 24 (BC24) and
42 (BC42) months, respectively. Some olive branches were also chopped in the same way
and stored under cover for 12 months. Occurrence of fermentation was evident in this sample
(BC12-F), which had been wetted by rain during chopping in the field and by infiltration
at the storage site. Olive branches were chopped 10±15 days after pruning and the product
was piled outside (without rainfall) a further 10±15 days (BC1). In all these samples, at the
end of storage, the leaf was mechanically separated from wood (10±15% of residual wood).
The mechanical separation of leaf and wood prior to the digestibility tests was carried
out using a winnowing machine (prototype of the Departamento de MecanizacioÂn
Agraria, University of CoÂrdoba).
2.1.2. In vivo digestibility determination and chemical composition
The digestibility of the eight samples of olive leaves after various conditions of storage
was measured using four or ®ve adult Segure~
na Merino sheep as described by GoÂmezCabrera et al. (1992). The amount of olive leaf offered to the sheep was 70 g DM kgÿ1
LW0.75 with 150 g of dehulled sun¯ower seed meal mixed with the leaf as a protein
supplement. The diet was fed twice a day (9 a.m. and 6 p.m.) at an intake level close to
maintenance. The adaptation period was 20 days (only 10 when two consecutive tests
were made) followed by 10 days of faeces collection. The animals were offered 20 g of a
mineral±vitamin corrector, STAY-DRY (18% Ca, 8% P, 10% Mg, 1.5% NaCl, 200 mg kgÿ1
Co, 400 mg kgÿ1 I, 3000 mg kgÿ1 Fe, 6000 mg kgÿ1 Mn, 4000 mg kgÿ1 Zn, 15 mg kgÿ1 Se,
300 UI kgÿ1 Vitamin A, 60 UI kgÿ1 Vitamin D3, 800 UI kgÿ1 Vitamin E).
In the leaf samples, gross energy (GE) content was measured in an adiabatic
calorimeter (Parr), dry matter (DM), ash and crude protein (CP) were measured according
to AOAC (1984) procedures and neutral detergent ®bre (NDF), acid detergent ®bre
(ADF) and acid detergent lignin (ADL) were determined by the method of Robertson and
Van Soest (1981). DM, ash and CP were determined for feed residues and faeces. Dry
matter digestibility (DMD), organic matter digestibility (OMD) and crude protein
digestibility (CPD) of the olive leaves were calculated by difference, taking into account
the following values already obtained for sun¯ower meal (g kgÿ1): DM 650, OM 700 and
CP 870 (GoÂmez-Cabrera et al., 1992).
2.2. Prediction of digestibility from chemical composition and in vitro studies
2.2.1. Collection and storage of olive leaves
Together with the formerly described eight samples of olive leaves, another eight
samples were analyzed (Table 1). Their digestibility and chemical parameters had been
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
191
determined earlier by our team. These samples had been kept initially in the laboratory in
airtight plastic bottles. Later, they were moved to a refrigerated room, where they were
stored at 108C for 4±6 years. Of these eight samples, six were analyzed by GoÂmezCabrera et al. (1992). Fresh leaves (sample FL), leaves oven dried at 60±658C for 16 h
(two samples obtained in 2 years, samples ODa and ODb), leaves dried on the branches
for 3 months under cover (two samples obtained in 2 years, samples B3a and B3b) and
leaves dried on the branches for 3 months outdoors (BOut3), exposed to rain and sun. The
other two samples (unpublished results) were from machine-chopped (DORCH)
branches. These were piled outside for 5±10 days. Wood material was separated out
using a 1 cm mesh sieve and a fan. Digestibility was determined in one half of the
material immediately (BC) and in the rest after this had been kept in evacuated airtight
plastic bags for a further month (BC2).
2.2.2. Chemical composition
Contents in ash, CP, NDF, ADF, ADL and GE were determined in all the olive leaf
samples of the set (16 in all).
2.2.3. In vitro studies
Two in vitro techniques were used, the rumen liquor±pepsin procedure of Tilley and
Terry (1963) and the modi®ed (HCl 0.1 N) pepsin±cellulase method (AufreÁre and
Michalet-Doreau, 1988). In both techniques, all samples were milled through a 1 mm
screen and DM solubilities were determined in triplicate or duplicate. Rumen liquor was
obtained from two mature cannulated ewes kept on alfalfa and the cellulase used was
extracted from Trichoderma viridae (cellulase Onozuka R10, Medicine Department
Yakult Honsha Co., Ltd.).
From in vivo and in vitro digestibility data and chemical composition of samples,
Pearson's correlation coef®cients and corresponding regression equations between in vivo
digestibilities and the other parameters were obtained. Analysis of variance was
performed using the SAS software package (SAS, 1982) with the following treatments.
PROC MEANS (to calculate means, standard deviation and ranks), PROC CORR (to
calculate correlations between variables), PROC GLM (to calculate variances and
regression) and MEANS/TUKEY (to calculate signi®cant differences between means).
3. Results and discussion
3.1. Effects of different drying methods on nutritive value of olive leaf
Chemical composition and in vivo digestibility values of the different leaves studied
are shown in Table 2.
The variations observed in the dry material are a re¯ection of the different degrees
of drying of the material and of the variation in atmospheric relative humidity during
the year. Ash content is affected and increased by both wood content (Alibes et al.,
1982) and soil contamination (Delgado-PertõÂnÄez, 1994). As there are only small
differences in wood content in the materials used, the differences in ash might have
192
Treatmenta
Chemical compositionb
Digestibility (%)b
DM
(% FM)
OM
(% DM)
CP
(% DM)
NDF
(% DM)
ADF
(% DM)
ADL
(% DM)
GE
(Mcal kgÿ1 DM)
DMD
OMD
Air-dried branches
B24
B42
93.6
92.1
91.3
91.5
12.9
11.0
44.5
44.6
34.5
33.4
23.3
19.2
5.1
4.9
39.5 f
39.0 f
43.1 f
43.1 f
9.9 ef
14.0 f
Baled branches
BB9
BB10
93.4
94.1
90.4
90.9
10.5
10.6
40.5
39.9
26.5
25.5
16.7
16.3
4.8
4.8
40.1 f
39.9 f
44.8 f
44.8 f
0.0 de
7.6 ef
Chopped branches
BC1
BC24
BC42
BC12-F
80.2
89.4
90.6
86.4
93.5
92.5
92.3
90.9
9.5
11.3
11.0
12.3
53.2
51.4
54.2
62.6
39.6
42.5
44.1
53.7
19.7
24.9
26.6
30.5
4.8
5.1
5.1
5.1
33.0
28.6
20.5
15.7
36.0
31.6
23.4
17.7
a
e
e
d
c
e
e
d
c
CPD
ÿ14.1
ÿ6.0
ÿ14.6
ÿ3.6
c
cd
c
d
Treatment codes, see Table 1.
FM, fresh matter; DM, dry matter; OM, organic matter; CP, crude protein; NDF, neutral detergent ®bre; ADF, acid detergent ®bre; ADL, acid detergent lignin; GE,
gross energy; DMD, dry matter digestibility; OMD, organic matter digestibility; CPD, crude protein digestibility; means within columns with different letters are
different (P 0:05).
b
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 2
Chemical composition and in vivo digestibility values of olive leaf samples dried under different conditions
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
193
been the result of contamination with dust/soil. Thus, the baled samples (without residual
wood) showed a higher ash content. This could also explain its lower content in gross
energy.
The CP level ranged between 9.5 and 12.9%, accords with previous results (GoÂmezCabrera et al., 1992), although, considering the residual wood content, it is somewhat
higher compared with other studies (GoÂmez-Cabrera et al., 1982; Alibes et al., 1982).
According to FernaÂndez Escobar (unpublished data), the CP content can be as high as
9.5% in leaves, but only 4.4% in stems. The same author observed variations close to 2%
units in leaf CP content with season, leaf age and foliar fertilization.
There was a wide variation in the content of NDF (39.9±62.6%), ADF (25.5±53.7%)
and ADL (16.3±30.5%), which was greater than in the sample set studied by GoÂmezCabrera et al. (1992). This may have been due in part to the residual wood content, which
was practically zero in the samples used by those authors. The proportion of ®bre
increased with storage time, as was clearly seen in the chopped samples. NDF and ADF
values in the chopped samples far exceeded those in the leaf from dried branches (airdried and baled branches). GoÂmez-Cabrera et al. (1992) obtained a higher ADF content
and a lower digestibility in leaves dried separately than in leaves dried on the branches.
The most signi®cant factor for ADL seemed to be temperature. The fermented leaves,
BC12-F, showed the highest value (30.5%), although a high level was also reached in
leaves from chopped branches stored for more than 24 months.
DM and OM digestibility decreased signi®cantly with increasing storage time, both for
leaves from dried branches and for leaves from chopped branches (Fig. 1). So, the
material dried on the branches lost approximately 20% of its initial value during the ®rst 2
years of storage, even when dry and sheltered from the rain. Such effect could be due to
changes in moisture content of the product resulting from seasonal changes in relative
humidity. In winter, water may condense on the stored product, possibly causing fungal
activity.
The loss of digestibility was particularly marked in the case of CP, whose value fell to
around 10% in leaves from dried branches and was zero in leaves from chopped branches
(Fig. 2). It can be seen that in all cases, the leaves from chopped branches had negative
values, indicating true digestibilities close to zero. This loss of digestibility was also
observed by GoÂmez-Cabrera et al. (1992). Delgado-PertõÂnÄez et al. (1998) studying a part
of the material used in this study, observed high levels of the seco-iridoid glycoside
oleuropein in materials freeze-dried or vacuum oven dried, disappearing during storage,
with a simultaneous reduction in CP digestibility. Probably the protein from stored leaves
remained complexed with the phenolic material because both increased their proportion
into the water or acetone±water insoluble residues.
Considerable differences in the digestibility were found between leaves from dried
branches and leaves from chopped branches. The fact that the branches were chopped
partially wetted and piled immediately would create a humid atmosphere between
individual leaves, leading to microbial or fungal activity and heating, particularly in the
centre of the pile. The darker coloring of these materials supports this hypothesis. It is
noteworthy that the leaves from chopped branches with obvious signs of fermentation
(BC12-F), caused by rainwater, presented the lowest digestibility value (DMD 15:7).
However, the chopped samples used in the digestibility test almost immediately after
194
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Fig. 1. In vivo organic matter digestibility (OMD) of olive leaf samples dried under different conditions
(Treatment codes, see Table 1).
being obtained (BC) or kept in evacuated airtight plastic bags (BC2) presented values
close to lowest quality leaves obtained from dried branches (DMD 38:3 for BC, 37 for
BC2).
3.2. Prediction of digestibility from chemical composition and in vitro studies
3.2.1. In vitro studies
Mean values of DM disappearance measured by pepsin±cellulase and rumen liquor±
pepsin, together with their standard errors are given in Table 3. When the results of
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
195
Fig. 2. In vivo crude protein digestibility (CPD) of olive leaf samples dried under different conditions
(Treatment codes, see Table 1).
digestibility in vitro were compared with those obtained in vivo, it was observed that with
the 16 samples used, the in vitro values far exceed the corresponding in vivo value.
A possible cause of the high in vitro value is the presence of substances having a toxic
or inhibitory effect on microbiological activity, such as phenolic compounds. A greater
dilution in the test tubes than in the rumen liquor could reduce the negative effect as
suggested by Maeng et al. (1971) and Ololade and Mowa (1975) in the case of cereal
straw treated with NaOH.
Hydrolyzable or condensed tannins have not been detected in olive leaves and have
been ruled out as a possible antinutritional factor (Delgado-PertõÂnÄez et al., 1998).
196
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 3
Dry matter in vitro Tilley and Terry (IVDMD), pepsin±cellulase (CELDMD) and in vivo (DMD) digestibilities
(%) of olive leaf samples dried under different conditions
Treatmenta
IVDMDb
CELDMc
DMD
Fresh leaves
FL
48.3 0.48
57.8 0.04
53.9
Dried branches (oven-dried)
ODa
ODb
47.2 0.44
53.1 0.47
57.3 0.74
61.7 0.20
47.5
47.2
Dried branches (air-dried)
B3a
B3b
B24
B42
BOut3
48.8
56.6
49.4
55.0
52.9
0.33
0.36
0.26
0.18
0.16
47.3
48.5
39.5
39.0
39.4
Dried branches (bales)
BB9
BB10
54.9 0.10
53.2 0.14
55.3 0.06
59.0 0.25
40.1
39.9
Chopped branches
BC
BC2
BC1
BC24
BC42
BC12-F
42.5
41.0
43.8
43.4
43.7
31.9
38.3
37.0
33.0
28.6
20.5
15.7
0.27
0.29
0.23
0.27
0.11
0.76
0.64
1.31
0.81
0.27
0.30
60.8
61.2
49.4
53.8
57.8
47.4
43.5
43.5
40.5
39.8
29.4
0.03
0.33
0.15
0.28
0.42
0.26
a
Treatment codes, see Table 1.
Mean and standard error of means of triplicate determinations.
c
Mean and standard error of means of duplicate determinations.
b
Similarly, though olive leaves are characterized by a high concentration of oleuropein (a
seco-iridoid glycoside which gives the fruit its bitter taste, Kubo et al., 1995), there is no
suggestion that oleuropein might be toxic to the rumen micro¯ora and the highest
digestibilities were recorded for the samples with the greatest oleuropein content
(Delgado-PertõÂnÄez et al., 1998).
The differences were generally smaller in the samples whose in vivo digestibility had
been determined in the earlier tests (by GoÂmez-Cabrera et al., 1992), so that the sample
used to determine in vitro digestibility had been stored for a long time. This could
indicate that such samples do not remain stable at these storage times under the given
conditions. A speci®c case is that of the fresh leaf sample (FL). For the determination of
in vitro digestibility, this leaf was not in its original state, but had been subjected to
drying, as was the oven-dried leaf of the same year (ODa). In fact, the in vitro
digestibility values obtained with the two methods were similar for the two types of
sample (48.3% for IVDMD and 57.8% for CELDMD in FL, versus 47.2% for IVDMD
and 57.3% for CELDMD in ODa).
It was observed that the pepsin±cellulase technique gave higher values than the rumen
liquor method with the samples of highest in vivo digestibility and lower values with
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
197
those of lowest digestibility. This is in accord with the results of Jones and Hayward
(1973) and of McQueen and Van Soest (1975) indicating that in samples of low
digestibility the enzymatic preparations do not solubilize as much organic matter as do
rumen micro-organisms.
3.2.2. Prediction of in vivo digestibility
In vivo digestibility of olive leaves studied was related to various chemical parameters
or in vitro disappearance by correlation (Table 4) and linear regression analysis.
Gross energy content did not reach signi®cant levels of correlation (P > 0:05) with any
of the chemical parameters and could not be predicted with suf®cient precision from
these chemical analyses.
In contrast, the digestibility values presented signi®cant correlations with certain
of the parameters analyzed. In the case of DM, both in vitro and in vivo and of OM,
highly signi®cant negative correlations were obtained with NDF, ADF and ADL,
while the correlation with ash or CP was not signi®cant. All the digestibilities were
highly inter-correlated. There was greater precision in the estimation of in vivo
digestibility with the pepsin±cellulase technique (RSD 4:48 and 4.63 for DMD and
OMD respectively) than with the rumen liquor method (RSD 7:66 and 7.85 for
DMD and OMD, respectively). This is in agreement with the results of Jones and
Hayward (1975) and of AufreÁre and Michalet-Doreau (1988), but contradict the results
found by others (Clark and Beard, 1977; Terry et al., 1978; Mcleod and Minson, 1978;
Carlier et al., 1979; Gasa et al., 1989). Such differences could be due, as indicated by
Van der Koelen and Van Es (1973) and by Aerts et al. (1977), to variability in the activity
of the rumen liquor.
In all cases, the highest correlation with chemical parameters was given by NDF
(r ÿ0:91 and RSD 4:34 for DMD, r ÿ0:91 and RSD 4:60 for OMD), with the
same degree of precision as obtained for in vitro digestibility with cellulase (r 0:90 for
DMD and 0.91 for OMD). However, Jarrige and Thivend (1969), Van der Koelen and Van
Es (1973), and Aerts et al. (1977), studying a wide range of grasses and legumes, and
Gasa et al. (1989), working with a set of by-products from the canning industry, show that
OMD can be more accurately predicted by biological methods (the two-stage
fermentation procedure of Tilley and Terry, the pepsin±cellulase solubility bioassay or
the in situ nylon bag technique).
The in vivo digestibility of CP was also correlated with these chemical parameters,
although with lower precision, both NDF (r 0:68) and in vitro digestibility with
cellulase (r 0:78). The inherent problem in the estimation of true digestibility of CP
could partly explain this lower precision.
It seems clear that the material used was non-uniform under the conditions in which it
is stored prior to analysis, affecting the relationship between the values of in vivo
digestibility and the analytical results. This is seen in the values of in vitro digestibility
obtained in many cases after years of sample storage (as were those obtained by GoÂmezCabrera et al., 1992). It is considered preferable in this type of estimation (and
speci®cally that performed with rumen liquor) to analyze all in a single series because of
the variability in conditions between different series (Van Es and Van der Meer, 1980).
More important is the fact that in vitro digestibility method not allow to distinguish the
198
DM
ASH
CP
NDF
ADF
ADL
GE
IVDMD
CELDMD
DMD
OMD
CPD
a
ASH
CP
NDF
ADF
ADL
GE
IVDMD
CELDMD
DMD
OMD
0.27
ÿ1.11
ÿ0.17
ÿ0.06
ÿ0.42
0.35
0.16
ÿ0.13
ÿ0.14
0.23
ÿ0.14
ÿ0.05
0.08
0.44
ÿ0.04
0.11
0.16
0.10
0.52*
0.97***
0.87***
0.16
ÿ0.81***
ÿ0.94***
ÿ0.91***
ÿ0.91***
ÿ0.68**
0.93***
0.33
ÿ0.87***
ÿ0.96***
ÿ0.89***
ÿ0.89***
ÿ0.65**
0.47
ÿ0.83**
ÿ0.91***
ÿ0.86***
ÿ0.87***
ÿ0.61*
ÿ0.52*
ÿ1.27
ÿ1.06
ÿ1.08
ÿ1.02
0.87***
0.69**
0.71**
0.60*
0.90***
0.91***
0.78***
0.99***
0.78***
0.77***
**
0.66
0.18
ÿ0.12
ÿ0.19
ÿ0.09
ÿ0.34
0.42
0.23
ÿ0.11
ÿ0.11
0.08
DM, dry matter; CP, crude protein; NDF, neutral detergent ®bre; ADF, acid detergent ®bre; ADL, acid detergent lignin; GE, gross energy; IVDMD, dry matter in
vitro Tilley and Terry digestibility; CELDMD, dry matter pepsin±cellulase digestibility; DMD, dry matter in vivo digestibility; OMD, organic matter in vivo
digestibility; CPD, crude protein in vivo digestibility.
***
P < 0:001; P < 0:01; P < 0:05.
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 4
Pearson's correlation coef®cients between different parametersa of olive leaf samples dried under various conditions
199
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 5
Correlation coef®cients (R) and standard error of regression (RSD) between in vivo digestibility of dry matter
(DMD) or organic matter (OMD) and different chemical parameters or in vitro dry matter disappearance
Itema
NDF
ADF
ADL
IVDMD
CELDMD
All the samples except fresh leaf (n 15)
Sample set evaluated in vivo in this work (n 8)
DMD
DMD
OMD
OMD
R
RSD
R
RSD
R
RSD
R
RSD
0.92
0.91
0.87
0.74
0.92
3.97
4.23
4.83
6.70
3.84
0.91
0.91
0.88
0.76
0.92
4.32
4.50
5.08
6.90
4.05
0.93
0.93
0.89
0.91
0.93
3.73
3.84
4.66
4.30
3.73
0.95
0.94
0.90
0.92
0.95
3.65
3.73
4.83
4.37
3.62
a
NDF, neutral detergent ®bre; ADF, acid detergent ®bre; ADL, acid detergent lignin; IVDMD, dry matter in
vitro Tilley and Terry digestibility; CELDMD, dry matter pepsin±cellulase digestibility.
effect of drying for the analysis, since the fresh leaves (FL) consumed by the sheep are
dried prior to in vitro analysis. As a result, the latter leaf was similar to that which was
oven-dried before being fed to the sheep.
Reducing the data set by (a) excluding the fresh leaf sample (n 15) and (b)
using only the eight samples evaluated in vivo in this work, the precision of the estimation increased (Table 5). This was especially signi®cant in the case of in vitro OM
digestibility of Tilley and Terry (RSD 6:9, with 15 samples and RSD 4:4 with eight
samples).
When a multiple regression was performed among the OMD or DMD and the chemical
or biological parameters, the variables included in the model were digestibility with
cellulase and ash. However, the introduction of a second parameter did not improve
signi®cantly the accuracy of the prediction.
4. Conclusions
1. There were marked differences in nutritive value between leaves from dried branches
and leaves dried off the branch, prior to being chopped. In the latter, the loss of
nutritive value during drying was signi®cantly greater.
2. Storage of dry leaf in piles, sheltered from the rain but exposed to light and air, did not
stop processes that reduce the nutritive value of the leaves.
3. Storage of the samples under laboratory conditions, in systems not protected from air
and/or light, also resulted in reduced in vitro digestibility.
4. Neutral detergent ®bre was the best procedure, at a practical laboratory level, for
predicting in vivo digestibility. It explains 83% of variability in organic matter,
although only 46% of that in protein. The equations obtained with NDF and with the
set of 15 samples (all except the sample of fresh leaf) were
DMD 98:21 ÿ 1:31 NDF %=DM r 0:92; RSD 3:97
OMD 105:65 ÿ 1:39 NDF %=DM r 0:91; RSD 4:32
200
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Acknowledgements
The authors thank the ComisioÂn Interministerial de Ciencia y TecnologõÂa for the
provision of funding (Project GAN 89-0289), the Centro de InvestigacioÂn y FormacioÂn
Agraria de CoÂrdoba for the samples, and Jaime PelaÂez for his assistance in preparing the
manuscript.
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87 (2000) 187±201
Predicting the nutritive value of the olive leaf
(Olea europaea): digestibility and chemical
composition and in vitro studies
Manuel Delgado-PertõÂnÄeza,*,
Augusto GoÂmez-Cabrerab, Ana Garridob
a
Dpto. Ciencias Agroforestales, Universidad de Sevilla, Ctra. Utrera Km. 1, 41013 Sevilla, Spain
b
Dpto. ProduccioÂn Animal, Universidad de CoÂrdoba, Apdo. 3048, 14080 CoÂrdoba, Spain
Received 17 November 1998; received in revised form 11 August 1999; accepted 10 August 2000
Abstract
Eight tests of olive leaf digestibility were carried out with sheep. The olive leaves were collected using
different procedures (four samples of leaves from dried branches and another four of leaves removed
from chopped branches and dried) and stored for varying times. Marked differences in OMD were
observed between the two types of procedures and between different times of storage. The greatest loss
of nutritive value was in the chopped samples. In the leaves from dried branches, with storage periods
longer than 9 months, the digestible organic matter content ranged between 431 and 448 g kgÿ1 OM,
while in the chopped samples, the value ranged between 360 g kgÿ1 OM (in a sample stored for only 1
month) and 177 g kgÿ1 OM (in a sample with obvious signs of fermentation).
The sample set was used together with another, evaluated prior to this work, to study the prediction of
in vivo digestibility (OMD, DMD and CPD) from chemical and biological (dry matter disappearance
after in vitro incubation with rumen liquor±pepsin or pepsin±cellulase solutions) parameters. In the case
of OMD, the best predictions were obtained with the pepsin±cellulase method (r 0:91, RSD 4:6)
and NDF (r ÿ0:91, RSD 4:6). # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Olive leaves; Nutritive value; Sheep; In vitro studies; Predicting in vivo digestibility
1. Introduction
In Mediterranean grassland ruminant production systems, the nutritive input from
natural grazing is much reduced. In Syria, the supply was estimated as between 14 and
*
Corresponding autor. Tel.: 34-423-3669; fax: 34-423-2644.
E-mail address: [email protected] (M. Delgado-PertõÂnÄez).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 9 5 - 4
188
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
35%, depending on the characteristics of the year (Treacher et al., 1993). Various
management strategies are used to complement the supply. They include long-range
(trashumance) and short-range ¯ock migration, but above all, the use of crop residues, in
particular those of cereals (Flamant, 1992).
Apart from cereals, an important crop in the Mediterranean basin is the olive, with the
region producing 98% of the world total (approximately 11 million tons) (FAO, 1995).
The use of by-products of this crop (leaf and olive pressings) has been part of the farming
tradition of the region (Sansoucy et al., 1985). However, the specialization in production
that has led to monocultivation of the olive in wide areas has also meant the absence of
animals that could directly consume the plentiful prunings. The prunings must therefore
be moved away from where they are produced, involving operations to increase their
density to make their transport and storage easier (Delgado-PertõÂnÄez et al., 1994).
Various reviews of work on the use of the olive leaf (Sansoucy et al., 1985; Nefzaoui
and Zidani, 1987) demonstrate a wide range in its nutritive value. It is therefore
essential to identify the factors responsible for such diversity and to obtain equations
for the determination of nutritive value, so that each batch can be assigned a correct
value.
Previous work (GoÂmez-Cabrera et al., 1992) has shown a minor effect of variety,
season and year in the nutritive value, but a greater importance of conservation by
drying the leaves, mainly when they were not attached to the branches. The main aim
of this work was to predict the nutritive value of the olive leaf from various chemical and
biological parameters. As there is no standard system of collection and manipulation,
emphasis has been placed on obtaining very different materials. Therefore we
included leaves dried on the branches or obtained after chopping the branches, to
con®rm our previous results (GoÂmez-Cabrera et al., 1992) and sometimes by increasing
storage time.
2. Materials and methods
2.1. Effects of different drying methods on nutritive value of olive leaf
2.1.1. Collection and storage of olive leaves
Mature branches were collected during routine pruning of Picual and Hojiblanca
cultivars of olive trees (Olea europaea L.).
A set of eight samples of olive leaves were evaluated (Table 1).
2.1.1.1. Dried branches
2.1.1.1.1. Leaves from air-dried branches. Leaves dried on pruned branches in the air
and under cover for 3 months. The branches were then crushed with a tractor and the
leaf was coarsely separated by hand and stored for 21 months (sample B24) and 39
months (sample B42) piled on the ground. Prior to the digestibility tests, the leaf was
mechanically separated from residual wood (of which some 15±20% remained at the end
of the process).
Treatment
Fresh leaves
Dried branches (oven-dried)
Item
a
FL
a,b
ODa/ODb
a
Short description of treatment
Storage time
Leaves from the branches
2±5 days
Branches oven dried at 60±658C
16 h
Dried branches (air-dried)
BOut3
B3a/B3ba,b
B24
B42
Branches air-dried outdoors
Branches air-dried under cover
Leaves from B3b were separated and stored for another 21 months
Leaves from B3b were separated and stored for another 39 months
3
3
3
3
Dried branches (bales)
BB9
BB10
Branches baled 7 days after pruning and stored under cover
Branches baled 17 days after pruning and stored under cover
7 days 9 months
17 days 9 months
Chopped branches
BCa
BC2a
BC24
BC42
BC1
BC12-F
Branches chopped and leaves stored in piles outside, without rain
Material BC stored in evacuated airtight plastic bags for a further month
Branches chopped and leaves stored in piles under cover
Branches chopped and leaves stored in piles under cover
Branches chopped and leaves stored in piles outside, without rain
Branches chopped and wetted leaves stored in piles inside (slight fermentation)
5±10 days
5±10 days 1 month
24 months
42 months
10±15 days
12 months
a
b
Material evaluated previously by our team (GoÂmez-Cabrera et al., 1992 and unpublished data).
Two samples obtained in 2 years.
months
months
21 months
39 months
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 1
Olive leaves obtained after different methods of drying
189
190
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
2.1.1.1.2. Leaves from baled branches. Branches of less than 3 cm in diameter at the base
were baled into high-pressure bales (0:6 m 0:4 m 0:3 m) using a LERDA machine.
Some samples were baled 7 days (sample BB9) or 17 days (sample BB10) after pruning
and stored under cover for 9 months. The leaves were then separated from the branches by
hand. Both samples were wetted by rain before they were baled and placed under cover
with sample BB9 receiving somewhat more rain.
2.1.1.2. Partially wetted chopped branches. Pruned olive branches were chopped while
fresh with a DORCH chopper (Kapinka) and stored in piles under cover for 24 (BC24) and
42 (BC42) months, respectively. Some olive branches were also chopped in the same way
and stored under cover for 12 months. Occurrence of fermentation was evident in this sample
(BC12-F), which had been wetted by rain during chopping in the field and by infiltration
at the storage site. Olive branches were chopped 10±15 days after pruning and the product
was piled outside (without rainfall) a further 10±15 days (BC1). In all these samples, at the
end of storage, the leaf was mechanically separated from wood (10±15% of residual wood).
The mechanical separation of leaf and wood prior to the digestibility tests was carried
out using a winnowing machine (prototype of the Departamento de MecanizacioÂn
Agraria, University of CoÂrdoba).
2.1.2. In vivo digestibility determination and chemical composition
The digestibility of the eight samples of olive leaves after various conditions of storage
was measured using four or ®ve adult Segure~
na Merino sheep as described by GoÂmezCabrera et al. (1992). The amount of olive leaf offered to the sheep was 70 g DM kgÿ1
LW0.75 with 150 g of dehulled sun¯ower seed meal mixed with the leaf as a protein
supplement. The diet was fed twice a day (9 a.m. and 6 p.m.) at an intake level close to
maintenance. The adaptation period was 20 days (only 10 when two consecutive tests
were made) followed by 10 days of faeces collection. The animals were offered 20 g of a
mineral±vitamin corrector, STAY-DRY (18% Ca, 8% P, 10% Mg, 1.5% NaCl, 200 mg kgÿ1
Co, 400 mg kgÿ1 I, 3000 mg kgÿ1 Fe, 6000 mg kgÿ1 Mn, 4000 mg kgÿ1 Zn, 15 mg kgÿ1 Se,
300 UI kgÿ1 Vitamin A, 60 UI kgÿ1 Vitamin D3, 800 UI kgÿ1 Vitamin E).
In the leaf samples, gross energy (GE) content was measured in an adiabatic
calorimeter (Parr), dry matter (DM), ash and crude protein (CP) were measured according
to AOAC (1984) procedures and neutral detergent ®bre (NDF), acid detergent ®bre
(ADF) and acid detergent lignin (ADL) were determined by the method of Robertson and
Van Soest (1981). DM, ash and CP were determined for feed residues and faeces. Dry
matter digestibility (DMD), organic matter digestibility (OMD) and crude protein
digestibility (CPD) of the olive leaves were calculated by difference, taking into account
the following values already obtained for sun¯ower meal (g kgÿ1): DM 650, OM 700 and
CP 870 (GoÂmez-Cabrera et al., 1992).
2.2. Prediction of digestibility from chemical composition and in vitro studies
2.2.1. Collection and storage of olive leaves
Together with the formerly described eight samples of olive leaves, another eight
samples were analyzed (Table 1). Their digestibility and chemical parameters had been
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
191
determined earlier by our team. These samples had been kept initially in the laboratory in
airtight plastic bottles. Later, they were moved to a refrigerated room, where they were
stored at 108C for 4±6 years. Of these eight samples, six were analyzed by GoÂmezCabrera et al. (1992). Fresh leaves (sample FL), leaves oven dried at 60±658C for 16 h
(two samples obtained in 2 years, samples ODa and ODb), leaves dried on the branches
for 3 months under cover (two samples obtained in 2 years, samples B3a and B3b) and
leaves dried on the branches for 3 months outdoors (BOut3), exposed to rain and sun. The
other two samples (unpublished results) were from machine-chopped (DORCH)
branches. These were piled outside for 5±10 days. Wood material was separated out
using a 1 cm mesh sieve and a fan. Digestibility was determined in one half of the
material immediately (BC) and in the rest after this had been kept in evacuated airtight
plastic bags for a further month (BC2).
2.2.2. Chemical composition
Contents in ash, CP, NDF, ADF, ADL and GE were determined in all the olive leaf
samples of the set (16 in all).
2.2.3. In vitro studies
Two in vitro techniques were used, the rumen liquor±pepsin procedure of Tilley and
Terry (1963) and the modi®ed (HCl 0.1 N) pepsin±cellulase method (AufreÁre and
Michalet-Doreau, 1988). In both techniques, all samples were milled through a 1 mm
screen and DM solubilities were determined in triplicate or duplicate. Rumen liquor was
obtained from two mature cannulated ewes kept on alfalfa and the cellulase used was
extracted from Trichoderma viridae (cellulase Onozuka R10, Medicine Department
Yakult Honsha Co., Ltd.).
From in vivo and in vitro digestibility data and chemical composition of samples,
Pearson's correlation coef®cients and corresponding regression equations between in vivo
digestibilities and the other parameters were obtained. Analysis of variance was
performed using the SAS software package (SAS, 1982) with the following treatments.
PROC MEANS (to calculate means, standard deviation and ranks), PROC CORR (to
calculate correlations between variables), PROC GLM (to calculate variances and
regression) and MEANS/TUKEY (to calculate signi®cant differences between means).
3. Results and discussion
3.1. Effects of different drying methods on nutritive value of olive leaf
Chemical composition and in vivo digestibility values of the different leaves studied
are shown in Table 2.
The variations observed in the dry material are a re¯ection of the different degrees
of drying of the material and of the variation in atmospheric relative humidity during
the year. Ash content is affected and increased by both wood content (Alibes et al.,
1982) and soil contamination (Delgado-PertõÂnÄez, 1994). As there are only small
differences in wood content in the materials used, the differences in ash might have
192
Treatmenta
Chemical compositionb
Digestibility (%)b
DM
(% FM)
OM
(% DM)
CP
(% DM)
NDF
(% DM)
ADF
(% DM)
ADL
(% DM)
GE
(Mcal kgÿ1 DM)
DMD
OMD
Air-dried branches
B24
B42
93.6
92.1
91.3
91.5
12.9
11.0
44.5
44.6
34.5
33.4
23.3
19.2
5.1
4.9
39.5 f
39.0 f
43.1 f
43.1 f
9.9 ef
14.0 f
Baled branches
BB9
BB10
93.4
94.1
90.4
90.9
10.5
10.6
40.5
39.9
26.5
25.5
16.7
16.3
4.8
4.8
40.1 f
39.9 f
44.8 f
44.8 f
0.0 de
7.6 ef
Chopped branches
BC1
BC24
BC42
BC12-F
80.2
89.4
90.6
86.4
93.5
92.5
92.3
90.9
9.5
11.3
11.0
12.3
53.2
51.4
54.2
62.6
39.6
42.5
44.1
53.7
19.7
24.9
26.6
30.5
4.8
5.1
5.1
5.1
33.0
28.6
20.5
15.7
36.0
31.6
23.4
17.7
a
e
e
d
c
e
e
d
c
CPD
ÿ14.1
ÿ6.0
ÿ14.6
ÿ3.6
c
cd
c
d
Treatment codes, see Table 1.
FM, fresh matter; DM, dry matter; OM, organic matter; CP, crude protein; NDF, neutral detergent ®bre; ADF, acid detergent ®bre; ADL, acid detergent lignin; GE,
gross energy; DMD, dry matter digestibility; OMD, organic matter digestibility; CPD, crude protein digestibility; means within columns with different letters are
different (P 0:05).
b
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 2
Chemical composition and in vivo digestibility values of olive leaf samples dried under different conditions
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
193
been the result of contamination with dust/soil. Thus, the baled samples (without residual
wood) showed a higher ash content. This could also explain its lower content in gross
energy.
The CP level ranged between 9.5 and 12.9%, accords with previous results (GoÂmezCabrera et al., 1992), although, considering the residual wood content, it is somewhat
higher compared with other studies (GoÂmez-Cabrera et al., 1982; Alibes et al., 1982).
According to FernaÂndez Escobar (unpublished data), the CP content can be as high as
9.5% in leaves, but only 4.4% in stems. The same author observed variations close to 2%
units in leaf CP content with season, leaf age and foliar fertilization.
There was a wide variation in the content of NDF (39.9±62.6%), ADF (25.5±53.7%)
and ADL (16.3±30.5%), which was greater than in the sample set studied by GoÂmezCabrera et al. (1992). This may have been due in part to the residual wood content, which
was practically zero in the samples used by those authors. The proportion of ®bre
increased with storage time, as was clearly seen in the chopped samples. NDF and ADF
values in the chopped samples far exceeded those in the leaf from dried branches (airdried and baled branches). GoÂmez-Cabrera et al. (1992) obtained a higher ADF content
and a lower digestibility in leaves dried separately than in leaves dried on the branches.
The most signi®cant factor for ADL seemed to be temperature. The fermented leaves,
BC12-F, showed the highest value (30.5%), although a high level was also reached in
leaves from chopped branches stored for more than 24 months.
DM and OM digestibility decreased signi®cantly with increasing storage time, both for
leaves from dried branches and for leaves from chopped branches (Fig. 1). So, the
material dried on the branches lost approximately 20% of its initial value during the ®rst 2
years of storage, even when dry and sheltered from the rain. Such effect could be due to
changes in moisture content of the product resulting from seasonal changes in relative
humidity. In winter, water may condense on the stored product, possibly causing fungal
activity.
The loss of digestibility was particularly marked in the case of CP, whose value fell to
around 10% in leaves from dried branches and was zero in leaves from chopped branches
(Fig. 2). It can be seen that in all cases, the leaves from chopped branches had negative
values, indicating true digestibilities close to zero. This loss of digestibility was also
observed by GoÂmez-Cabrera et al. (1992). Delgado-PertõÂnÄez et al. (1998) studying a part
of the material used in this study, observed high levels of the seco-iridoid glycoside
oleuropein in materials freeze-dried or vacuum oven dried, disappearing during storage,
with a simultaneous reduction in CP digestibility. Probably the protein from stored leaves
remained complexed with the phenolic material because both increased their proportion
into the water or acetone±water insoluble residues.
Considerable differences in the digestibility were found between leaves from dried
branches and leaves from chopped branches. The fact that the branches were chopped
partially wetted and piled immediately would create a humid atmosphere between
individual leaves, leading to microbial or fungal activity and heating, particularly in the
centre of the pile. The darker coloring of these materials supports this hypothesis. It is
noteworthy that the leaves from chopped branches with obvious signs of fermentation
(BC12-F), caused by rainwater, presented the lowest digestibility value (DMD 15:7).
However, the chopped samples used in the digestibility test almost immediately after
194
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Fig. 1. In vivo organic matter digestibility (OMD) of olive leaf samples dried under different conditions
(Treatment codes, see Table 1).
being obtained (BC) or kept in evacuated airtight plastic bags (BC2) presented values
close to lowest quality leaves obtained from dried branches (DMD 38:3 for BC, 37 for
BC2).
3.2. Prediction of digestibility from chemical composition and in vitro studies
3.2.1. In vitro studies
Mean values of DM disappearance measured by pepsin±cellulase and rumen liquor±
pepsin, together with their standard errors are given in Table 3. When the results of
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
195
Fig. 2. In vivo crude protein digestibility (CPD) of olive leaf samples dried under different conditions
(Treatment codes, see Table 1).
digestibility in vitro were compared with those obtained in vivo, it was observed that with
the 16 samples used, the in vitro values far exceed the corresponding in vivo value.
A possible cause of the high in vitro value is the presence of substances having a toxic
or inhibitory effect on microbiological activity, such as phenolic compounds. A greater
dilution in the test tubes than in the rumen liquor could reduce the negative effect as
suggested by Maeng et al. (1971) and Ololade and Mowa (1975) in the case of cereal
straw treated with NaOH.
Hydrolyzable or condensed tannins have not been detected in olive leaves and have
been ruled out as a possible antinutritional factor (Delgado-PertõÂnÄez et al., 1998).
196
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 3
Dry matter in vitro Tilley and Terry (IVDMD), pepsin±cellulase (CELDMD) and in vivo (DMD) digestibilities
(%) of olive leaf samples dried under different conditions
Treatmenta
IVDMDb
CELDMc
DMD
Fresh leaves
FL
48.3 0.48
57.8 0.04
53.9
Dried branches (oven-dried)
ODa
ODb
47.2 0.44
53.1 0.47
57.3 0.74
61.7 0.20
47.5
47.2
Dried branches (air-dried)
B3a
B3b
B24
B42
BOut3
48.8
56.6
49.4
55.0
52.9
0.33
0.36
0.26
0.18
0.16
47.3
48.5
39.5
39.0
39.4
Dried branches (bales)
BB9
BB10
54.9 0.10
53.2 0.14
55.3 0.06
59.0 0.25
40.1
39.9
Chopped branches
BC
BC2
BC1
BC24
BC42
BC12-F
42.5
41.0
43.8
43.4
43.7
31.9
38.3
37.0
33.0
28.6
20.5
15.7
0.27
0.29
0.23
0.27
0.11
0.76
0.64
1.31
0.81
0.27
0.30
60.8
61.2
49.4
53.8
57.8
47.4
43.5
43.5
40.5
39.8
29.4
0.03
0.33
0.15
0.28
0.42
0.26
a
Treatment codes, see Table 1.
Mean and standard error of means of triplicate determinations.
c
Mean and standard error of means of duplicate determinations.
b
Similarly, though olive leaves are characterized by a high concentration of oleuropein (a
seco-iridoid glycoside which gives the fruit its bitter taste, Kubo et al., 1995), there is no
suggestion that oleuropein might be toxic to the rumen micro¯ora and the highest
digestibilities were recorded for the samples with the greatest oleuropein content
(Delgado-PertõÂnÄez et al., 1998).
The differences were generally smaller in the samples whose in vivo digestibility had
been determined in the earlier tests (by GoÂmez-Cabrera et al., 1992), so that the sample
used to determine in vitro digestibility had been stored for a long time. This could
indicate that such samples do not remain stable at these storage times under the given
conditions. A speci®c case is that of the fresh leaf sample (FL). For the determination of
in vitro digestibility, this leaf was not in its original state, but had been subjected to
drying, as was the oven-dried leaf of the same year (ODa). In fact, the in vitro
digestibility values obtained with the two methods were similar for the two types of
sample (48.3% for IVDMD and 57.8% for CELDMD in FL, versus 47.2% for IVDMD
and 57.3% for CELDMD in ODa).
It was observed that the pepsin±cellulase technique gave higher values than the rumen
liquor method with the samples of highest in vivo digestibility and lower values with
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
197
those of lowest digestibility. This is in accord with the results of Jones and Hayward
(1973) and of McQueen and Van Soest (1975) indicating that in samples of low
digestibility the enzymatic preparations do not solubilize as much organic matter as do
rumen micro-organisms.
3.2.2. Prediction of in vivo digestibility
In vivo digestibility of olive leaves studied was related to various chemical parameters
or in vitro disappearance by correlation (Table 4) and linear regression analysis.
Gross energy content did not reach signi®cant levels of correlation (P > 0:05) with any
of the chemical parameters and could not be predicted with suf®cient precision from
these chemical analyses.
In contrast, the digestibility values presented signi®cant correlations with certain
of the parameters analyzed. In the case of DM, both in vitro and in vivo and of OM,
highly signi®cant negative correlations were obtained with NDF, ADF and ADL,
while the correlation with ash or CP was not signi®cant. All the digestibilities were
highly inter-correlated. There was greater precision in the estimation of in vivo
digestibility with the pepsin±cellulase technique (RSD 4:48 and 4.63 for DMD and
OMD respectively) than with the rumen liquor method (RSD 7:66 and 7.85 for
DMD and OMD, respectively). This is in agreement with the results of Jones and
Hayward (1975) and of AufreÁre and Michalet-Doreau (1988), but contradict the results
found by others (Clark and Beard, 1977; Terry et al., 1978; Mcleod and Minson, 1978;
Carlier et al., 1979; Gasa et al., 1989). Such differences could be due, as indicated by
Van der Koelen and Van Es (1973) and by Aerts et al. (1977), to variability in the activity
of the rumen liquor.
In all cases, the highest correlation with chemical parameters was given by NDF
(r ÿ0:91 and RSD 4:34 for DMD, r ÿ0:91 and RSD 4:60 for OMD), with the
same degree of precision as obtained for in vitro digestibility with cellulase (r 0:90 for
DMD and 0.91 for OMD). However, Jarrige and Thivend (1969), Van der Koelen and Van
Es (1973), and Aerts et al. (1977), studying a wide range of grasses and legumes, and
Gasa et al. (1989), working with a set of by-products from the canning industry, show that
OMD can be more accurately predicted by biological methods (the two-stage
fermentation procedure of Tilley and Terry, the pepsin±cellulase solubility bioassay or
the in situ nylon bag technique).
The in vivo digestibility of CP was also correlated with these chemical parameters,
although with lower precision, both NDF (r 0:68) and in vitro digestibility with
cellulase (r 0:78). The inherent problem in the estimation of true digestibility of CP
could partly explain this lower precision.
It seems clear that the material used was non-uniform under the conditions in which it
is stored prior to analysis, affecting the relationship between the values of in vivo
digestibility and the analytical results. This is seen in the values of in vitro digestibility
obtained in many cases after years of sample storage (as were those obtained by GoÂmezCabrera et al., 1992). It is considered preferable in this type of estimation (and
speci®cally that performed with rumen liquor) to analyze all in a single series because of
the variability in conditions between different series (Van Es and Van der Meer, 1980).
More important is the fact that in vitro digestibility method not allow to distinguish the
198
DM
ASH
CP
NDF
ADF
ADL
GE
IVDMD
CELDMD
DMD
OMD
CPD
a
ASH
CP
NDF
ADF
ADL
GE
IVDMD
CELDMD
DMD
OMD
0.27
ÿ1.11
ÿ0.17
ÿ0.06
ÿ0.42
0.35
0.16
ÿ0.13
ÿ0.14
0.23
ÿ0.14
ÿ0.05
0.08
0.44
ÿ0.04
0.11
0.16
0.10
0.52*
0.97***
0.87***
0.16
ÿ0.81***
ÿ0.94***
ÿ0.91***
ÿ0.91***
ÿ0.68**
0.93***
0.33
ÿ0.87***
ÿ0.96***
ÿ0.89***
ÿ0.89***
ÿ0.65**
0.47
ÿ0.83**
ÿ0.91***
ÿ0.86***
ÿ0.87***
ÿ0.61*
ÿ0.52*
ÿ1.27
ÿ1.06
ÿ1.08
ÿ1.02
0.87***
0.69**
0.71**
0.60*
0.90***
0.91***
0.78***
0.99***
0.78***
0.77***
**
0.66
0.18
ÿ0.12
ÿ0.19
ÿ0.09
ÿ0.34
0.42
0.23
ÿ0.11
ÿ0.11
0.08
DM, dry matter; CP, crude protein; NDF, neutral detergent ®bre; ADF, acid detergent ®bre; ADL, acid detergent lignin; GE, gross energy; IVDMD, dry matter in
vitro Tilley and Terry digestibility; CELDMD, dry matter pepsin±cellulase digestibility; DMD, dry matter in vivo digestibility; OMD, organic matter in vivo
digestibility; CPD, crude protein in vivo digestibility.
***
P < 0:001; P < 0:01; P < 0:05.
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 4
Pearson's correlation coef®cients between different parametersa of olive leaf samples dried under various conditions
199
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Table 5
Correlation coef®cients (R) and standard error of regression (RSD) between in vivo digestibility of dry matter
(DMD) or organic matter (OMD) and different chemical parameters or in vitro dry matter disappearance
Itema
NDF
ADF
ADL
IVDMD
CELDMD
All the samples except fresh leaf (n 15)
Sample set evaluated in vivo in this work (n 8)
DMD
DMD
OMD
OMD
R
RSD
R
RSD
R
RSD
R
RSD
0.92
0.91
0.87
0.74
0.92
3.97
4.23
4.83
6.70
3.84
0.91
0.91
0.88
0.76
0.92
4.32
4.50
5.08
6.90
4.05
0.93
0.93
0.89
0.91
0.93
3.73
3.84
4.66
4.30
3.73
0.95
0.94
0.90
0.92
0.95
3.65
3.73
4.83
4.37
3.62
a
NDF, neutral detergent ®bre; ADF, acid detergent ®bre; ADL, acid detergent lignin; IVDMD, dry matter in
vitro Tilley and Terry digestibility; CELDMD, dry matter pepsin±cellulase digestibility.
effect of drying for the analysis, since the fresh leaves (FL) consumed by the sheep are
dried prior to in vitro analysis. As a result, the latter leaf was similar to that which was
oven-dried before being fed to the sheep.
Reducing the data set by (a) excluding the fresh leaf sample (n 15) and (b)
using only the eight samples evaluated in vivo in this work, the precision of the estimation increased (Table 5). This was especially signi®cant in the case of in vitro OM
digestibility of Tilley and Terry (RSD 6:9, with 15 samples and RSD 4:4 with eight
samples).
When a multiple regression was performed among the OMD or DMD and the chemical
or biological parameters, the variables included in the model were digestibility with
cellulase and ash. However, the introduction of a second parameter did not improve
signi®cantly the accuracy of the prediction.
4. Conclusions
1. There were marked differences in nutritive value between leaves from dried branches
and leaves dried off the branch, prior to being chopped. In the latter, the loss of
nutritive value during drying was signi®cantly greater.
2. Storage of dry leaf in piles, sheltered from the rain but exposed to light and air, did not
stop processes that reduce the nutritive value of the leaves.
3. Storage of the samples under laboratory conditions, in systems not protected from air
and/or light, also resulted in reduced in vitro digestibility.
4. Neutral detergent ®bre was the best procedure, at a practical laboratory level, for
predicting in vivo digestibility. It explains 83% of variability in organic matter,
although only 46% of that in protein. The equations obtained with NDF and with the
set of 15 samples (all except the sample of fresh leaf) were
DMD 98:21 ÿ 1:31 NDF %=DM r 0:92; RSD 3:97
OMD 105:65 ÿ 1:39 NDF %=DM r 0:91; RSD 4:32
200
M. Delgado-PertõÂnÄez et al. / Animal Feed Science and Technology 87 (2000) 187±201
Acknowledgements
The authors thank the ComisioÂn Interministerial de Ciencia y TecnologõÂa for the
provision of funding (Project GAN 89-0289), the Centro de InvestigacioÂn y FormacioÂn
Agraria de CoÂrdoba for the samples, and Jaime PelaÂez for his assistance in preparing the
manuscript.
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