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Quercetin and flaxseed included in the diet of fattening lambs: effects on immune response, stress during road
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transport and ruminal acidosis
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J. Benavides, M. Martínez-Valladares, M.L. Tejido, F.J. Giráldez, R. Bodas1, N. Prieto, V. Pérez, S. Andrés†
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Instituto de Ganadería de Montaña (CSIC-Universidad de León). Finca Marzanas. E-24346 Grulleros, León (Spain).
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Corresponding author: Sonia Andrés, Instituto de Ganadería de Montaña (CSIC-ULE). Finca Marzanas. E-24346
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Grulleros, León (Spain). Tel. +34 987 317 156 Fax +34 987 317 161 E-mail: sonia.andres@eae.csic.es
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1Present
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Agricultura y Ganadería, Junta de Castilla y León. Finca Zamadueñas. Ctra. Burgos, km 119. 47071 Valladolid,
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Spain.
address: Instituto Tecnológico Agrario – Subdirección de Investigación y Tecnología. Consejería de
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Abstract
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Thirty two lambs were divided in 4 groups with 2 replicates each (8 batches in total) according to their body weights.
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The lambs were fed a total mixed ration (TMR) formulated either with palm oil (CTRL; 2 replicates, 4 animals per
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replicate; 34 g palm oil kg-1 TMR) or flaxseed (FS, 2 replicates, 4 animals per replicate; 85 g flaxseed kg-1 TMR).
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Four more batches were fed the same TMRs but enriched with quercetin (QCT, 2 replicates, 4 animals per replicate,
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34 g palm oil plus 2 g quercetin kg-1 TMR; FS-QCT, 2 replicates, 4 animals per replicate, 85 g flaxseed plus 2 g
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quercetin kg-1 TMR). Three weeks after starting with the experimental diets, the animals were immunised by a
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subcutaneous injection of ovalbumin, and blood samples were collected at days 0, 4, 9, 14 and 17 post-immunization
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to measure antibodies against ovalbumin. Afterwards, all of the lambs were subjected to a 4-h transportation-stress
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period to study the evolution of haematological and biochemical parameters during road transport and, finally,
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slaughtered. Mean Ova-specific IgG titres were significantly lower in the lambs fed FS on days 14 and 17 when
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compared to CTRL group (FS×DAY, P=0.033). Additionally, FS reduced white blood cells counts and tissue damage
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(creatine phosphokinase, P<0.05) during road transport. Consequently, flaxseed showed some immunological
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properties and protection against tissue damage during road transport. Regarding ruminal acidosis, both quercetin and
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flaxseed seemed to be adequate to reduce the level of parakeratosis.
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Keywords: immune response, acidosis and oxidative stress, quercetin, flaxseed, lamb
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Introduction
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Several studies performed during the last two decades have evidenced a strong relationship between nutrition and
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immune response (Carroll and Forsberg, 2007). Lately, some substances such as polyunsaturated fatty acids (PUFAs)
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or phenolic compounds which are being included in the diet of animals in order to improve meat or milk quality
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attributes also have demonstrated immunomodulator properties.
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Most of these studies have been performed in vitro or in small monogastrics such as rodents or rabbits (Wallace
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et al., 2001; Arita et al., 2005). However, in spite of its relevance, there is a lack of experiments which have been
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focused on the effects of nutrition on the immune response in livestock animals. Moreover, the few studies which can
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be found in the literature have described different or contradictory effects attending to physiological, nutritional or
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environmental conditions. For example, n−3 PUFAs in fish oil seem to be related to a lower lymphocyte proliferation
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(anti-inflammatory effect) in periparturient cows (Lessard et al., 2003) whereas under high temperature conditions
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n−3 PUFAs seem to promote a pro-inflammatory response (Caroprese et al., 2009). These contradictory results might
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be explained by different antioxidant requirements promoted by stress conditions, since these PUFAs are more
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sensitive to oxidative stress than saturated fats (Carroll and Forsberg, 2007). Additionally, it must be considered that
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cells of the immune system are highly reactive. Consequently immune cells, which have a high percentage of PUFAs
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in their plasma membranes, are dependent particularly on high levels of antioxidants to protect them from cell and
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membrane damage promoted by reactive oxygen species (ROS) (Nockels, 1996).
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Although the body produces a number of endogenous antioxidants as a defense mechanism against ROS, under
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conditions of high oxidative stress (e.g., PUFAs rich diets, road transport, etc.) the ability to eliminate ROS can be
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exceeded. Then, dietary sources of antioxidants can be beneficial in reducing the deleterious effects of ROS on the
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highly reactive cells of the immune system (Carrol and Forsberg, 2007; Hamer, 2007). In this sense, plants provide a
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potential source of phytochemicals with antioxidant and immunomodulatory properties such as phenolic compounds,
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so the interest in these substances has increased in the last decade in human and animal medicine, as they are
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perceived by the consumer as a natural approach to treat diseases and preserve food safety. In this sense quercetin is a
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flavonoid with demonstrated antioxidant, antiviral and anticarcinogenic properties in monogastrics (Nair et al., 2002).
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However, there is little information about the effectiveness of this compound when included in the diet of ruminants.
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The objective of this study was to test, on the one hand, whether flaxseed (rich in PUFAs) and quercetin allow
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modifying immune response (antibody, and haematological parameters) in fattening lambs when included in the diet.
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Also, changes related to ruminal/metabolic acidosis and the fatigue of animals when stressed by road transport were
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measured.
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Material and Methods
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Animals and diets
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Two weeks before the commencement of the trial, 32 male Merino lambs were treated with Ivermectin (Ivomec,
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Merial Labs, Barcelona, Spain) and vaccinated against enterotoxaemia (Miloxan, Merial Labs, Barcelona, Spain).
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After stratification on the basis of body weight (average body weight (BW), 15.5 ± 2.12 kg), the lambs were
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allocated randomly to 4 different groups (2 replicates per dietary treatment, 8 batches in total). All of the groups were
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fed their corresponding total mixed ration (TMR) as described below: two replicates of the control group (CTRL, 4
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animals per replicate; 34 g palm oil kg-1 TMR), two replicates fed ground flaxseed (FS, 4 animals per replicate; 85 g
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flaxseed kg-1 of TMR), two replicates fed control diet plus quercetin (Shaanxi Sciphar Biotechnology Co., Ltd, Xi'an,
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China) (QCT, 4 animals per replicate; 34 g palm oil plus 2 g quercetin kg-1 TMR), and two replicates fed ground
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flaxseed plus quercetin (FS-QCT, 4 animals per replicate; 85 g flaxseed plus 2 g quercetin kg-1 TMR). The four
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TMRs were formulated to be isoenergetic and isoproteic. The ingredients and chemical composition of TMR are
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shown in Table 1. All handling practises followed the recommendations of the European Council Directive
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86/609/EEC for the protection of animals used for experimental and other scientific purposes and all the animals were
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able to see and hear other animals.
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[INSERT TABLE 1 NEAR HERE, PLEASE]
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After 7 days of adaptation to the basal diet, all of the lambs were fed the corresponding TMR (CTRL, FS, QCT
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and FS-QCT groups) ad libitum during the experimental period (at least 5 weeks, until the animals reached the
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intended BW, approx. 25 kg). The TMR was weighed and supplied ad libitum at 9:00 a.m. every day, and fresh
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drinking water was always available. Samples of feed offered and orts (approximately 20% of total offered) were
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taken daily, pooled to an individual composite sample every week, oven-dried at 55°C for at least 72 h (to constant
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weight), ground to pass through a 1-mm screen using a Willey mill (Arthur H. Thomas, Philadelphia, PA), and stored
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until analyses. Only one computation of DM intake, average daily gain and feed conversion ratio was computed for
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each batch of animals.
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OVA immunization and anti-OVA ELISA
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Three weeks after beginning the test, the animals were immunised by a subcutaneous injection of 1 mg chicken egg
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albumin (day 0; ovalbumin, OVA) (Sigma, Madrid, Spain) emulsified in Montanide ISA 206 VF (Seppic, Paris,
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France) at 1 mg/ml.
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For the detection of the antibodies against OVA, slightly modified methods explained in Wattegedera et al.
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(2008) were followed. Briefly, high affinity-binding 96-well flat-bottom ELISA plates (Thermo Fisher Scientific,
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Madrid, Spain) were coated overnight at 4 °C with 1 μg/ml OVA diluted in carbonate-bicarbonate buffer. As a
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positive control, and in order to standardize the absorbance obtained in the different plates, 100 μl of a 1:1000
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dilution of a monoclonal anti-chicken egg albumin (Clone OVA-14, Sigma, Madrid, Spain) was used in all the plates.
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Aliquots from the same serum obtained from a non-immunised sheep diluted 1:100 were used as a negative control in
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all the plates. Plates were incubated with 100 μl test sera, diluted 1:100, in duplicate; horseradish peroxidase
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conjugate protein G (BioRad Laboratories, Richmond, Ca, USA) at 1:1500 dilution was added as secondary Ab and
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ABTS, 2,2'-azino-di-3-ethylbenzthiazoline sulfonic acid (Boheringer Mannheim GmbH, Mannheim, Germany) was
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added to each well as enzymatic substrate. The absorbance values were measured spectrophotometrically using an
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ELX800 ELISA reader (Bio-Tek Instruments, Winooski, USA) at 450 nm. Results were expressed as a quotient of
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the mean optical density (O.D.) of each serum and the mean O.D. of the positive control sample in each plate.
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Blood samples were collected at days 0, 4, 9, 14 and 17 post-immunization by jugular venipuncture into non-
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heparinised vacutainers (Becton Dickinson, Madrid, Spain). They were allowed to clot and the serum was stored at
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−20 °C until used for serological analyses.
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Stress induction
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Road transportation was used as a quantifiable source of stress (Averós et al., 2009). After having collected the last
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blood sample for anti-ova ELISA analysis (day 17 after ovalbumin injection, that is to say 5 weeks after having
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started with the experimental diets), all of the animals were subjected to a 4 h-transportation period, at a space
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allowance of 0.30 m2 per animal in a truck with a non-slip metal floor that was covered with straw bedding. Before
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the road transport (PRE) and immediately after the 4 h-transportation period (POST) blood samples were collected
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by jugular venipuncture into two 10 ml vacutainers containing either no anticoagulant or EDTA. Biochemical
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(cortisol, glucose, creatine phosphokinase (CPK), lactate, and lactate dehydrogenase (LDH)) and haematological
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analyses were performed as explained by López-Campos et al. (2010).
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Rumen and liver sampling
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Finally, all of the lambs were weighed (24.8 ± 1.05 kg), stunned, slaughtered by exsanguinations from the jugular
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vein, eviscerated and skinned. Liver was weighed and a piece of liver was cut and kept at -80 ºC until thiobarbituric
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acid reactive substances (TBARS) analysis (Bodas et al., 2012). Rumen was weighed emptied, rumen fluid was
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strained through 4 layers of cheesecloth and pH was determined immediately. Then, two square samples of 3×2 cm
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from different locations of ruminal wall (posterior dorsal and anterior ventral areas) were placed into histological
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cassettes, washed under tap water and then fixed by immersion in 10% buffered formalin for one week. Digital gross
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pictures of the ruminal mucosa were taken to measure the colour of the ruminal epithelium as an indicator of the
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degree of keratinisation. Pictures from all the samples (image size 6mp) were taken with the same camera (Lens:
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Nikkor 105 mm f/2.8 D AF Micro. Camera: Nikon D100; Nikon Europe BV, Badhoevedorp, The Netherlands),
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where focus, lens aperture and exposure time were set to manual, adjusted at the first sample and then kept
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unchanged until all the pictures were taken. During the whole process, the camera was attached to a stand with
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incandescent bulbs for illumination; the position of the camera and the bulbs remained unchanged for all the pictures.
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There was a scale on a known size in all the pictures so measurements could be carried out.
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Once downloaded into the computer, a 1×1 cm representative area of the region of interest located over a
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representative area of each sample of ruminal epithelium was selected and the ruminal papillae within this area were
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counted. The picture was then converted to grey values and the mean grey value (ranging from 0 to 255, where 0 is
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black and 255 is white) of the selected region of interest was measured. A mean value was obtained for the two areas
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studied from each rumen. All these operations were performed with the ImageJ 1.43 software (Rasband, W.S.,
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ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA).
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Statistical analyses
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Individual animal data of rumen (including number of papillae and colour) and liver characteristics, and batch data of
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dry matter intake (DMI), average daily gain (ADG) and feed to gain ratio (FC), were subjected to a two way analysis
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of variance, using the GLM procedure of SAS (SAS Inst. Inc.) according to the following model:
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yijk = μ + FSi + QCTj + (FS×QCT)ij + εijk
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where yijk is the dependent variable, μ is the overall mean, FS is the effect of flaxseed addition, QCT is the effect
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of quercetin addition, FS×QCT is the effect of the interaction between quercetin and flaxseed, and ε ijk is the residual
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error. Least square means were generated and separated using the PDIFF option of SAS for main or interactive
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effects, with the level of significance being determined at P<0.05.
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Data of antibody production and the evolution of biochemical parameters and white blood cells during road
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transport were analysed as a repeated measures design using the MIXED procedure of SAS (SAS Inst. Inc.), with
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flaxseed and quercetin addition and day as fixed effects and animal within treatment as the experimental unit,
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according to the following model:
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yijklm = μ + FSi + QCTj + (FS×QCT)ij + TIMEk + FS×TIMEik + QCT×TIMEjk + FS×QCT×TIMEijk +
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ANIMAL(FS×QCT)ijl + εijklm
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where yijklm is the dependent variable, μ is the overall mean, FS is the effect of flaxseed, QCT is the effect of
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quercetin addition, TIME is the effect of the time (sampling day or hour), ANIMAL(FS×QCT) the animal within
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treatments as the experimental unit and εijklm is the residual error. Different covariance matrixes were evaluated on the
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basis of Schwarz´s Bayesian information model fit criteria. Least square means were generated and separated using
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the PDIFF option of SAS for main or interactive effects, with the level of significance being determined at P<0.05.
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For reasons of clarity reasons, tables only show the diet interaction; diet by day interactions are mentioned in the text
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when they were or tended to be statistically significant (P<0.05 or P<0.10, respectively).
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Results
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Intake, average daily gain and feed-to-gain ratio
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Table 2 summarizes the production parameters of the lambs. There were no differences between the groups in terms
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of TMR intake. However, a trend toward significantly higher values (P=0.077) in the ADG was observed for the
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lambs being fed flaxseed (FS and FS-QCT groups).
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[INSERT TABLE 2 NEAR HERE, PLEASE]
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Anti-OVA antibody response
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Anti-Ova IgG was detectable in the sera of all lambs from day 9 post immunisation until the end of the experiment
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with values increasing with time (Table 3). Mean Ova-specific IgG titres, measured as a quotient, in the flaxseed-
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supplemented groups (FS and FS-QCR) tended to be lower at day 9 post immunization and were significantly lower
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(FS×DAY, P=0.033) on days 14 and 17 when compared to CTRL group.
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[INSERT TABLE 3 NEAR HERE, PLEASE]
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Evolution of haematological and biochemical parameters during road transport
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As expected, no effect of the diet on cortisol or glucose levels was observed before (PRE) or after (POST) road
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transport (data not shown). However, a significant effect of flaxseed (P=0.027) was observed, animals being fed
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flaxseed (FS and FS-QCT groups) presenting lower levels of CPK in blood before (PRE, 186 and 222 U/L,
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respectively) or after road transport (POST, 218 and 224 U/L, respectively) when compared to the CTRL and QCT
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lambs (PRE, 216 and 234 U/L, respectively; POST, 253 and 284 U/L, respectively). The rest of parameters [LDH or
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lactate], which are indicators of tissue damage during road transport (Jiang and Ames, 2003), showed no differences
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among groups (data not shown).
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Table 4 summarizes the changes in white blood cell (WBC) counts in the lambs as a consequence of road
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transport. Before road transport (PRE) the lambs being fed flaxseed (FS and FS-QCT groups) presented lower levels
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of total WBC counts when compared to CTRL and QCT groups, whereas quercetin did not seem to have an effect on
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this parameter. After road transport (POST) a significant decrease in total WBC counts was detected in all of the
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groups (P<0.001); however, the lowest levels still occurred in the FS and FS-QCT groups. Regarding the differential
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counts of WBCs, lymphocytes were lower in the lambs being fed flaxseed (FS and FS-QCT groups) before (PRE)
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and after road transport (POST), whereas granulocytes and monocytes counts did not seem to be affected either by
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dietary flaxseed or quercetin.
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[INSERT TABLE 4 NEAR HERE, PLEASE]
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Rumen and liver characteristics
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Table 5 summarizes the results regarding ruminal and liver characteristics obtained after slaughter. All of the groups
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presented similar ruminal pH values, with no significant differences being observed among them (P>0.05). However,
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the lambs from the CTRL group showed a darker ruminal epithelium and a heavier empty rumen weight when
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compared to the animals from the other groups (P<0.05). There were no differences in the number of papillae
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between the different groups (mean value of all the animals, 88.5±3.51 papillae/cm2). A trend towards significantly
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higher liver weights was observed in the animals being fed quercetin (QCT and FS-QCT lambs) when compared to
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the CTRL and FS groups (P=0.062), whereas the lambs being fed flaxseed (FS and FS-QCT) showed a trend towards
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significantly lower levels in liver TBARS values (P=0.060).
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[INSERT TABLE 5 NEAR HERE, PLEASE]
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Discussion
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Intake, average daily gain and feed-to-gain ratio
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Although all of the animals were fed ad libitum, no significant differences were observed in feed intake, ADG or FC
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(Table 2), as diets were isoenergetic and isoproteic.
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Anti-OVA antibodies response
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Quercetin had no effect on antibody production (QCT, P=0.586, Table 3), which is in agreement with the data
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previously reported by other authors (Exon et al., 1998; Sforcin et al., 2005). Recently it has been demonstrated the
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low bioavailability of quercetin aglycone in cows after intraruminal (or oral) application, unlike in monogastric
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species (Berger et al., 2012), which might have been the cause of the lack of clear systemic effects in the present
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study.
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However, those animals supplemented with FS (FS and FS-QCT groups) showed lower anti-OVA antibodies titers
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than CTRL and QCT groups (FS, P=0.012, Table 3). Supplementation of diets with n−3 fatty acids (such as those
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provided by flaxseed) have been shown to decrease the inflammatory response (both serum antibodies and local
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subcutaneous inflammation) of type I hypersensitivity reactions involving OVA sensitization in murine models
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(Hogenkamp et al., 2011; de Matos et al., 2012). This might be due to the fact that the fatty acid composition of
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lymphocytes and other immune cells is modified according to the fatty acid composition of the diet, which alters the
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capacity of those cells to produce eicosanoids involved in immunoregulation (Calder, 2007). Omega-3 fatty acids
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such as α-linolenic acid (C18:3n−3) are metabolized to eicosapentaenoic acid (EPA), which leads to an anti-
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inflammatory environment, thus reducing serum antibodies through inhibition of the secretion of several pro-
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inflammatory cytokines, such as IL-2 or TNF-α (Mullen et al., 2010; de Matos et al., 2012).
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Stress induction
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Biochemical parameters during road transport indicated that all of the groups were stressed similarly as a
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consequence of this management practice (data not shown). Thus, no differences among diets on cortisol levels or
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serum glucose concentration (which is related to the activity of glucocorticoides and catecholamines) were apparent
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either before or after road transport. However, there was a significant lowering effect of flaxseed (P=0.027) on CPK
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levels before and after road transport, which may be indicating either a lower level of tissue damage or physical
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fatigue of FS and FS-QCT lambs (Kramer and Hoffmann, 1997). In this regard, it must be noted that flaxseed is
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characterised by higher levels of vitamin E than the palm oil present in the CTRL group (Schwartz et al., 2008), so
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this potent antioxidant might have exerted some protection against stress produced by road transport (Morán et al.,
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2012) thus preserving the welfare of the lambs. The tendency to lower TBARS values in liver observed in FS lambs
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when compared to the rest of groups (Table 5) seems to confirm this theory.
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The mechanism by which n−3 PUFA affected the total WBC counts was not investigated in the current study,
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but the results obtained are in agreement with other in vitro and animal feeding studies suggesting that saturated fatty
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acids have a limited effect on immune function, whereas feeding plants rich in n−3 fatty acids such as flaxseed shows
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immunomodulator properties (Calder et al., 2002; Lessard et al., 2003). Thus, the decrease in WBC counts observed
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in the FS and FS-QCT lambs before the stress (PRE) in the present study (Table 4) would be in agreement with the
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inhibition of T lymphocyte proliferation by n−3 FA reported previously in rats fed diets containing large amounts of
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linseed oil compared with rats fed diets rich in hydrogenated coconut oil (Marshall and Johnston, 1985). Additionally,
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dietary n−3 PUFAs have shown to decrease phagocytosis and lymphocyte proliferation in broiler chickens (Al-
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Khalifa et al., 2012), and influence the transendothelial migration of leukocytes and leukocyte trafficking in general
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(Grimm et al., 2002). However, due to the inherent complexity of the immune response, further experiments should
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clarify whether these changes in the haematological profile of the lambs represent improved adaptation to the stress
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promoted by road transport.
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Finally, the descent in WBC counts (total, lymphocytes and granulocytes) observed in all of the groups after
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road transport (POST) would be in agreement with physiological changes (neutrophilia and lymphopenia) observed
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in stressed animals (Kannan et al., 2000).
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Ruminal/metabolic acidosis
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The partial substitution of cereal grains by dietary fat (palm oil or flaxseed) and the TMR (concentrate plus 15%
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chopped barley straw, Table 1) fed to the fattening lambs might have reduced the incidence of ruminal acidosis and
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the development of metabolic acidosis in the present study (DeVries and von Keyserlingk, 2009; González et al.,
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2012; Jenkins, 1993). This is in agreement with the lack of significant differences in the ruminal pH values (Table 5),
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all the lambs having normal physiological values for this parameter. However, in the CTRL group the ruminal
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mucosa was significantly darker, corresponding to brown colour appreciated at slaughter, which is an indirect
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indicator for ruminal parakeratosis related with an increased thickness of the stratum corneum of the ruminal
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epithelium (Alvarez-Rodriguez et al., 2012). This effect also might explain the increase in the weight of the empty
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rumen (Table 5). This stratum has a protective function, and the number of cell layers present in it could be increased
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as a consequence of several situations such as low ruminal pH, increased propionate:acetate ratios, and increased
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molar proportions of butyrate and propionate (Baldwin, 1998). Since the composition, and specifically the
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carbohydrate content, of the four diets was very similar and none of the groups developed ruminal acidosis, we are
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uncertain about the cause of the darker colours in the CTRL group. Nevertheless, it must be borne in mind that either
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only one measure or the average pH over a day is sometimes considered a poor indicator of ruminal acidosis (Sauvant
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et al., 1999) because it does not reflect the large daily fluctuations in pH (Dragomir et al., 2008). However, our results
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suggest that both additives, quercetin and flaxseed, somehow protected the ruminal mucosa against the development
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of parakeratosis. Regardless of the mechanism of this protection, it is important to highlight this result, as
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parakeratosis could be indicative of subacute ruminal acidosis, a condition caused by transient falls of ruminal pH to
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ranges below pH 5.5, and with several negative consequences to the animal such as loss of condition, interference
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with normal rumen microbiota or predisposition to septicaemia and abscesses formation in the liver (Kleen et al.,
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2003; Steele et al., 2009).
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Conclusions
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Flaxseed supplemented to fattening lambs exhibited some immunomodulator properties, although the biological
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significance of these effects remains to be determined in further studies. Additionally flaxseed supplementation
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tended to reduce the increments of a blood tissue damage indicator during road transport, thus preserving the health
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of the animal under stress conditions. Moreover, parakeratosis in fattening lambs was reduced by both, quercetin and
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flaxseed when included in the diet. Finally, it must be stated that the combination of both, quercetin and flaxseed in
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the diet of fattening lambs does not seem to improve the benefits achieved with any of these ingredients when
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included separately in the diet.
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Acknowledgments
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Financial support received from ‘Consejería de Educación de la Junta de Castilla y León’ (Project CSI185B11-2) is
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gratefully acknowledged. Julio Benavides, María Martinez-Valladares, María L. Tejido, Raúl Bodas, and Nuria Prieto
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(JAE-Doc contracts) were supported by the programme ‘Junta para la Ampliación de Estudios’ (CSIC-European
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Social Fund).
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