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Naringin and vitamin E influence the oxidative stability and
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lipid profile of plasma in lambs fed fish oil
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R. Bodas*, N. Prieto, O. López-Campos, F. J. Giráldez and S. Andrés
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Instituto de Ganadería de Montaña (CSIC-ULE). Finca Marzanas, s/n. E-24346
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Grulleros (León, Spain).
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* CORRESPONDING AUTHOR: Raúl Bodas, Instituto de Ganadería de Montaña
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(CSIC-ULE). Finca Marzanas, s/n. 24346 Grulleros, León (Spain). Tel. +34 987 307
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054 Fax +34 987 317 161. E-mail: raul.bodas@eae.csic.es
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Abstract
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Thirty two Merino lambs (15 weeks old) fed barley straw and fish oil enriched
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concentrate were used to assess the effect of vitamin E (6 g kg-1 DM) and naringin (1.5
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and 3 g kg-1 DM) on plasma lipid peroxidation (TBARS), total antioxidant status (TAS),
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immune response, plasma cholesterol, and triglycerides. After 21 days feeding the
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experimental diets, lambs were subjected to a 4-h transportation stress period and then
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held 4 more hours without feed. TBARS values before stress were lower for animals
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consuming vitamine E and naringin when compared to control lambs (P<0.05).
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However, after stress all groups presented similar levels of TBARS. TAS decreased
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(P<0.05) in all groups in response to stress with values recovering (P<0.05) to pre-stress
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values following 4 h of rest. A rise (P<0.05) in serum concentrations of triacylglycerol
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following 21 d of fish oil supplementation was dampened in lambs consuming vitamin
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E or naringin. Both pre-stress TBARS and triacylglycerol-reducing effects of naringin
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added to fish oil enriched concentrate for fattening lambs are reported.
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Keywords: flavonoid; naringin; lamb; cholesterol; antioxidant; TBARS
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Introduction
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Flavonoids are a group of naturally occurring compounds ubiquitous in the plant
40
kingdom and known to have strong antioxidant effects (Kim et al, 2004). Thus, in order
41
to improve meat and milk quality, supplementing ruminants with these compounds may
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be useful particularly when they are fed unsaturated fatty acids. For example, n – 3 fatty
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acids are highly susceptible to peroxidation both in plasma and tissues. Transporting
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animals to the slaughterhouse can increase this susceptibility and, therefore, the values
45
of TBARS and pigment oxidation in meat (Kannan et al, 2000; Renerre, 2000; Young et
46
al, 2003). Furthermore, lipid peroxidation has been implicated in deterioration of
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physiological functions that include growth and reproduction, as well as immunity,
48
leading to a higher susceptibility to infectious diseases (Gladine et al, 2007).
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Consequently, a supply of antioxidants is recommended to preserve both, the health of
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animals supplemented with oil (McDowell et al, 1996) and the oxidative stability of
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their products (Wood and Enser, 1997; Kannan et al, 2000). In this sense, different
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studies have demonstrated an insufficient ability of Vitamin E to inhibit
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lipoperoxidation when n – 3 fatty acids intake is increased (Miret et al, 2003).
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Nevertheless, the antioxidant effect of plant extracts rich in polyphenols (flavonoids)
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needs further investigation under the same conditions (Kim et al, 2004 and 2006). This
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information has timely implications, since the European Commission may lift the ban
57
on feeding fish meal and oils to ruminants (Stevenson 2005).
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Additionally, the incremental increase in plasma concentrations of cholesterol and
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triacylglycerols (TAG) when animals are fed fish oil might be counteracted by naringin,
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a type of grapefruit and citrus flavonoid. In fact, naringin is a potent plasma
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triacylglycerol and cholesterol-lowering agent in monogastrics (Casaschi et al, 2002;
3
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Jeon et al, 2004). It is unknown whether ruminants fed with fish oil can benefit from the
63
same effect of naringin. Moreover, some authors suggest that polyphenol compounds
64
may have antimicrobial activity and some other beneficial effects on certain immune
65
parameters, such as inhibition of inflammation or modulation of the activation of B and
66
T lymphocytes (Tripoli et al., 2007; Hamer, 2007).
67
The present study was conducted to assess the effect of naringin on the lipid
68
peroxidation (TBARS), the total antioxidant status (TAS) and the immunological
69
response of fattening lambs fed fish oil and subjected to transportation stress. In
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addition, biochemical parameters such as serum cholesterol (total, HDL and LDL) and
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triglycerides (TAG) have been considered.
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Material and Methods
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2.1. Animals and diets
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Thirty two Merino lambs (initial age 14-16 weeks) were used in this experiment.
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Lambs were kept with their mothers until weaning, allowing free access to a
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commercial starter concentrate, barley straw and alfalfa hay, until the commencement of
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the trial. Animals were dewormed with Ivomec (Merial Labs., Spain) and vaccinated
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against enterotoxaemia (Miloxan, Merial Labs., Spain).
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After random stratification on the basis of body weight (mean 28.4 kg), the lambs
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were allocated to one of four treatments (8 per treatment) prior to housing individually.
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All handling practices followed the recommendations of European Council Directive
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86/609/EEC for protection of animals used for experimental and other scientific
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purposes.
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After 5 days of adaptation to the basal diet (barley straw and basal concentrate feed),
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all the lambs were fed barley straw and the concentrate feed enriched with fish oil (30
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g·kg-1) alone (Control group) or supplemented with either vitamin E (6 g kg-1, VitE
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group) or different doses of naringin (1.5 g kg-1, Nar15 group; and 3 g kg-1, Nar30
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group) for 21 days. Concentrate and forage were supplied in separate feeding troughs at
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9:00 a.m. every day, and fresh drinking water was always available. The ingredients and
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chemical composition of the feeds are shown in Table 1. The straw (200 g day-1) and
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concentrate offered (30 g kg-1 BW day-1) were weighed daily. The orts were also
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weighed daily with sub-samples collected for subsequent analyses. Since guidelines for
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naringin supplementation of ruminants do not exist, two doses were selected based on
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previous research (Shin et al, 1999; Jeon et al, 2001; Gladine et al, 2007), where
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chronic doses of 1.0 and 0.5 g kg-1 and an acute single dose of 100 g kg-1 were used,
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respectively. In the present experiment interaction between ruminal bacterial
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community and naringin was expected (Gladine et al., 2007), so two chronic doses
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higher than those used in monogastric animals were selected (1.5 and 3 g kg-1).
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[PLEASE, INSERT TABLE 1 NEAR HERE]
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Three weeks later the animals were subjected to a 4-h transportation period and then
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held in pens 4 more hours with fresh drinking water to simulate preslaughter conditions.
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2.2. Blood sample collection
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All the animals were blood sampled by jugular venipuncture before supplying the
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experimental concentrate (day 0) and three weeks later before the transport period (day
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21, 0 h), immediately after a 4-h transportation period (day 21, 4 h), and again 4 hours
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later (day 21, 8 h). Blood samples were collected into two Vacutainer tubes (10 ml) with
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and without sodium heparin.
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Blood samples collected in the sodium heparin tubes (day 21) were immediately
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placed in iced water and centrifuged at 1000 × g for 10 min at 4ºC. Then plasma was
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separated and stored at -80ºC until required for antioxidant analyses. Those samples in
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tubes with no anticoagulant (day 0 and day 21) were allowed to clot for 30 minutes at
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room temperature and centrifuged at 2000 × g for 15 min at 4ºC. Thereafter, serum was
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stored at -20ºC until used to measure biochemical parameters.
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2.3. Immune response
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Fifteen days after having started this experiment, phytohemagglutinin (PHA, 1 mg,
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Sigma–Aldrich, Spain) dissolved in 1 ml of sterile saline solution was injected
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intradermally into a 2 cm wide circle marked on armpit. Skin-fold thickness was
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determined just before PHA injection and 24 h later with a calliper. The increase in
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skin-fold thickness for each animal was computed using these two measurements.
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2.4. Antioxidant parameters
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Lipid peroxidation was analysed in the plasma samples using the TBARS Assay Kit
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(Thiobarbituric Acid Reactive Substances) provided by Cayman Chemical (Michigan,
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USA), whereas total antioxidant status (TAS) was measured according to the Trolox-
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Equivalent Antioxidant Capacity (TEAC) assay (Cayman Chemical, Michigan, USA).
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Both analyses were performed in plasma samples according to manufacturer’s
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instructions.
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2.5. Biochemical parameters
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Total cholesterol, LDL, HDL and triacylglyceride (TAG) concentrations in serum
130
samples were determined by an automated enzymatic colorimetric principle with test
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kits from Roche Diagnostics on Cobas Integra 400 (Roche Diagnostic System).
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Serum
cortisol
levels
were
determined
by
a
solid-phase,
competitive
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chemiluminescent enzyme immunoassay using a commercially available assay kit
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(Immulite® 1000 Cortisol, Siemens Healthcare, Munich, Germany; inter and intra-assay
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CV of 6.7 and 7.3%, respectively)
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2.6. Statistical analyses
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Data of feed intake and immune response were subjected to analysis of variance
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using the GLM procedure of SAS package (SAS, 1999). Data corresponding to
139
antioxidant and biochemical parameters were analysed as a complete randomised,
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repeated measures design using the MIXED procedure of SAS (Littell et al, 1998) with
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individual lamb as the experimental unit. Least square means were generated and
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separated using the PDIFF option of SAS for main or interactive effects, significance
143
being determined at P<0.05. The tables show the residual standard deviation (RSD)
144
comparing all the possible combinations between treatments or times.
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Results and discussion
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No differences could be observed among groups in the concentrate or barley straw
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intake (average: 639 ± 17.7 and 114 ± 5.8 g·animal-1·day-1, respectively; P > 0.05) and
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body weight gain (average: 108 ± 9.9 g·animal-1·day-1; P > 0.05).
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Table 2 summarizes changes in plasma cortisol, lipoperoxidation (TBARS) and
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antioxidant status (TAS) in response to stress. Cortisol levels are used in the welfare
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assessment of farmed livestock, being regarded as reliable indicators of acute and
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chronic stress (Fisher et al, 1997). Serum concentrations of cortisol were increased (P <
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0.01) in response to transportation, and concentrations returned to baseline (P < 0.01) in
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all animals following 4 h of rest (average values of 0.63±0.079, 0.89±0.158 and
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0.51±0.071 µg·dl-1 for 0, 4 and 8 h, respectively).
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[PLEASE, INSERT TABLE 2 NEAR HERE]
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Stress suffered by the animal when transported to the slaughterhouse exacerbates the
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lipoperoxidation of long chain n – 3 fatty acids consumed with the fish oil (Kannan et
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al, 2000). Lipid peroxides, derived mainly from polyunsaturated fatty acids, can be
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measured by TBARS procedure since these compounds are unstable and decompose to
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form reactive carbonyl compounds such as malondialdehyde (MDA). In the present
163
study, animals consuming diets supplemented with either vitamin E or naringin showed
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lower TBARS values before stress (P<0.05) when compared to Control lambs (Table 2:
165
0 h). However, transportation stress increased TBARS values in all lambs and
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differences were not detected (P > 0.05) among treatment groups.
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These data demonstrate lipoperoxidation was suppressed by vitamin E and naringin
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prior to transportation likely due to the high dose of antioxidants used in the present
169
study when compared to those (0.1 and 0.5 g of vitamin E kg-1 diet) recommended by
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Weber et al (1997) or Demirel et al (2004). Naringin or vitamin E supplementation
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were unable to maintain low plasma concentrations of TBARS following stress.
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In order to determine the contribution of the four different diets to the oxidative
173
stability of plasma samples, the analysis of total antioxidant status (TAS) was
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performed, as it may provide more relevant biological information compared to that
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obtained by the measurements of endogenous (antioxidant enzymes) or food-derived
176
(e.g. vitamin E or naringin) components (Prior and Cao, 1999). All the groups showed a
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decrease (P < 0.001) in TAS following stress, the values being increased again four
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hours later (average values of 5.06±0.432, 3.49±0.295 and 5.78±0.440 mM Trolox for
8
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0, 4 and 8 h, respectively). These results may indicate an insufficient antioxidant effect
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of either vitamin E or naringin when administered to lambs fed fish oil under stress
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conditions. Indeed, Gladine et al (2007) observed a significant reduction of plasma
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susceptibility to lipoperoxidation when a supranutritional dose (10% of dry matter
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intake) of grape polyphenols was put directly into the rumen of sheep fed linseed oil.
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These authors suggested that the ruminal metabolism caused the hydrolysis of the
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naringin, thus allowing the absorption of the aglycone fraction (naringenin), the only
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metabolite detected in the plasma of sheep. Apart from the increase in antioxidant
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enzyme activities (SOD and CAT), the ability of polyphenols (and their metabolites) to
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spare or recycle vitamin E would also partially explain their antioxidant properties
189
(Kannan et al, 2007; Jeon et al, 2001 and 2002) and the reduction of plasma
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susceptibility to lipoperoxidation. This reduction in susceptibility to oxidation may
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affect meat characteristics, protecting the lipids and proteins from oxidation or
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discoloration (Renerre, 2000, Young et al, 2003). Nonetheless, the bitter taste of
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naringin may affect palatability of the diet at the higher levels (10% of dry matter
194
intake) as described by Gladine et al (2007).
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The generation of inflammatory mediating T lymphocytes in response to PHA has
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been recognized as an indicator of immunological competence in ewes subjected to
197
chronic stress (Albenzio et al, 2003). The response to PHA injection did not differ by
198
diet, as the average increase in skin-fold thickness did not differ (overall mean = 8.04
199
mm, residual standard deviation = 1.911, P = 0.236) among groups. Although the lack
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of differences could be due to the absence of a diet effect, lambs were not subjected to
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any physiological or physical stress when immune response was tested by PHA
202
injection.
9
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Generally speaking, the supplementation of fish oil for 21 days promoted an
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incremental increase in total, HDL and LDL cholesterol, although the HDL values
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corresponding to Control and Nar30 lambs were kept closer to the basal levels than
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Nar15 and VitE lambs (Table 3).
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[PLEASE, INSERT TABLE 3 NEAR HERE]
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Previous experiments in rabbits (Jeon et al, 2004), rats (Kim et al, 2004 and 2006)
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and humans (Jung et al, 2003; Baba et al, 2007) have demonstrated that
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hypercholesterolemic subjects consuming flavonoids evidence a reduction of total
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cholesterol and LDL cholesterol plus an incremental increase of HDL cholesterol,
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whereas no changes were found in normocholesterolemic subjects. In the present study
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we had normocholesterolemic lambs (day 0) being fed a high cholesterol diet (fish oil)
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during 21 days, but we did not observe a clear cholesterol lowering effect in naringin as
215
compared to Control lambs. In this sense, Jung et al (2003) have described an inhibitory
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effect of naringin on hepatic cholesterol biosynthesis [hepatic 3-hydroxy-3-methyl-
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glutaryl CoA reductase (HMG-CoA reductase)] and esterifying enzymes [acylCoA:
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cholesterol acyltransferase (ACAT)] in hypercholesterolemic subjects following 8
219
weeks of flavonoid supplementation.
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Regarding to TAG, this parameter was increased in the Control group at the end of
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the experiment (day 21), whereas the rest of groups were able to keep TAG
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concentration closer to basal levels (day 0) despite the fish oil consumption. Our results
223
are in agreement with those observed in the hepatic lipid profile of ethanol treated rats
224
after being fed naringin for 6 weeks (Seo et al, 2003). These results have been attributed
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at least in part to an inhibitory effect of flavonoids on diacylglycerol acyltransferase
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(DGAT1) activity, one of the main enzymes involved in TAG synthesis, thus suggesting
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these compounds may be used in the clinical treatment of hypertriglyceridemia
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(Casaschi et al, 2002; Borradaile et al, 2003).
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Neither vitamin E nor naringin were able to maintain low levels of lipid
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peroxidation following transportation stress. The effects of naringin on immune
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response and plasma cholesterol remain unclear. Results of the present study may be
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limited due to either the briefness of our experiment (just 3 weeks) when compared to
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previous studies (between 6 and 8 weeks), or inadequate doses of naringin.
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Conclusions
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Dietary naringin decreased TBARS prior to stress in fish oil supplemented fattening
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lambs.
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Acknowledgments
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239
This investigation was funded by the CSIC ‘Intramural’ Project 200840I116 and
supported by Junta de Castilla y León (GR158).
240
241
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15
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Table 1. Ingredients and chemical composition of the experimental feeds.
Control
VitE
Nar15
Nar30
Barley straw
Barley
534
531
532.5
532
Soy bean meal
214
213
214
213
Corn
165
164
165
165
Molasses
29
29
29
29
Mineral vitamin premix
28
28
28
28
Fish oil
30
30
30
30
Vitamin E
0
6
0
0
Naringin
0
0
1.5
3
Dry matter (DM, g·kg-1 fresh)
877
877
875
877
908
Crude protein
179
177
178
178
29
Neutral detergent fibre
168
161
163
163
846
Ash
65
61
62
64
46
Ingredients (g·kg-1 as feed)
Chemical composition (g·kg-1
DM)
339
340
16
341
Table 2. Least square means of plasma cortisol and antioxidant parameters on day 21 before the
342
transport period (0 h), immediately after a 4-h transportation period (4 h) and 4 hours after
343
having finished the transport (8 h) of lambs fed with concentrate enriched with fish oil (30 g·kg-
344
1
345
Nar15; and 3 g kg-1, Nar30).
) alone (Control) or supplemented with either vitamin E (6 g kg-1, VitE) or naringin (1.5 g kg-1,
P value1
Diets
Control
Cortisol (µg/dL)
0h
0.726
4h
0.830
8h
0.527
VitE
Nar15
Nar30
D
H
D*H
RSD
0.599
1.098
0.381
0.665
0.772
0.518
0.519
0.868
0.593
0.967
0.003
0.425
0.319
<0.001
0.032
0.851
0.606 <0.001
0.910
3.123
Thiobarbituric Acid Reactive Substances (μM Malondialdehyde)
0h
3.88b
2.40a
2.74a
2.13a
4h
3.50b
4.21b
3.98b
3.86b
0.376
b
b
b
b
8h
4.32
4.03
3.98
3.85
Total antioxidant status (mM Trolox)
0h
4.80
5.27
4.67
4h
3.45
3.68
3.10
8h
5.86
6.40
4.72
346
1
347
a, b
5.50
3.73
6.15
P values for diet (D), hour (H) and their interaction (D*H).
Different superscripts in the same row or column indicate a significant interaction D*H
348
17
349
Table 3. Least square means of blood biochemical parameters on days 0 and 21 of lambs fed
350
with concentrate enriched with fish oil (30 g·kg-1) alone (Control) or supplemented with either
351
vitamin E (6 g kg-1, VitE) or naringin (1.5 g kg-1, Nar15; and 3 g kg-1, Nar30).
Day
352
353
Control
VitE
Diets
Nar15
Nar30
D
P value1
T
D*T
RSD
Total cholesterol (mmol/L)
0
1.05
1.01
21
1.31
1.39
1.01
1.22
1.10
1.32
0.894
<0.001
0.498
0.301
HDL cholesterol (mmol/L)
0
0.628a
0.608a
21
0.702ab
0.829b
0.615a
0.738b
0.691ab
0.775b
0.733
<0.001
0.043
0.145
LDL cholesterol (mmol/L)
0
0.392
0.376
21
0.618
0.573
0.390
0.481
0.405
0.553
0.873
<0.001
0.548
0.226
0.361
0.042
3.703
Triacylglycerol (mg/dl)
0
15.1a
15.6ab
13.9a
16.0ab
0.230
b
ab
a
a
21
19.4
15.9
13.4
14.5
1
P values for diet (D), time (T) and their interaction (D*T)
a, b
Different superscripts in the same row or column indicate a significant interaction D*T
18