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Metabolic acidosis corrected by including antioxidants
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in diets of fattening lambs
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Lara Morán, F. Javier Giráldez*, Raúl Bodas1, Julio Benavides, Nuria
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Prieto, and Sonia Andrés
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Instituto de Ganadería de Montaña (CSIC-Universidad de León). Finca Marzanas. E-
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24346 Grulleros, León (Spain).
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*CORRESPONDING AUTHOR: F. Javier Giráldez, Instituto de Ganadería de Montaña
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(CSIC-ULE). Finca Marzanas. E-24346 Grulleros, León, Spain. Tel. +34 987 317 156
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Fax +34 987 317 161 E-mail: j.giraldez@eae.csic.es
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Present address: Instituto Tecnológico Agrario, Junta de Castilla y León. Finca Zamadueñas. Ctra.
Burgos, km. 119. 47071 Valladolid (Spain).
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Abstract
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Thirty-two Merino lambs fed barley straw and a concentrate alone (CONTROL) or
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CONTROL enriched with vitamin E (0.6 g kg-1 of concentrate, VITE006) or carnosic
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acid (0.6 g kg-1 of concentrate CARN006; 1.2 g kg-1 of concentrate, CARN012) were
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used to assess the effect of these antioxidant compounds on metabolic acidosis. After 7
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wk of being fed the experimental diets, the pH of blood samples was lower (P=0.012)
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for the CONTROL lambs than groups fed antioxidants. Metabolic acidosis in fattening
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lambs was corrected by both antioxidants (carnosic acid and vitamin E).
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Keywords: carnosic acid; vitamin E; acidosis; oxidative stress; lamb
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1. Introduction
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With intensive production systems, animals may be stressed by acidogenic diets
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(Berkemeyer, 2009) fed to meet high metabolic demands. Under this situation, reactive
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oxygen species (ROS) production at the mitochondrial level might be increased,
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whereas the mitochondrial energy production (ATP) would be partially inhibited
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(Boveris et al., 2010). Therefore, pyruvate would be partially directed towards lactic
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acid production and converted to CO2;, thus hypercapnia would be corrected by
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activation of the respiratory control system (Berkemeyer, 2009). However, experiments
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in rats have demonstrated that high levels of ROS might disrupt the respiratory control
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(Mulkey et al., 2003), avoid compensation for hypercapnia and worsen metabolic
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acidosis promoted by the diet. To our knowledge, no studies have been carried out in
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ruminants. Consequently, the aim of the present study was to determine if including
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antioxidants (vitamin E or carnosic acid, a potent antioxidant extracted from
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Rosmarinus officinalis L., an herb which is commonly used as a flavouring and
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medicinal plant widely around the world) in the diet for fattening lambs might
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neutralize the negative consequences of ROS, thus correcting the metabolic acidosis
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caused by the high-grain diets.
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2. Materials and methods
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2.1. Animals and diets
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After stratification on the basis of body weight (mean body weight (BW), 15.2 ±
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0.749 kg), 32 male Merino lambs were allocated randomly to four different groups: a
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control group (CONTROL), a group fed vitamin E (α- tocopheryl acetate) at a rate of
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0.6 g kg-1 of concentrate (VITE006, equivalent to 900 IU of vitamin E kg-1 of
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concentrate), a third group fed the same dose (0.6 g kg-1 of concentrate, CARN006) of
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carnosic acid (Shaanxi Sciphar Biotechnology Co., Ltd, Xi'an, China) and a fourth
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group fed double the low dose of carnosic acid (1.2 g carnosic acid kg-1 of concentrate,
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CARN012). The animals were individually penned. All handling practices followed the
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recommendations of the European Council Directive 86/609/EEC for the protection of
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animals used for experimental and other scientific purposes.
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The basal diet was composed of concentrate [dry matter (DM) 888 g kg-1, crude
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protein (CP) 178 g kg-1 DM, neutral detergent fibre (NDF) 134 g kg-1 DM, and ash 56 g
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kg-1 DM] and barley straw (DM 913 g kg-1, CP 35 g kg-1 DM, NDF 757 g kg-1 DM, ash
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55 g kg-1 DM). After 7 days of adaptation to the basal diet (barley straw and the
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concentrate with no antioxidants), all lambs were fed barley straw plus one of the four
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concentrates above for 7 wk. The concentrate (35 g kg-1 BW day-1) and forage (200 g
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day-1) were weighed and supplied in separate feeding troughs at 9:00 h every day; fresh
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drinking water was always available. The orts (<5% of the total diet) were weighed
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daily. The lack of significant differences in feed intake and performance of the animals
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[concentrate (P=0.912) or barley straw (P=0.715) intake; average daily gain (P=0.691)
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and feed to gain ratio (P=0.566)] is addressed in Morán et al. (2012a).
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2.2. Blood, liver and rumen sampling
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After 7 wk of being fed the experimental diets blood samples collected by jugular
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venipuncture into lithium heparin tubes were assayed in a Stat Profile pHOx Plus blood
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analyser (Nova Biomedical, Waltham, MA, USA) for pH, bicarbonate (HCO3-), CO2
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pressure (pCO2), anion Gap, total CO2 (tCO2), and Na+, K+ and Cl- concentrations an
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hour after being sampled. Finally, all lambs underwent food withdrawal during 6 h
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before being slaughtered, then they were weighed, stunned, slaughtered by
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exsanguination from the jugular vein, eviscerated and skinned (Morán et al., 2012b). A
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piece of liver was excised and held at -80 ºC for TBARS analysis (Bodas et al., 2012).
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Rumen fluid was strained through four layers of cheesecloth and pH was determined
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immediately. Two pieces of the rumen wall [posterior dorsal (PDR) and anterior ventral
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(AVR) areas] were placed in 10% formalin for further anatomical study (López-Campos
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et al., 2012).
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2.3. Statistical analysis
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Data were subjected to one-way analysis of variance with the fixed effect of
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treatment as the only source of variation, using the GLM procedure of the SAS package.
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Orthogonal contrasts were performed to test the effects of antioxidants supplementation
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(CONTROL vs. CARN006+CARN012+VITE006), vitamin E vs. carnosic acid
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(VITE006 vs. CARN006+CARN012) and level of carnosic acid (CARN006 vs.
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CARN012).
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3. Results
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3.1. Blood gases, acid-base parameters and liver antioxidant status (TBARS)
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Table 1 presents the blood gases, acid base profile and liver antioxidant status
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(TBARS) of the lambs. The lambs in the CONTROL group had lower blood pH values
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when compared to the lambs fed antioxidants (P=0.012). Differences (P<0.05) between
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the CONTROL and the antioxidants groups also were observed for pCO2, anion gap, K+
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and TBARS. In addition, lambs fed antioxidants had higher blood pH and lower pCO2,
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anion Gap and K+ when compared to the CONTROL group. Lambs fed vitamin E had
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lower TBARS than the average of lambs fed carnosic acid. Compared to the low level
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of carnosic acid (CARN006), the high level decreased blood HCO3-, tCO2 and Na+.
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[INSERT TABLE 1 NEAR HERE, PLEASE]
3.2. Ruminal pH and anatomopathological study of ruminal wall
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The ruminal pH values (overall mean values 5.52 ± 0.082) reflected ruminal acidosis
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for all of the animals of the present study. Moreover, the interior of the rumen wall was
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grey to dark in all of the animals (characteristic of parakeratosis). This lack of effect of
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antioxidants on inflammation of ruminal ephitelium was corroborated by the lack of
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differences in the mucose (AVR, overall mean values 0.68 ± 0.253 mm; PDR, overall
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mean values 0.82 ± 0.161 mm) and submucose thickness (AVR, overall mean values
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0.69 ± 0.099 mm; PDR, overall mean values 0.71 ± 0.115 mm) and the anastomosis and
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number of papillae (AVR, overall mean values 81.1 ± 4.80 papillae cm-2; PDR, overall
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mean values 95.9 ± 8.10 papillae cm-2).
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4. Discussion
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Feeding high-grain diets to growing lambs to achieve maximum growth rates often is
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associated with ruminal acidosis (local), as was apparent for all the lambs in the present
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study (anatomopathological study of the ruminal wall). Absorbed into the bloodstream,
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the acid (H+) reduces blood pH (normal range of 7.36–7.44) to a more acidic state
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(metabolic acidosis, which is not local but systemic), so blood pH can be considered as
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an indicator of ruminal acidosis (Horn et al., 1979). In the present study only the lambs
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of the CONTROL group showed latent metabolic acidosis (Table 1), that is to say, a
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blood pH constantly at the low end of the normal range and a high anion Gap
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(Berkemeyer, 2009). In contrasts, lambs fed antioxidants, despite ruminal acidosis, did
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not exhibit metabolic acidosis (Table 1). These results are consistent with those of
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Vestweber et al. (1974) who reported mean blood pH of 7.44 and 7.36, respectively,
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before and after producing acute acidosis in sheep.
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Reduction in liver TBARS for lambs receiving CARN012 and VITE006 highlights
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the ability of these compounds to improve the antioxidant status (Table 1). The
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relationship between metabolic acidosis and oxidative stress (Mobbs, 2007) is apparent
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from a mitochondrial point of view; the electron transport chain and the oxidative
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phosphorylation (O2 is consumed to produce H2O and ATP) are coupled by a proton
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(H+) gradient across the inner mitochondrial membrane known as the proton-motive
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force (∆p). This gradient is used by the FOF1 ATP-synthase complex to make ATP via
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oxidative phosphorylation. However, 1% of the mitochondrial electron flow leads to the
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formation of ROS (O2•–) under normal physiological conditions. This percentage is
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increased when H+ leakage (passive diffusion of H+ with no ATP production) increases
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due to accumulation of H+ (Boveris et al., 2010), as happens during metabolic acidosis.
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In acidosis, O2 is channeled to ROS production, which reacts with various cellular
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targets changing membrane excitability, synaptic transmission, gene induction and
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cellular metabolism.
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As a result, ATP production is reduced when H+ leaks during metabolic acidosis
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(Berkemeyer, 2009). Pyruvate is diverted toward lactate production and most of the
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lactate is converted into CO2, promoting hypercapnia and respiratory acidosis. To
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compensate for this situation, the hypoxic/hypercapnic chemoreflex is activated;
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however, if hyperventilation takes place (as happens when ROS are augmented, as will
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be explained below), the binding tenacity of haemoglobin for oxygen increases, and less
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oxygen is released for cells (the Bohr Effect). Consequently, cells produce lactic acid
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and the body continues to be exposed to organic acid with no recovery (lactic acid trap;
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Berkemeyer, 2009). These effects would explain the high pCO2 (hypercapnia) and low
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pH (latent acidosis) observed in the CONTROL lambs (Table 1).
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As previously stated, hypercapnia is one stimulus but not the only for the respiratory
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control system (Mulkey et al., 2003). In fact, the respiratory control might be disrupted
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by oxidation stress of the brain stem respiratory control centre (possibly CO2/H+
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chemosensitive neurons). For example, hyperoxia (Mulkey et al., 2003) and chemical
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oxidants (Lipton et al., 2001) increase respiration rate, whereas antioxidants can block
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the effects of hyperoxia (Mulkey et al., 2003). Consequently the lambs in the
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CONTROL group presumably suffered from hyperventilation (lactic acid trap), while
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antioxidant fed lambs would have neutralized the negative effects of ROS at the
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respiratory control centre.
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The low concentrations of plasma K+ in the antioxidant groups when compared to the
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CONTROL corroborate this theory (Table 1). Neurons are stimulated by a free radical-
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dependent mechanism, possibly inhibiting a K+ channel taking charge of maintaining
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low extracellular potassium concentrations, which are necessary to preserve potassium
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gradients across cell membranes (essential for nerve excitation) (Hoshi and Heinemann,
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2001; Mulkey et al., 2003). Specifically, the oxidation of methionine to methionine
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sulfoxide during ROS production is one of the main factors involved in the inactivation
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process of the ion channels (Hoshi and Heinemann, 2001). Thus, the high concentration
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of plasma potassium in the CONTROL group may indicate that cell uptake of potassium
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was impaired by ROS production. On the other hand, the normal values of pH, anion
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Gap, pCO2 and K+ in the lambs fed antioxidants indicate that these compounds helped
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to detoxify ROS, and attenuate the deleterious consequences of metabolic acidosis.
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5. Conclusions
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Including antioxidants in the diet of fattening lambs had no detectable effect on
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ruminal acidosis. However, metabolic acidosis was corrected when either carnosic acid
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or vitamin E was added to the concentrate being fed. Consequently, supplements of both
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compounds should help preserve acid-base status of fattening lambs fed high-grain
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diets.
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Acknowledgments
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Financial support received project AGL2010-19094 is gratefully acknowledged. Raúl
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Bodas, Nuria Prieto, Julio Benavides (JAE-Doc contracts) and Lara Morán (JAE pre-
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doc grant) were supported by the programme ‘Junta para la Ampliación de Estudios’
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(CSIC-European Social Fund).
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References
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Berkemeyer, S., 2009. Acid-base balance and weight gain: are there crucial links via
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protein and organic acids in understanding obesity? Med. Hypotheses 73, 347-356.
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Bodas, R., Prieto, N., Jordán, M.J., López-Campos, Ó., Giráldez, F.J., Morán, L.,
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Andrés, S., 2012. The liver antioxidant status of fattening lambs is improved by
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naringin dietary supplementation at 0.15% rates but not meat quality. Animal 6,
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863-870.
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Boveris, A., Carreras, M.C., Poderoso, J.J., 2010. The Regulation of Cell Energetics and
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Mitochondrial Signaling by Nitric Oxide, in: Ignarro, L.J., (Ed.), Nitric Oxide:
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Biology and Pathobiology. Academic Press, San Diego, CA, pp. 441 - 482
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Horn, G.W., Gordon, J.L., Prigge, E.C., Owens, F.N., 1979. Dietary buffers and ruminal
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and blood parameters of subclinical lactic acidosis in steers. J. Anim. Sci. 48, 683–
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691.
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Hoshi, T., Heinemann, S.H., 2001. Regulation of cell function by methionine oxidation
and reduction. J. Physiol. 531, 1-11.
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Lipton, A.J., Johnson, M.A., McDonald, T., Lieverman, M.W., Gozal, D., Gaston, B.,
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2001. S-nitrosothiols signals the ventilatory response to hypoxia. Nature 413, 171-
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174.
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López-Campos, Ó., Bodas, R., Prieto, N., Giráldez, F:J., Pérez, V., Andrés, S., 2010.
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Naringin dietary supplementation at 15% rates does not provide protection against
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sub-clinical acidosis and does not affect the responses of fattening lambs to road
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transportation. Animal 4, 958-964.
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Mobbs, C., 2007. Oxidative stress and acidosis, molecular responses to, in: Finck, G.,
(Ed.), Encyclopedia of Stress. Academic Press, San Diego, CA, p. 49.
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Morán, L., Andrés, S., Bodas, R., Benavides, J., Prieto, N., Pérez, V., Giráldez, F.J.,
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2012a. Antioxidants included in the diets of fattening lambs: effects on immune
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response, stress, welfare and distal gut microbiota. Anim. Feed Sci. Technol. 173,
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177-185.
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Morán, L., Andrés S., Bodas, R., Prieto, N., Giráldez, F.J., 2012b. Meat texture and
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antioxidant status are improved when carnosic acid is included in the diet of
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fattening lambs. Meat Sci. 91, 430-434.
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Mulkey, D.K., Henderson, R.A., Putnam, R.W., Dean, J.B., 2003. Hyperbaric oxygen
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and chemical oxidants stimulate CO2/H+-sensitive neurons in rat brain stem slices.
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Vestweber, J.G.E., Leipold, H.W., Smith, J.E., 1974. Ovine ruminal acidosis: clinical
studies. Am. J. Vet. Res. 35, 1587-1590.
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Table 1
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Blood gases, acid base profile and liver antioxidant status (TBARS) in not supplemented fattening lambs (CONTROL) or supplemented either
with carnosic acid (0.6 and 1.2 g kg-1 concentrate) or vitamin E (0.6 g kg-1 concentrate)
Parameter1
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Orthogonal contrasts2
Dietary treatment (n=8)
CONTROL
CARN006
CARN012
VITE006
sed
C vs. AO
VE vs. CA
C06 vs. C12
pH (ven)
7.38a
7.42b
7.42b
7.41b
0.013
0.001
0.482
0.635
HCO3- (ven) (mmol/L)
25.3
26.8
25.2
26.3
0.74
0.218
0.581
0.045
pCO2 (ven) (mmHg)
46.6b
44.1ab
42.3a
44.6ab
1.37
0.022
0.246
0.194
Anion Gap (mmol/L)
15.4b
13.5a
14.0ab
13.3a
0.67
0.004
0.382
0.468
tCO2 (ven) (mmol/L)
26.7
28.1
26.4
27.7
0.77
0.295
0.521
0.045
Na+ (mmol/L)
146
147
145
145
0.7
0.484
0.217
0.042
K+ (mmol/L)
4.87b
4.56a
4.38a
4.38a
0.126
0.001
0.418
0.176
Cl- (mmol/L)
110
111
110
110
0.6
0.944
0.095
0.214
TBARS (µg MDA g-1 liver)
1.65b
1.58b
1.21ab
0.77a
0.244
0.053
0.010
0.207
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HCO3-: bicarbonate; pCO2: CO2 pressure; tCO2: total CO2
Orthogonal contrasts: C vs. AO = CONTROL vs. CARN006+CARN012+VITE006; VE vs. CA = VITE006 vs. CARN006+CARN012; C06 vs.
C12 = CARN006 vs. CARN012.
a, b
: different subscripts in the same row indicate significant differences (P<0.05) between diets.
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