Application of KRL test to assess total antioxidant activity in pigs: Sensitivity to dietary antioxidants

Research in Veterinary Science 94 (2013) 372–377

Contents lists available at SciVerse ScienceDirect

Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc

Application of KRL test to assess total antioxidant activity in pigs: Sensitivity to dietary antioxidants Raffaella Rossi ⇑, Grazia Pastorelli, Carlo Corino Università degli Studi di Milano, Department of Veterinary Science for Health, Animal Production and Food Safety, Via Celoria 10, 20133 Milan, Italy

a r t i c l e

i n f o

Article history: Received 25 January 2012 Accepted 12 August 2012

Keywords: Dietary antioxidant Total antioxidant activity KRL test Pig

a b s t r a c t The application of Kit Radicaux Libres (KRL) test to assess total blood antioxidant activity in pigs was evaluated. The KRL has been validated and is widely used in humans for assessing the effectiveness of natural or pharmaceutical treatments, and in vitro to evaluate the antioxidant activities of natural or synthetic antioxidants. In this study the sensitivity of the KRL test in assessing the effectiveness of dietary antioxidant supplementation (vitamin E and plant extract) was evaluated in two different phases of pig breeding. The first trial, in post-weaned piglets (40 piglets/group) fed dietary vitamin E supplementation for 60 days, indicated that there was a higher total antioxidant activity (P = 0.032) of whole blood and of red blood cells (P = 0.001) than for control pigs. The second trial indicated that long-term supplementation of water soluble plant extract (20 pigs/group) from the leaves of Verbenaceae (Lippia spp.) tended (P = 0.091) to increase antioxidant activity in the whole blood of treated, rather than control pigs. These results indicate that the KRL might be recommended as one of efficient means for evaluating antioxidant activity of dietary ingredients fed to pigs. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction In recent years increasing experimental data has provided evidence for the involvement of oxidative stress in the development of many diseases. In farm animals, oxidative stress may be involved in several pathological disorders, including those relevant to health and animal production (Lykkesfeldt and Svendsen, 2007). Reactive oxygen species (ROS) have biological functions essential for normal physiology, and production of ROS may be increased considerably in response to various stimuli (Machlin and Bendich, 1987). These include extracellular factors signaling through plasma membrane receptors, such as hormones, proinflammatory cytokines and physical and environmental factors like UV stress, drug and pathogen invasion (Bashan et al., 2009). Deficiencies in antioxidant substances or excess exposure to stimulators of ROS production may result in oxidative stress, defined as an impaired homeostasis between oxidants and antioxidants at a cellular level (Finkel and Holbrook, 2000). Oxidative stress can be measured directly by detecting free radical production, or indirectly by detecting antioxidant molecules or oxidative damage biomarkers. In general, free radical production can be readily detected, but it is extremely difficult ⇑ Corresponding author. Address: Università degli Studi di Milano, Department of Health, Animal Science and Food Safety, Via Trentacoste, 2, 20134 Milan, Italy. Tel.: +39 02 50315758; fax: +39 02 50315746. E-mail address: [email protected] (R. Rossi). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.08.005

to assessing in vivo the total antioxidant activity. Antioxidant may be present within cells, cells membranes and in extracellular fluids. Intracellular antioxidants include glutathione which can quench free radicals and enzymes, such as catalase, glutathione peroxidase, and superoxide dismutase, which can decompose the oxidants. Membrane bound antioxidant include a-tocopherol, b-carotene and ubiquinone (Halliwell and Gutteridge, 1989). The antioxidant system in plasma is mostly accounted for by low molecular weight antioxidants (ascorbic acid, glutathione, tocopherols, phenolic compounds) of a dietary origin (Evans and Halliwell, 2001). The concentration of individual antioxidants is generally not representative of the total antioxidant activity due to the synergistic and antagonistic interactions among antioxidants. Thus, total antioxidant activity may give more biologically relevant information than merely measuring concentrations of individual antioxidants (Somogyi et al., 2007). There are many methods for assessing oxidative stress (Monaghan et al., 2009). Table 1 shows several methods that assess the total antioxidant capacity in plasma/serum. The Kit Radical Libres (KRL) test is a biological test that evaluates the antioxidant status of an organism by testing the antioxidant defence systems of both plasma and red blood cells (RBC) (Prost, 1992). The KRL test evaluates the total antioxidant activity of blood by measuring the time required to haemolyse 50% of the RBC exposed to a controlled free radical attack. The KRL test has been validated for humans and has several in vivo and in vitro applications. In humans, the test has been used to study the effectiveness of natural or

373

R. Rossi et al. / Research in Veterinary Science 94 (2013) 372–377 Table 1 Methods to assess antioxidant capacity. Method

Sample

Principle

References

OXY-adsorbent test

Plasma

Iorio (2004)

Total antioxidant activity (TAS) Oxygen radical absorbance capacity (ORAC)

Plasma/serum

Trapping antioxidant parameter (TRAP)

Plasma

Ferric reducing antioxidant power (FRAP)

Plasma

Total oxidant scavenging capacity (TOSC)

Plasma/serum

Cupric reducing antioxidant capacity (CUPRAC) KRL test

Plasma/serum

Colorimetric assessment of ability of the anti-oxidant barrier to cope with the oxidant action of hypochlorous acid Colorimetric assessment of sample capacity to quench and decolorize chromogenic-free radical Colorimetric assessment of sample antioxidant capacity in presence of 2,20 -azobis(2-amidinopropane) dihydrochloride Measure of oxygen consumption during a controlled lipid peroxidation reaction induced by thermal decomposition of an azo-compound Colorimetric assessment of sample antioxidant potential by measuring its ferric reducing ability, in presence of ferric chloride (FeCl3) Gas chromatographic assessment capacity of antioxidants against hydroxyl radicals, peroxyl radicals and peroxynitrite Colorimetric assessment of sample antioxidant potential in the presence of bis(neocuproine) copper(II) chelate Time needed to haemolyse 50% of red blood cells when sample is under a free radical attack (AAPH)

Plasma/serum

Whole blood/ red blood cell

pharmaceutical treatments (Girodon et al., 1997). In this respect, the KRL test has been used to distinguish between low dose supplementation of different antioxidant vitamins in elderly patients (Lesgards et al., 2002). Moreover the antioxidant capacity of mononuclear cells was also evaluated (Caspar-Bauguil et al., 2009). For in vitro studies, the test has been used to assess the antioxidant potential of synthetic and natural substances (Blache et al., 1991; Lesgards et al., 2005; Rossi et al., 2009b). Some studies on the antioxidant status of animals, using the KRL test, have been published, particularly on rats (Durand and Blache, 1996; Blache et al., 2002; Boukortt et al., 2004; Taleb-Senouci et al., 2009), birds (Alonso-Alvarez et al., 2004; Bertrand et al., 2006), and rabbits (Brzeziska-lebodziska, 2001). However, there have been few studies on intensively reared animals. The objective of this study was to determine the sensitivity of the KRL test in pigs fed dietary supplementation of liposoluble and water soluble antioxidants in different breeding phases. 2. Material and methods Two independent trials were performed to evaluate the sensitivity of the KRL test for identifying the effects of fat and water soluble antioxidant supplements on total blood antioxidant activity of pigs. In the first trial, dietary supplementation with vitamin E was performed during the post-weaning period. In the second trial, long-term supplementation with plant antioxidants from weaning to slaughter was evaluated. The dosage of plant extract in the feed was chosen on the basis of our previous study in pig (Corino et al., 2007). The dosage of vitamin E in the feed was chosen on the basis of KRL test results that compared the antioxidant activity of Vitamin E and plant extract (Rossi et al., 2009b). All procedures involving animals were carried out in accordance with European Communities Council Directive (86/609/EEC, 1986) and approved by the Italian Ministry of Health (L. n. 116/92). 2.1. Liposoluble antioxidant supplementation Eighty weaned Dalland piglets (24 days of age), 40 castrated males and 40 females, weighing 7 ± 0.5 kg, were randomly selected and allotted to two dietary treatments: control diet (C, 175 mg/kg of a-tocopheryl acetate) and a diet supplemented with the control level plus 54 mg/kg of a-tocopheryl acetate (Vit E, 225 mg/kg of

Rice-Evans and Miller (1994) Re et al. (1999) Cao and Prior (1998)

Wayner et al. (1985)

Benzie and Strain (1996)

Winston et al. (1998)

Apak et al. (2005) Prost (1992)

a-tocopheryl acetate). The experimental groups were balanced for body weight and sex. The animals were individually identified and divided into 10 pens (8 piglets/pen) and reared in an environmentally-controlled room. The experimental diets were formulated to meet or a little exceed the requirements for all nutrients (NRC, 1998), and were presented for ad libitum consumption. Weight and feed consumption were recorded and the average daily gain (ADG) and feed conversion ratio (FCR) were calculated. On 10 randomly selected castrated male piglets per treatment (two piglets/pen), fasting blood samples were obtained at weaning, 15 and 60 days post-weaning by anterior vena cava puncture. The blood samples were collected in 10 mL vacutainer glass tubes containing EDTA (VenojectÒ, Terumo Europe N.V., Leuven, Belgium), and immediately stored at 4 °C. Analyses were performed within 24 h of collection. 2.2. Water soluble antioxidant supplementation In the second trial, the effect of plant antioxidant supplementation was evaluated in castrated male pigs from weaning to 99.7 ± 0.5 kg LW on total blood antioxidant activity. Forty weaned Dalland piglets (24 days of age), 20 castrated males and 20 females, weighing 7 ± 0.4 kg, were randomly selected and allotted to two dietary treatments: control diet (C) and an experimental diet supplemented with 1 kg/t of plant extract (PE), titrated in phenyl propanoid glycosides expressed as verbascoside. The antioxidant supplement contained a water-soluble extract of Verbenaceae (Lippia spp.) leaves, prepared on an industrial scale by a standardised procedure that included ultrasonic extraction with 60% aqueous ethyl alcohol followed by spray-drying with maltodextrins as an excipient. To avoid oxidation in the complete feed, the supplement was microencapsulated within a protective matrix of hydrogenated vegetable lipids using spray cooling technology (Sintal Zootecnica, Isola Vicentina, Vicenza, Italy). The experimental groups were balanced for body weight and sex. The animals were individually identified and divided into 10 pens (four pigs/pen) and reared in an environmentally-controlled room. The experimental diets were formulated to meet or a little exceed the requirements for all nutrients (NRC, 1998), and were presented for ad libitum consumption. Piglets weight and feed consumption were recorded and the average daily gain (ADG) and feed conversion ratio (FCR) were calculated. On 10 randomly selected castrated male pig per treatment (2 pigs/pen), fasting blood sam-

374

R. Rossi et al. / Research in Veterinary Science 94 (2013) 372–377

ples were obtained at weaning, 15, 60, 90 and 150 days after weaning by anterior vena cava puncture. The blood samples were collected in 10 mL vacutainer glass tubes containing EDTA (VenojectÒ, Terumo Europe N.V., Leuven, Belgium) and immediately stored at 4 °C. Analyses were performed within 24 h of collection.

2.3. KRL test The principle of the KRL test is to submit whole blood to thermocontrolled free radical aggression in order to mobilize all families of any free radical scavengers present in the blood to neutralize the oxidation processes (Pieri et al., 1996; Girodon et al., 1997; Stocker et al., 2003). All the chemical and enzymatic antioxidant systems of the sample were triggered to protect cells integrity until lysis. The total antiradical activity of whole blood and RBC for each pig from both trials was evaluated using the KRL biological test (Laboratoires Spiral, France). Whole blood and RBC samples were submitted in an isotonic saline solution to organic free radicals produced at 37 °C from the thermal decomposition of a solution of 2.20 -azobis (2-amidinopropane) dihydrochloride (AAPH) (Kirial International, Dijon, France). Haemolysis was recorded using a 96-well microplate reader by measuring the optical density decay at 450 nm. For each well, absorbance measurements were performed 75 times, once every 150 s. Results were expressed as the time required to reach 50% of maximal haemolysis (half-haemolysis time – HT50 – in minutes), which refers to the whole blood resistance to free-radical attack. Intra and inter-assay coefficients of variation of the KRL test were 2.5% and 4%, respectively.

2.4. Statistical analysis All analyses were performed using SPSS software (SPSS/PC Statistics 18.0, SPSS Inc., Chicago, IL, USA). The growth performances were analysed using one way ANOVA with treatment as the main effect. In order to determine the effects of the dietary treatments and the sampling time on the blood analyses, the data were submitted to repeated measure ANOVA. For blood analyses, total antioxidant activity of whole blood and RBC measured at weaning were used as covariates. Value from each pen was considered as the experimental unit for growth performances. Each pig was considered as the experimental unit for blood analyses. Data are preTable 2 Growth performance of piglets fed control (C) or vitamin E (Vit E) supplemented diet.1

Initial weight, kg Final weight, kg ADG, g/day FCR, kg/kg

C

Vit E

6.9 ± 0.5 27.4 ± 0.9 341 ± 16 1.26 ± 0.03

7.1 ± 0.4 28.2 ± 1.2 351 ± 39 1.18 ± 0.02

1 Means ± SE; n = 5; No significant effects of dietary treatment on growth performances parameters was observed (P > 0.05).

sented as means ± SE, and a value of P < 0.05 was used to indicate statistical significance. 3. Results 3.1. Liposoluble antioxidant supplementation The data on growth performances in piglets fed the control and vitamin E supplemented diet are shown in Table 2. There was no significant effect (P > 0.05) of dietary vitamin E on ADG and FCR. Data on the antioxidant activity of whole blood and RBC in piglets fed the control and vitamin E supplemented diet for 60 days after weaning are shown in Table 3. The antioxidant activity was significantly higher (P = 0.032) in the whole blood of piglets fed with the vitamin E supplement. The same result was observed in the RBC (P = 0.01). No significant effect of sampling time (P = 0.235 and P = 0.212) or the dietary treatment by sampling time interaction (P = 0.239 and P = 0.124) were determined for whole blood and RBC, respectively. 3.2. Water soluble antioxidant supplementation The long-term feeding with plant antioxidant supplement had no significant effect on ADG and on FCR, as shown in Table 4. The results of the antioxidant activity of whole blood and RBC in pig fed control and plant antioxidant supplemented diet for 150 days after weaning are reported in Fig. 1. The antioxidant activity tended to be significantly higher (P = 0.091) in the whole blood of pigs fed with the plant antioxidants. No significant difference was reported in the RBC antioxidant activity (P = 0.227). No significant effect of sampling time (P = 0.799 and P = 0.463) or the dietary treatment by sampling time interaction (P = 0.918 and P = 0.703) were determined for whole blood and RBC, respectively. 4. Discussion In some experimental studies erythrocytes are employed to assess oxidative stress, due to their membranes which are rich in polyunsaturated fatty acids and thus susceptible to lipid peroxidation (Devasena et al., 2001). In fact, the resistance of erythrocytes to peroxidative lysis has been used for many years to evaluate

Table 4 Growth performance of piglets fed control (C) or plant extract (PE) supplemented diet.1

Initial weight, kg Final weight, kg ADG, g/day FCR, kg/kg

C

PE

7.1 ± 0.4 99.5 ± 0.7 618 ± 13.4 2.36 ± 0.4

7.1 ± 0.6 99.7 ± 0.8 616 ± 13.5 2.52 ± 0.11

1 Means ± SE; n = 5; No significant effects of dietary treatment on growth performances parameters was observed (P > 0.05).

Table 3 Total antioxidant activity in whole blood and red blood cells in piglets fed control (C) or vitamin E (Vit E) supplemented diet.1

15 days post weaning 60 days post weaning

Whole blood half-haemolysis time, min2

Red blood cells half-haemolysis time, min2

C

Vit E

C

Vit E

83.69 ± 1.97 72.67 ± 1.91

91.42 ± 2.19 77.95 ± 2.2

58.12 ± 1.49 51.71 ± 1.65

61.76 ± 1.37 58.89 ± 1.52

1 Means ± SE; n = 10; C and Vit E treatments differ (P = 0.032) for whole blood and (P = 0.010) for red blood cells. No significant effect of sampling time (P = 0.235 and P = 0.212) or the dietary treatment by sampling time interaction (P = 0.239 and P = 0.124) was determined for whole blood and RBC, respectively. 2 Half haemolysis time of whole blood and red blood cells at weaning were used as the covariate in the respective analyses.

R. Rossi et al. / Research in Veterinary Science 94 (2013) 372–377

Fig. 1. Total antioxidant activity in whole blood (A) and red blood cells (B) in pigs fed control (C) or plant extract (PE) supplemented diet from weaning to slaughter. HT50, min; time for half haemolysis of blood and RBC samples. Data are reported as means ± SE; n = 10; C and PE treatments differ for: P = 0.091 for whole blood and P = 0.227 for red blood cells. No significant effect of sampling time (P = 0.799 and P = 0.463) or the dietary treatment by sampling time interaction (P = 0.918 and P = 0.703) was determined for whole blood and RBC, respectively. Half haemolysis time of whole blood and red blood cells at weaning were used as the covariate in the respective analyses.

the status of antioxidant defences (Gordon et al., 1955; Kellogg and Fridovich, 1977). More recently, an alternative approach using a KRL test has been used to evaluate the total antioxidant defences of blood and RBC (Blache et al., 1991). A negative correlation between the plasma lipid peroxidation rate and erythrocyte resistance to free radical attack (KRL test) was found in rabbits (Brzeziska-lebodziska, 2001). This result indicates that the plasma antioxidant defence system is associated with red blood cells, and that the erythrocytes may be a good model for oxidative stress studies. Furthermore other experimental studies have reported that antioxidant defences of erythrocytes are usually used to estimate oxidative stress (Kurata et al., 1993; Cimen, 2008). In fact, in the KRL test, free radicals are generated in plasma and react with RBC membranes. Thus, some liposoluble antioxidants efficiently inhibit the lipid peroxidation of the RBC membranes and haemolysis, and at the same time water soluble antioxidants are integrated in a biological pathway, contributing to the protection of the cells integrity (Lesgards et al., 2005). The KRL test, as shown in this study, discriminates between the dietary supplementation of both lipids and water soluble antioxidants. In both the experimental trials, the detected KRL values were in line with the reference values of castrated male post-weaned piglets (Pastorelli et al., 2009).

375

Profitable swine production requires a rapid increase in body weight and lean tissue, raising the demand on the metabolic system of these animals, which can lead to increased oxidative stress unless antioxidant systems are enhanced through nutrition. The more common antioxidant source employed in pig diets is synthetic vitamin E, although natural antioxidants, such as phenolic compounds have been evaluated for their efficacy as antioxidants. It has been previously reported that vitamin E supplementation reduces oxidative stress, which is particularly high in weaned piglets (Lauridsen and Jensen, 2005; Sauerwein et al., 2005). In post-weaned piglets, an increase of plasma and tissue a-tocopherol concentrations was observed as the dietary level of a-tocopheryl acetate increased (Moreira and Mahan, 2002; Wilburn et al., 2008). Weiss and Mahan (2008) reported that the mortality of weaned piglets was greatly reduced when they were given parenteral selenium and vitamin E. It has also been observed in sows that a lower antioxidant status may be associated with higher mortality (Mahan et al., 2007). The higher total antiradical activity observed in this study in the vitamin E supplemented animals indicated a higher resistance to oxidative stress. This might lead to lowering incidence of related diseases, such as enteritis, pneumonia and sepsis (Lykkesfeldt and Svendsen, 2007). The results on growth performance are in agreement with previous studies on post-weaned piglets (Chung et al., 1992; Wilburn et al., 2008) which reported no improvement in the ADG or FCR due to dietary level of a-tocopheryl acetate. In our previous study a marked decrease in both whole blood and RBC total antioxidant activity after weaning was observed (Rossi et al., 2009a). The present results show that a-tocopheryl acetate supplementation (225 mg/kg diet) for 60 days increased both blood and RBC antioxidant activity in post-weaned piglets. This was in agreement with a recent study, which showed an increase in the total antioxidant activity of whole blood in piglets fed dietary vitamin E (150 mg/kg feed) and selenium for 39 days (Guillou et al., 2009). In a previous study Frankicˇ et al. (2010) did not observe differences in TAS (total antioxidant status) values of piglets fed dietary vitamin E (90 g/kg feed) for 14 days. This was also observed by Flis et al. (2010) in pigs fed different amounts of vitamin E (0–100 mg/kg), although plasma vitamin E concentration was approximately four times higher. The disagreement of these results might be due to the different dietary vitamin E levels used in the studies mentioned or to the different methods employed to evaluate the oxidative stress. In the literatures no data have been reported on the effects of long-term supplementation of plant extracts on the total antioxidant status of pigs from weaning to slaughter. Some studies have been conducted in post-weaned or in the finishing phase to enhance oxidative stress responses and to improve the oxidative stability of meat and meat products, respectively (Haak et al., 2006; Corino et al., 2007; Lahucky et al., 2010). In a recent study an improvement in drip and cooking losses and sensory characteristics of Longissimus Dorsi muscle was observed in pig fed a natural extract mixture (Kołodziej-Skalska et al., 2011). Moreover an enhancement of oxidative stability was observed in raw chops, cooked ham and dried-ham of pigs supplemented with high level of n-3 polyunsaturated fatty and plant extracts rich in polyphenols (Mairesse et al., 2010). The growth performance of pigs was not influenced by dietary plant antioxidant, agreeing with a previous study using pigs fed plant antioxidant extracts rich in polyphenols from 60 to 100 kg of live weight (Habeanu et al., 2009). The present results show that total blood antioxidant activity tended to be higher in pigs receiving a long-term supplementation of plant antioxidant from weaning to slaughter than controls. This was also observed in an our previous study on post-weaned piglets in which dietary supplementation with water-soluble extracts of

376

R. Rossi et al. / Research in Veterinary Science 94 (2013) 372–377

Verbenaceae improved the oxidative status measured using the derivatives of Reactive Oxygen Metabolites test (Corino et al., 2007). The same results were observed in suckling lambs (Casamassima et al., 2009) and Italian hares (Lepus corsicanus) fed with the same plant antioxidants (Palazzo et al., 2011). This is related to the increased serum levels of vitamins A and E and to the ability of water-soluble extracts of Verbenaceae to strengthen the endogenous antioxidant system (Palazzo et al., 2011). In conclusion, the KRL may have effectiveness for assessing antioxidant activity of dietary fat and water soluble antioxidants fed to pigs. Supplemental dosage of vitamin E increased the total antioxidant activity in blood and RBC, suggesting a better resistance to oxidative stress in the post-weaning phase. Long-term supplementation with plant extracts from weaning until slaughter tended to increase blood total antioxidant activity. Overall these results indicate that the KRL test could be considered as a new method to assess total antioxidant activity in pigs. Additional studies are needed to examine other possible causes such as genetic type or housing conditions on variation in blood total antioxidant activity in pigs. Conflict of interest statement All the authors involved in this work declare that there are no financial or other contractual agreements that might cause a conflict of interest, or any financial and personal relationships with other people or organisations that could inappropriately influence their work. Acknowledgments This work is part of a research project called ‘‘Nuove metodologie per la valutazione oggettiva del benessere animale nella specie suina’’ financed by Regione Lombardia – Agricultural Department, according to the Plan of Research and Development ‘‘2008’’. References Alonso-Alvarez, C., Bertrand, S., Devevey, G., Gaillard, M., Prost, J., Faivre, B., Sorci, G., 2004. An experimental test of the dose-dependent effect of carotenoids and immune activation on sexual signals and antioxidant activity. American Naturalist 164, 651–659. Apak, R., Güçlü, K., Özyürek, M., Karademr, S.E., Altun, M., 2005. Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: The CUPRAC method. Free Radical Research 39 (9), 949–961. Bashan, N., Kovsan, J., Kachko, I., Ovadia, H., Rudich, A., 2009. Positive and negative regulation of insulin signaling by reactive oxygen and nitrogen species. Physiological Review 89, 27–71. Benzie, I.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: The FRAP assay. Analytical Biochemistry 239, 70–76. Bertrand, S., Alonso-Alvarez, C., Devevey, G., Faivre, B., Prost, J., Sorci, G., 2006. Carotenoids modulate the trade-off between egg production and resistance to oxidative stress in zebra finches. Oecologia 147, 576–584. Blache, D., Prost, M., Raffi, J., 1991. In vitro biological test of resistance to oxidation: Application to identification of irradiated food. In: Potential New Methods of Detection of Irradiated Food. Commission of the European Communities, Luxembourg, pp. 105–116. Blache, D., Durand, P., Prost, M., Loreau, N., 2002. (+)-Catechin inhibits platelet hyperactivity induced by an acute iron-load in vivo. Free Radical Biology and Medicine 33, 1670–1680. Boukortt, F.O., Girard, A., Prost, J.L., Ait-Yahia, D., Bouchenak, M., Belleville, J., 2004. Fish protein improves the total antioxidant status of streptozotocin-induced diabetes in spontaneously hypertensive rat. Medical Science Monitor 10, BR397–BR404. Brzeziska-lebodziska, E., 2001. Erythrocyte osmotic fragility test as the measure of defence against free radicals in rabbits of different age. Acta Veterinaria Hungarica 49, 413–419. Cao, G., Prior, R.L., 1998. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clinical Chemistry 44, 1309–1315. Casamassima, D., Palazzo, M., Presutti, T., Colella, G.E., 2009. Productive performances, plasmatic oxidative status and some blood parameters in suckling lambs supplemented with verbascoside. In: Proceedings of the ASPA XVIII Congress, 668.

Caspar-Bauguil, S., Maestre, N., Segafredo, C., Galinier, A., Garcia, J., Prost, M., Périquet, B., Pénicaud, L., Salvayre, R., Casteilla, L., 2009. Evaluation of whole antioxidant defenses of human mononuclear cells by a new in vitro biological test: Lack of correlation between erythrocyte and mononuclear cell resistance to oxidative stress. Clinical Biochemistry 42, 510–514. Chung, Y.K., Mahan, D.C., Lepine, A.J., 1992. Efficacy of dietary D-alpha-tocopherol and DL-alpha-tocopheryl acetate for weanling pigs. Journal of Animal Science 70, 2485–2492. Cimen, M.Y., 2008. Free radical metabolism in human erythrocytes. Clinica Chimica Acta 390, 1–11. Corino, C., Rossi, R., Musella, M., Cannata, S., Pastorelli, G., 2007. Growth performance and oxidative status in piglets supplemented with verbascoside and teupolioside. In: Proceedings of the ASPA XVII Congress, pp. 292–294. Devasena, T., Lalith, S., Padma, K., 2001. Lipid peroxidation, osmotic fragility and antioxidant status in children with acute post-streptococcal glomerulonephritis. Clinica Chimica Acta 308, 155–161. Durand, P., Blache, D., 1996. Enhanced platelet thromboxane synthesis and reduced macrophage-dependent fibrinolytic activity related to oxidative stress in oral contraceptive-treated female rats. Atherosclerosis 121, 205–216. European Communities Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Available at: . Evans, P., Halliwell, B., 2001. Micronutrients: Oxidant/antioxidant status. British Journal of Nutrition 85, S67–S74. Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239–247. Flis, M., Sobotka, W., Antoszkiewicz, Z., Lipin´ski, K., Zdun´czyk, Z., 2010. The effect of grain polyphenols and the addition of vitamin E to diets enriched with alinolenic acid on the antioxidant status of pigs. Journal of Animal and Feed Sciences 19, 539–553. Frankicˇ, T., Levart, A., Salobir, J., 2010. The effect of vitamin E and plant extract mixture composed of carvacrol, cinnamaldehyde and capsaicin on oxidative stress induced by high PUFA load in young pigs. Animal 4, 572–578. Girodon, F., Blache, D., Monget, A.L., Lombart, M., Brunet-Lecompte, P., Arnaud, J., Richard, M.J., Galan, P., 1997. Effect of two-years supplementation with low dose antioxidant vitamins and/or minerals in elderly subjects on levels of nutrients and on antioxidant defense parameters. Journal of the American College of Nutrition 16, 357–365. Gordon, H.H., Nitowsky, H.M., Cornblath, M., 1955. Studies of tocopherol deficiency in infants and children. Haemolysis of erythrocytes in hydrogen peroxide. American Journal of Diseases in Children 90, 669–681. Guillou, D., Launay, C., Durand, P., Prost, M., 2009. Effet de la supplémentation en Vitamine E et en sélénium organique sur la mesure du potentiel global de défense anti-radicalaire des porcelets. Journées de la Recherche Porcine 41, 195–196. Haak, L., Raes, K., Smet, K., Claeys, E., Paelinck, H., De Smet, S., 2006. Effect of dietary antioxidant and fatty acid supply on the oxidative stability of fresh and cooked pork. Meat Science 74, 476–486. Habeanu, M., Hebean, V., Taranu, I., Marin, D., Ropota, M., Tamas, V., 2009. Effects of the dietary camelina oil on large white pigs’ meat quality. Archiva Zootechnica 12, 31–45. Halliwell, B., Gutteridge, J.M.C., 1989. Free Radicals in Biology and Medicine, second ed. Clarendon Press, Oxford. Iorio, E.L. (Ed.), 2004. D-ROMs Test and Global Assessment of Oxidative Stress. Diacron International, Grosseto, Italy. Kellogg, E.W., Fridovich, I., 1977. Liposome oxidation and erythrocyte lysis by enzymatically generated superoxide and hydrogen peroxide. Journal of Biological Chemistry 262, 6121–6128. Kołodziej-Skalska, A., Rybarczyk, A., Matysiak, B., Jacyno, E., Pietruszka, A., Kawecka, M., 2011. Effect of dietary plant extracts mixture on pork meat quality. Acta Agriculturae Scandinavica Section A 61, 80–85. Kurata, M., Suzuki, M., Agar, N.S., 1993. Antioxidant systems and erythrocyte lifespan in mammals. Comparative Physiology and Biochemistry B 106, 477– 487. Lahucky, R., Nuernberg, K., Kovac, L., Bucko, O., Nuernberg, G., 2010. Assessment of the antioxidant potential of selected plant extracts – In vitro and in vivo experiments on pork. Meat Science 85, 779–784. Lauridsen, C., Jensen, S.K., 2005. Influence of supplementation of all-rac-alfatocopheryl acetate preweaning and vitamin C postweaning on alfa-tocopherol and immune responses of piglets. Journal of Animal Science 83, 1274–1286. Lesgards, J.F., Durand, P., Lassarre, M., Stocker, P., Lesgards, G., Lanteaume, A., Prost, M., Lehucher-Michel, M.P., 2002. Assessment of lifestyle effects on the overall antioxidant capacity of healthy subjects. Environmental Health Perspectives 110, 479–487. Lesgards, J.F., Lehucher-Michel, M.P., Vidal, N., Prost, M., Stocker, P., 2005. Assessment of antioxidative activity of lipid-and water soluble vitamins in humans blood. Comparative analyses between a biological test and chemical methods. International Journal for Vitamin and Nutrition Research 75, 11–18. Lykkesfeldt, J., Svendsen, O., 2007. Oxidants and antioxidants in disease: Oxidative stress in farm animals. The Veterinary Journal 173, 502–511. Machlin, L.J., Bendich, A., 1987. Free radical tissue damage: Protective role of antioxidant nutrients. FASEB Journal 1, 441–445. Mahan, D.C., Peters, J.C., Hill, G.M., 2007. Are antioxidants associated with pig and sow mortalities? In: Proceeding of Swine Nutrition Conference, pp. 13–21.

R. Rossi et al. / Research in Veterinary Science 94 (2013) 372–377 Mairesse, G., Benet, M., Meteau, K., Juin, N., Durand, D., Mourot, J., 2010. L’ajout d’antioxydants vegetaux dans l’alimentation des porcs permet-il de limiter la peroxydation des viandes enrichies en acides gras poly-insatures n-3? Journées de la Recherche Porcine 42, 197–203. Monaghan, P., Metcalfe, N.B., Torres, R., 2009. Oxidative stress as a mediator of life history trade-offs: Mechanisms, measurements and interpretation. Ecology Letters 12, 75–92. Moreira, I., Mahan, D.C., 2002. Effect of dietary levels of vitamin E (all rac-tocopheryl acetate) with or without added fat on weanling pig performance and tissue atocopherol concentration. Journal of Animal Science 80, 663–669. NRC, 1998. Nutrient Requirements of Swine, 10th rev. ed. Natl. Acad. Press, Washington, DC. Palazzo, M., Vizzarri, F., Cinone, M., Corino, C., Casamassima, D., 2011. Assessment of a natural dietary extract, titrated in phenylpropanoid glycosides, on blood parameters and plasma oxidative status in intensively reared Italian hares (Lepus corsicanus). Animal 5, 844–850. Pastorelli, G., Rossi, R., Cannata, S., Corino, C., 2009. Total antiradical activity in male castrated piglets blood: Reference values. Italian Journal of Animal Science 8 (Suppl. 2), 640–642. Pieri, C., Moroni, F., Marra, M., 1996. Food restriction increases the protection of erythorcytes against the haemolysis induced by peroxyl radicals. Mechanisms of Ageing and Development 87, 15–23. Prost, M., 1992. Process for the determination by means of free radicals of the antioxidant properties of a living organism or potentially aggressive agents. In: US patent 5,135,850. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine 26, 1231–1237. Rice-Evans, C., Miller, N.J., 1994. Total antioxidant status in plasma and body fluids. Methods Enzymology 234, 279–293. Rossi, R., Pastorelli, G., Cannata, S., Corino, C., 2009a. Effect of weaning on total antiradical activity in piglets. Italian Journal of Animal Science 8 (Suppl. 2), 673.

377

Rossi, R., Corino, C., Pastorelli, G., Durand, P., Prost, M., 2009b. Assessment of antioxidant activity of natural extracts. Italian Journal of Animal Science 8 (Suppl. 2), 655–657. Sauerwein, H., Schimtz, S., Hiss, S., 2005. Acute phase protein haptoglobin and its relation to oxidative stress in piglet undergoing weaning-induced stress. Redox Report 10, 295–302. Somogyi, A., Rosta, K., Pusztai1, P., Tulassay, Z., Nagy, G., 2007. Antioxidant measurements. Physiological Measurement 28, R41–R55. SPSS/PC Statistics 18.0, SPSS Inc., Chicago, IL, USA. Stocker, P., Lesgards, J.F., Vidal, N., Chalier, F., Prost, M., 2003. ESR study of a biological assay on whole blood: Antioxidant efficiency of various vitamins. Biochimica et Biophysica Acta 1621, 1–8. Taleb-Senouci, D., Ghomari, H., Krouf, D., Bouderbala, S., Prost, J., Lacaiile-Dubois, M.A., Bouchenak, M., 2009. Antioxidant effect of Ajuga iva aqueous extract in streptozotocin-induced diabetic rats. International Journal of Phytotherapy and Phytopharmacology 16, 623–631. Wayner, D.D.M., Burton, G.W., Ingold, K.U., Locke, S., 1985. Quantitative measurement of the total, peroxyl radical trapping antioxidant capability of human blood plasma by controlled peroxidation. The important contribution made by plasma proteins. FEBS Letters 18, 33–37. Weiss, W.P., Mahan, D.C., 2008. Oxidative stress during the lifecycle of animals. Journal of Animal Science 86 (E-suppl. 2/J), 383. Wilburn, E.E., Mahan, D.C., Hill, D.A., Shipp, T.E., Yang, H., 2008. An evaluation of natural (RRR-a-tocopheryl acetate) and synthetic (all-rac-a -tocopheryl acetate) vitamin E fortification in the diet or drinking water of weanling pigs. Journal of Animal Science 86, 584–591. Winston, G.W., Regoli, F., Dugas Jr, A.J., Fong, J.H., Blanchard, K.A., 1998. A rapid gas chromatographic assay for determining oxyradical scavenging capacity of antioxidants and biological fluids. Free Radical Biology and Medicine 24, 480– 493.