ANTIOXIDANT STATUS IN DAIRY COWS DURING LACTATION

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1 Buletin USAMV-CN, 63/2006 ( ) ISSN ANTIOXIDANT STATUS IN DAIRY COWS DURING LACTATION Pintea Adela, Daniela Zinveliu, R. Al. Pop, Sanda Andrei, Ervin Kiss Department of Chemistry and Biochemistry, University of Agricultural Sciences and Veterinary Medicine, 3-5 Manastur street, , Cluj-Napoca, Romania, Key words: dairy cows, SOD, GPX, catalase, MDA, uric acid, carotenoids Abstract: During the transition period important metabolic changes occur in dairy cows, which can also experience oxidative stress. The study was carried out to asses the activity of antioxidant enzymes, the level of lipid peroxidation ant the level of serum antioxidants, carotenoids and uric acid. SOD activity in cows was situated around 1365 U/g Hb in the first week after parturition, 1890 U/g Hb in the second week, 2290 c in the third week, 1896 U/g Hb in the fourth week, and 1905 U/g Hb in the sixth week after parturition, value close to that of the cows in late lactation (2005 U/g Hb). GPX activity in cows was situated around 129 U/g Hb in the first week after parturition, and than increased to reach 166 U/g Hb in the sixth week after parturition, value comparable with that of cows in late lactation (160 U/g Hb). The same pattern of enzyme activity modification was observed for blood catalase. The maximum level of lipid peroxidation was observed in the first week after parturition (62 µmols/l), comparing with the level of MDA in the cows in late lactation (35 µmol/l). In the second week after parturition the level of MDA decrease significantly, and remains at approximatively same values during the experiment. The high level of MDA in the first week after parturition correlates with a low activity of antioxidant enzymes (SOD, GPX, catalase) and a low level of antioxidants. The experimental data show that during parturition and onset of lactation oxidative stress appears in dairy cows, but these modifications are of short-therm. INTRODUCTION Parturition and the onset of lactation represent periods of drastic physiological changes and intense metabolic demands in dairy cows. Important changes occur during parturition and early lactation, especially in the lipids and proteins metabolism [Marcos et al., 1990; Grum et al., 1996 ; Minor et al., 1998, Bell, 1995; Drackley, 1999; Doepel et al., 2002; Park, et al., 2002; Turk et al., 2004; Castillo, 2006]. The transition period is under strict hormonal control, which coordinate the activity of all tissues and organs in order to respond to this metabolic demands [Castillo et al, 2005 ; Vernon, 2002; Castillo et al. 2006]. The intense metabolic processes are accompanied by modification of energetic metabolism and by an increase of oxygen consumption. This lead to an increase in the generation of reactive oxygen species (ROS), which are normally neutralized by enzymatic and non-enzymatic defense systems of living organisms. The imbalance between the rate of ROS production and their neutralization lead to the oxidative stress [Frei et al., 1988; Sies, 1991; Trevisan, 2001]. It was shown that during transition period, oxidative stress can occur and it can contribute to some periparturient disorders or metabolic diseases [Miller and Madsen, 1994; Brezezinska- Slebodzinska et al., 1994; Ronchi et al., 2002 ; Bernabucci et al, 2002; Bernabucci et al, 2005]. The aim of this study was the evaluation of antioxidant status during early lactation period, first 6 weeks after parturition, comparing with late lactation. The activity of main antioxidant enzymes: superoxiddismutase (SOD), erythrocyte glutathion peroxidase (GPX), catalase was determined, together with the level of lipid peroxidation level (malondialdehyde 130

2 assay MDA). Uric acid and carotenoids, known as non-enzymatic antioxidants, were also determined in serum [Hicks et al., 1993; Krinsky, 1994]. MATERIALS AND METHODS The experiment was carried in U.S.A.M.V Cluj-Napoca farm, on a group of ten cows Baltata Romaneasca, in a period of six weeks (end of March May). All the cows gave birth in the same period and were fed with the same diet during the experiment. SOD was determined in the erythrocytes lysate using the kit RANSOD, from Randox Laboratories. It is based on the inhibition of I.N.T. (a tetrazolium salt) oxidation by superoxid anion generated in xanthine/xanthine oxidase system., in the presence of blood SOD. GPX activity assay is based on the method of Paglia and Valentine and uses the Ransel kit from Randox Laboratories. For both enzymes the hemoglobin content in blood was measured and the activity expressed as Units/g Hemoglobin. Catalase activity was determined by a titrimetric method, using potasium permanganate. The level of lipid peroxidation was determined using the spetrophotometric assay with thiobarbituric acid. Total carotenoids were estimated by spectrophotometric assay, at 450 nm, of an hexanic extract obtained from plasma. Uric acid was determined using an enzymatic method with uricase, provided by Hospitex Diagnostic. RESULTS AND DISCUSSIONS Superoxiddismutase (SOD) accelerates the dismutation of the toxic superoxide radical to hydrogen peroxide and is considered the first intracellular defense against reactive oxygen species. The cytosol of all eukaryotic cells contains CuZn-SOD. Determination of SOD is important in the evaluation of antioxidant status, under physiological or pathological conditions. As can be seen in Fig. 1, the SOD activity has an average of 2045 U/g Hb for cows in late lactation. A significant decrease of SOD activity can be observed in dairy cows in the first week after parturition. The activity increased starting with the second week, and was close to the control after six weeks Glutathione peroxidase (GPX) plays an important role in cellular antioxidant defense. It uses glutathione as reducing agent and reduces hydrogen peroxide and other peroxides to water, or respectively to the corresponding alcohol. Determination of GPX activity might be beneficial in the evaluation of selenium status and in the evaluation of antioxidant status. For the cows in late lactation the average GPX activity was situated around 169 U/g Hb. One week after parturition a decrease of GPX activity was observed, followed by a significant increase in the second week, and a normalization after six weeks (Fig. 2). The high values of GPX and SOD in the second, and respectively in the third week can be interpretate as an effort of the organism to adapt to a high level of ROS production. The same pattern of enzyme activity modification was observed for blood catalase. 131

3 Fig. 1. Activity of blood superoxidedismutase (SOD) during lactation U/g Hb late lactation week 1 week 2 week 3 week 4 week late lactation week 1 week 2 week 3 week 4 week 6 Malondyaldehyde (MDA) is considered the final product of lipid peroxidation and a marker of oxidative stress. As can be observer in the Table 1, the maximum level of lipid peroxidation was observed in the first week after parturition (62 µmols/l), comparing with the level of MDA in the cows in late lactation (35 µmol/l). The total lipid serum concentration is also lower in the first week, and this is explained by an intense lipolisys and mobilization of fatty acids in order to sustain the lactogenesis. A similar pattern was observed in recent studies regarding MDA and TAS (total antioxidant status) in dairy cows [Castillo et al., 2005, Castillo et al., 2006]. The modification is of short-therm, since in the second week after parturition the level of MDA decrease significantly, and remains at approximatively same values during the experiment. The high level of MDA in the first week after parturition correlates with a low activity of antioxidant enzymes (SOD, GPX, catalase). Total carotenoids and uric acid concentration in serum were also low in the first week after parturition (Table 1). All these data show that the parturition and onset of lactation can induce, beside the important modification metabolic status, an oxidative stress. 132

4 Fig. 2. Activity of erythrocyte glutathione peroxidase (GPX) during lactation U/g Hb late lactation week 1 week 2 week 3 week 4 week late lactation week 1 week 2 week 3 week 4 week 6 Fig. 3. Serum MDA level during lactation micromols MDA/l late lactation week 1 week 2 week 3 week 4 week late lactation week 1 week 2 week 3 week 4 week 6 133

5 Table 1. Concentration of total lipids, MDA, carotenoids and uric acid during lactation Sample Total lipids mg/100 ml Total carotenoids µg/100 ml Uric acid mg/100 ml Late lactation 500.6± ± ±0.08 Week ± ± ±0.06 Week ± ± ±0.09 Week ± ± ±0.08 Week ± ± ±0.07 Week ± ± ±0.10 We have to remark that the inter-individual variations on MDA values, as well as for enzyme activities are rather high and this can be linked to the adaptative capacity of each organism, in a order to optimize the consumption of oxygen and neutralization of free radicals. CONCLUSIONS Besides metabolic parameters, the antioxidant status in dairy cows changes during parturition and early lactation. The activity of antioxidant enzymes: SOD, GPX, catalase is lower in first week after parturition and increase afterward, reaching the normal value for dairy cows in six weeks. It correlates with low level of serum antioxidants: carotenoids and uric acid, and with a high level of lipid peroxidation (MDA concentration). The experimental data show that during parturition and onset of lactation oxidative stress appears in dairy cows, but these modifications are of short therm. BIBLIOGRAPHY 1. Bell, A.W., 1995, Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation, Journal of Animal Sciences, 73, Bernabucci, U., B. Ronchi, N. Lacetera, A. Nardone, 2002, Markers of oxidative status in plasma and erythrocytes of transition dairy cows during hot season, Journal of Dairy Science, 85, Bernabucci, U., B. Ronchi, N. Lacetera, A. Nardone, 2002, Influence of body condition score on relathionships between metabolic status and oxidative stress in periparturient dairy cows, Journal of Dairy Science, 88, Brezezinska-Slebodzinska, E.J., J.K. Miller, J.D. Quigley, J.R. Moore, 1994, Antioxidant status of dairy cows supplemented prepartum with vitamin E and selenium, J. Dairy Sci. 77, Castillo, C., J. Hernandez, A. Bravo, M. Alonso-Lopez, V., Pereira, J.L. Benedito, 2005, Oxidative status during late pregnancy and early lactation in dairy cows, The Veterinary Journal, 169, Castillo, C., J. Hernandez, I. Valverde, V. Pereira, J. Sotillo, M. Alonso-Lopez, J.L. Benedito, 2006, Plasma malonaldehyde (MDA) and total antioxidant status (TAS) during lactation in dairy cows, Research in veterinary Science, 80, Doepel, L., H. Lapierre, J.J. Kenelly, 2002, Peripartum performance and metabolism in dairy cows in response to prepartum energy and protein intake, Journal of Dairy Science, 85, Drackley, J.K., 1999, Biology of dairy cows during the transition period: The final frontier? J. Dairy Sci. 82, Frei, B.R., R. Stocker, B.N. Ames, 1988, Antioxidant defenses and lipid peroxidation in human blood plasma., Proc. Natl. Acad. Sci.USA, 85, Grum, D.E., J.K. Drackley, L.R. Hansen, J.D. Jr Cremin, 1996, Production, digestion and hepatic lipid metabolism of dairy cows fed incresead energy from fat or concentrate, Journal of Dairy Science, 79,

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