EFFECTS OF SELENIUM SUPPLEMENTATION ON SERUM CORTISOL IN REPEATEDLY HANDLED CATTLE

This study evaluated the relationship between Se supplementation and serum cortisol in repeatedly handled cattle. Sixty Nellore calves were randomly distributed into four experimental groups, which were fed a mineral-protein mixture added with Se to achieve individual daily supplementation of 0 (Gc), 3.6 (G3.6), 5.4 (G5.4) and 6.4 (G6.4) mg Se. Stressful handling procedures and blood sampling were performed on days 0, 15, 30, 60, 90 and 120. Serum cortisol increased until day 90 and decreased on day 120, irrespective of Se supplementation. This finding shows that cattle reached a stress state but adapted. Cortisol and Se levels were not markedly correlated. Serum Se increased over the course of the experiment in the supplemented groups. However, because serum Se decreased in Gc, it may have mobilized to form the selenoproteins needed to remove metabolic wastes from handling-related oxidative stress.


INTRODUCTION
Handling cattle in the corral is a common practice that causes physical, psychological and metabolic and activation of the hypothalamus-pituitary-adrenal axis (HPA) and consequent release of cortisol, adrenalin and noradrenalin by the adrenal gland.It can promote oxidative stress, thereby increasing hydroperoxide and lipid peroxide production.These free radicals are detoxified by the glutathione peroxidase (GSH-Px), which can convert them into oxygen and water or non-toxic fatty acid hydroxides (Carroll and Forsberg, 2007).

MATERIALS AND METHODS
Sixty uncastrated male Nellore calves (Bos taurus indicus) aged 10 to 12 months and acclimatized to an extensive pasture system in Brachiaria decumbens pasture for at least 30 days were tested.They were equally and randomly distributed into four experimental groups that received ad libitum protein-mineral mixture supplemented with Se at different concentrations.Considering a daily consumption of 200 g of mixture, individual Se supplementation provided by each diet was 0 mg (Gc, Se-free mixture), 3.6 mg (G 3.6 , 18 mg Se/kg feed), 5.4 mg (G 5.4 , 27 mg Se/kg feed) and 6.4 mg (G 6.4 , 32 mg Se/kg feed).The protein mineral mixture (Top Line Recria ® , Matsuda Sementes e Nutrição Animal) contained calcium, phosphorus, magnesium, sodium, cobalt, copper, sulphur, iron, iodine, manganese, nickel, zinc, vitamin A, vitamin D, vitamin E, buffer, 400 kcal metabolizable energy, 140 mg non-protein nitrogen (maximum equivalent protein), 650 mg fluorine (maximal), 95% phosphorous solubility in citric acid 2% (minimum).Cattle groups were held in paddocks of similar topography.They were moved from one paddock to another every 30 days.The paddocks had stocking density of 100 kg live weight per hectare.Pasture samples were collected at grazing height and stored at -5°C for Se determination.
On days 0, 15, 30, 60, 90 and 120, the cattle was submitted to handling followed by blood sampling.Blood was collected from the jugular vein in vacuum tubes without anticlotting agent and centrifuged at 2500 rpm for 10 min.Serum samples were stored at -20°C until analyzed using a commercial kit for solid phase radioimmunoassay (DPC-Diagnostic Products Corporation, USA).Se concentration in serum (days 0, 60 and 120) was determined by graphite furnace atomic absorption spectrophotometry.The involved stressors were: conveyance of the animals to the corral; entry into a new environment (corral); stay in the corral; forced exercise during handling in the corral; presence of people speaking loudly and restraint in the stunning box for 5 min for blood sampling.
Cortisol levels were compared among the groups and among the observation days by mean of repeated measures using ANOVA followed by the LSD test.Data on Se levels violated normality predictions of distribution (Shapiro-Wilk test) and were analyzed using Kruskal-Wallis analysis (H) for comparisons among the groups and Friedman (χ 2 r ) for comparisons among the days.Significant differences were compared by the Nemenyi multiple comparisons test.Associations between Se and cortisol levels were tested by Spearman's Rank Correlation test (r s ).The alpha error was set at 5%.

RESULTS
Cortisol levels were neither affected (p= 0.79) by Se supplementation (data pooled for each day) nor by any interactions between Se concentration and time (p= 0.14) (figure 1).However, they increased gradually over the experiment (p<0.01;data pooled for each group); a peak on day 90 was followed by a cortisol drop on day 120 (figure 2).
A comparison of groups showed that Se level was higher in Gc than in G 5.4 and G 6.4 only on day 0 (p= 0.005).Over the course of the experiment, Se decreased only in Gc from day 60 on (p<0.004).In G 3.6 , it increased progressively (p<0.01).In G 5.4 , Se level was higher on day 120 than on day 0 (p<0.01),whereas in G 6.4 it was high from day 60 on (p<0.002)(figure 3).Of 12 associations tested, we found a negative correlation only between cortisol and Se levels in G 6.4 on day 0 (rs= -0.514; p>0.050).
Se levels in the pasture were similar among the paddocks (0.04 mg Se/kg dry matter).

DISCUSSION
Cortisol is a good indicator of chronic stress in cattle.Therefore, the increased levels of this hormone over the experiment indicate that the repeated handling procedures adopted, stressed the cattle.At the beginning of the experiment, the cattle was likely not stressed because cortisol levels were nearly 2.50 µg/dL.This is lower than the baseline cortisol values reported earlier for Zebu (3.29 µg/dL; Vásquez and Herrera, 2003) and Nellore cattle (3.68 µg/dL; Reis et al., 2006).As indicated by the cortisol levels, corral handling did not stress the cattle in the first 2 weeks of the experiment, but after day 15, serum cortisol increased gradually to a peak on day 90.This finding corroborates Andrade et al. (2001) and Reis et al. (2006), who also considered handling inside the corral as a cattle stressor.
On day 120, serum cortisol concentrations dropped significantly (p<0.01;figure 2), possibly because the cattle adapted to the handling procedures even though baseline cortisol levels had not recovered.Other studies report cattle adaptation to handling procedures (Solano et al., 2004;Figure 2. Effect of handling stressor on serum cortisol concentrations (± sd) after repeated handling on days 0, 15, 30, 60, 90 and 120.Means followed by different letters indicate significant differences (p<0.01).Data from groups Gc, G 3.6 , G 5.4 and G 6.4 were pooled.
An intriguing result was observed in the non-supplemented group.Serum Se decreased in Gc cattle (figure 3).This can be expected in Se-deficient pastures such as the Brachiaria decumbens analyzed.Se levels in this forage were in fact more than twofold lower than that recommended by the NRC (2000) for beef cattle.However, we can not rule out the hypothesis that serum Se in this group was decreased because this element was mobilized to form the selenoproteins needed to remove metabolic wastes from handling-related oxidative stress.This is an interesting topic for further study.
On the basis of the experimental conditions and the results obtained we conclude that: Se supplementation with 3.6; 5.4 and 6.4 mg per animal per day does not affect serum cortisol concentrations in cattle; repeated handling inside the corral is a stress agent; cattle adapt to the handling procedures after 120 days, but serum cortisol concentration does not return to baseline values after 120 days.