Effect of dietary supplementation of trace elements on the lipid peroxidation in broiler meat assessed after a refrigerated and frozen storage *

The aim of this study was to determine the effect of dietary supplementation of Cu, Zn, Fe, Mn and Se on the lipid peroxidation in broiler meat assessed after the refrigerated and frozen storage. Broiler chicks Ross 308 were randomly divided at the day of hatching into 4 groups and fed for 42 days with the diets containing a different amount and form of Cu, Fe, Zn, Mn and Se. The diets for the 1st and 2nd group of birds were supplemented with the equivalent amounts of trace elements in an inorganic form (Cu sulphate, 5 mg/kg; Fe sulphate, Zn oxide and Mn oxide, 50 mg/kg), however sodium selenite or selenized yeast were given in a dose of Se 0.3 mg/kg, respectively. The 3rd and 4th group received the same feed as chickens in the 1st and 2nd group but with the highly reduced amount of supplemented nutrients in the organic “proteinated” form (Bioplex Cu, 2.5 mg/kg; Bioplex Fe, Bioplex Zn and Bioplex Mn, 10 mg/kg), except of selenium which was given in a dose of Se 0.3 mg/kg as sodium selenite or selenized yeast, respectively. The diet supplemented with the trace elements in the proteinated forms that were restricted to 50% (Cu), 20% (Fe, Zn and Mn) and on its regular level (Se) had the same effect on the carcass weight, weight of breast and thigh parts and the concentration of trace minerals in a muscle (except of Se) as the feeding of diet with the recommended dose of inorganic nutrients. The selenized yeast was shown to be more effective in the formation of Se deposit in the muscle of broilers than sodium selenite. On the other hand, the feeding of diet supplemented only with inorganic forms of Cu, Fe, Zn, Mn and Se influenced the quality of broiler meat assessed after the refrigerated and frozen storage. The peroxide value and malondialdehyde value increased significantly in breast and thigh muscle of chickens in this 500 LIPID PEROXIDATION IN BROILER MEAT AFTER STORAGE group, what could be possibly explained by the negative interactions between sodium selenite and transition metal ions when supplemented in the form of inorganic salts.


INTRODUCTION
The naturally favourable fatty acids profile in the chicken meat is a reason of increasing interest at the expense of the beef cuts (Carnevale de Almeida et al., 2006).Chicken meat contains a higher level of polyunsaturated fatty acids (Pípová et al., 1995), which have a beneficial effect on the human health.A big demerit of this meat is that during its storage a large degradation of lipid fractions is promoted, whose severity positively correlated with the concentration of polyunsaturated fatty acids (Cortinas et al., 2005).The processes causing the oxidative degradation of lipids are called lipid peroxidation (LPO) and are initiated by free radicals.
The free radicals are involved in the uncontrolled chain reaction that primarily affect the phospholipids.The cell membrane phospholipids are particularly susceptible to the oxidative damage.During the lipoperoxidative process, the peroxyl radicals are generated and prolong the subsequent chain reaction leading to lipohydroperoxides and lipid radicals, which form new radicals and make from these reactions a vicious circle.The lipohydroperoxides are easily decomposed to aldehydes, ketones, alcohols and lactones, which could significantly affect the organoleptic characteristics of poultry meat after their accumulation (Higgins et al., 1999), being potentially toxic for humans.Lipohydroperoxides, peroxyl radicals and hydroperoxides generated within the initiation and propagation of LPO can induce a further damage of proteins and DNA (Tirosh and Reznick, 2000).Therefore, the determination of malondialdehyde and other secondary products of LPO is routinely used for the assessment of meat deterioration.
It is widely established that a deficient as well as excessive intake of trace elements could cause an oxidative stress (e.g., lipid peroxidation) in poultry (Surai, 2002), but the negative consequences of LPO can be overcome by the adequate supplementation of antioxidants into the diet.Cu, Zn, Fe, Mn and Se are considered as antioxidant nutrients, Fe as a part of catalase, Cu, Zn and Mn as a part of superoxide dismutase and Se as a part of glutathione peroxidase and all have an interdependent role in the antioxidant protection of organism by the termination of LPO reactions.On the contrary, the same elements as the trasitional metal ions have the ability to generate highly reactive hydroxyl radical through their interaction with reactive oxygen species (Aruoma et al., 1991).
The aim of this study was to determine the effect of dietary supplementation of Cu, Zn, Fe, Mn and Se on the lipid peroxidation in broiler meat assessed after 24 PETROVIČ V. ET AL. and 72 h of refrigerated as well 2 and 6 months of frozen storage, respectively.

Animals, diets and treatments
Two hundred unsexed broilers Ross 308, were randomly divided at the day of hatching into 4 groups (50 per group) and were fed for 42 days with the diets containing a different amount and form of Cu, Fe, Zn, Mn and Se.The diets were fed ad libitum during a whole experimental period for the healthy development of broiler chickens with free access to water.The broiler feed "Starter" was fed for the 1 st to 21 st day, the broiler feed "Grower" for the 21 st to 35 th day and the broiler feed "Finisher" for the 35 th to 42 nd day, respectively.The diets and Bioplex Cu, Bioplex Fe, Bioplex Zn, Bioplex Mn and Sel-Plex were prepared and purchased from Alltech Inc. (Belgium).All premixes were fortified with phytase and xylanase, and a coccidiostatic preparation was complemented into the diets for the 1 st to 35 th day of the broiler´s life.The composition of diets fed to the broiler chickens during the entire experimental period is presented in Table 1. 2 2 2 L-lysine 1.5 1.5 1.5 DL-methionine 2 2 2 L-threonine 0.5 0.6 0.6 1 kg of basal diet contained, g: dry matter 879, crude protein 201, crude fat 73, ash 45, crude fibre 31, lysine 11.3, methionine 4.8, methionine+cystine 8.2, threonine 7.6, tryptophan 2.4, valine 9, arginine 12.9, histidine 5, Ca 6.8, P 4.5, Na 1.5, K 8.6, Cl -2, Cu 10; mg: Fe 150, Zn 35, Mn 15, Se 0.1; ME broiler 12.31 MJ; 1 premix supplied per kg of basal diet, IU: vit.A, 10000, vit.D 3 3000, vit.E 20; mg: vit.K 3, thiamine 2, riboflavin 8, niacin 15, cholinchlorid 50, pantothenic acid 50, pyridoxine 5, folic acid 2, biotin 0.2, I 2, Co 1; μg: cyanocobalamin 30 During the whole experiment, the broiler chickens in the 1 st and 2 nd group were fed with the diet supplemented with Cu 5 mg/kg DM (Cu sulphate), Fe, Zn and Mn 50 mg/kg DM (Fe sulphate, Zn oxide, Mn oxide), and Se 0.3 mg/kg DM in a form of sodium selenite or selenized yeast, respectively.The 3 rd and 4 th group received the same feed supplemented with highly reduced dose of trace elements (except of Se) in the proteinated forms: Cu, 2.5 mg/kg DM (Bioplex Cu), Fe, Zn and Mn, 10 mg/kg DM (Bioplex Fe, Bioplex Zn, Bioplex Mn) and Se, 0.3 mg/kg DM in the form of sodium selenite or selenized yeast (Sel-Plex), respectively.
At the 1 st day of life the broiler chicks were placed in one-level cages.The lighting regime was 23L:1D till the end of experiment.The initial room temperature 32°C was reduced every week by 3°C to a final temperature of 22-23°C.From 2 nd to 5 th day of age, the birds received an antibiotic (Enroxyl 5% premix, UNI Biotech) at the dose 0.5 ml per litre and Newcastle disease vaccine (AVIPEST Lyof., Mevak a.s.) was applied on the 18 th day of life.Both preparations were given into the drinking water.
The protocol was approved by the Local Ethical and Scientific Authorities.

Sample analysis
On the 42 nd day of experiment, the broiler chickens were slaughtered by decapitation.Bleeding of chickens followed after the stunning in accordance with the rules established for the slaughtering of animals and it was performed by the responsible veterinary surgeon (Bugarský et al., 2003).For the detection of lipid peroxidation (LPO) during chilling, the carcasses of 10 birds from each group were immediately trimmed for breast and thigh meat by removing skin, bones and connective tissues.Subsequent, the breast and thigh meat within each group was separately sliced, over-wrapped in the transparent oxygen-permeable polyvinyl chloride film and stored at 4°C for 24 and 72 h.A second part of samples of breast and thigh muscles was packed in the polyethylene sacks and was stored in freezer at -18°C during 2 and 6 months.Ahead of LPO analysis, the frozen samples were defrost at the chilling conditions (4°C) for 12 h, homogenized and analysed immediately.
Peroxide value examination was done according to the Veterinary Laboratory Methods (Edict of Ministry of Agriculture of Slovak Republic, 2003, No. 149/1/2003-100).
After preceding the wet mineralization in the microwave oven MLS 1200, the concentration of copper, zinc and iron in all samples was analysed by the flame AAS method (Perkin Elmer, AAnalyst 100), whereas the concentration of manganese was determined by the flameless AAS method (Perkin Elmer, 4100 ZL) and the concentration of selenium was measured by the same instrument equipped with a hydride generation system (Perkin Elmer, 4100 ZL with a quartz cuvette).

Statistical analysis
Statistical analysis was done using one-way analysis of variance (ANOVA) with the post hoc Tukey's multiple comparison test by the statistical software GrafPad Prism, Version 4.00 (2003).

RESULTS AND DISCUSSION
The differences in the carcass weights (g: 1803 ± 210.1, 1721 ± 155.1, 1817±153.2and 1839±122.7,respectively), breast parts in % of carcass weights (24.93, 25.53, 23.97 and 25.08, respectively) and thigh parts in % of carcass weights (29.33, 29.68, 28.3 and 28.11, respectively) found in all experimental groups of chickens after the slaughter, were not statistically significant.Nollet et al. (2007Nollet et al. ( , 2008) ) found no significant differences in the body weight between the groups of broilers fed the diets supplemented with 100, 67, 50, 33 and 17% of recommended dose of the organic form of minerals (Bioplex Cu, Bioplex Fe, Bioplex Zn, Bioplex Mn).The addition of trace elements (Cu, Fe, Zn, Mn and Se) in the organic "proteinated" form into the diet could be reduced to 33% of regular levels with comparison to the supplementation of their inorganic salts, without compromising the body weight of broilers (Peric et al., 2006;Nollet et al., 2007).These findings could be in accordance with our results especially when we considere that there is a linear correlation between the body weight and carcass weight.Rossi et al. (2007) observed that the body weight of broiler chickens was not affected by the form of Zn and Se presented in the diet but the carcass quality was improved only in the groups of birds fed the diet supplemented with the organic form of trace elements.
The concentration of Cu, Fe, Zn and Mn in the breast and thigh muscle was not significantly different within the groups of chickens but the concentration of iron and zinc was approximately two times higher in the thigh muscle compared to the breast muscle (Table 2).This could be explained by the different nature of both muscles as mentioned thereinafter.The concentration of Se in the breast and thigh muscle shown the pattern of Se deposition only in the groups of birds supplemented with the selenized yeast.Schrauzer (2000) found the proportion of selenomethionine, that is escaping metabolism to H 2 Se, is non-specifically incorporated into the structural proteins of muscles, where it replaces common methionine.On the other hand, sodium selenite is unable to build significant Se deposits in the muscle of poultry (Kuricová et al., 2003;Petrovič et al., 2006) and is rapidly excreted with other trace elements in the inorganic form via the faeces of birds (Nollet et al., 2007).From the results found in the 1 st group of birds it is apparent that the supplementation of diet only with the inorganic forms of trace elements (Cu, Fe, Zn, Mn and Se) significantly influences the lipid peroxidation (LPO) by increasing the peroxide number (Figure 1, Table 3) as well as the TBARS  4) in the poultry meat.In recent experiments, the increased MDA levels in the organs of chickens revealed the pro-oxidative properties of sodium selenite (Balogh et al., 2004;Petrovič et al., 2006).The formation of superoxide anion during the reduction of selenite into H 2 Se (selenide) by glutathione in the cells induced LPO (Kobayashi et al., 2001).Moreover, the superoxide anion formed during this redox reaction could cause that the transition metals from the supplemented inorganic salts are not bound to the proteins and instead catalyse the formation of hydroxyl radical what subsequently increase LPO.This statement could be considered as a pure speculation but can explain the increased peroxide value and malondialdehyde value in muscles of chickens in 1 st experimental group.On the other hand, the negative interactions between the elements declined when their proteinated forms were supplemented into the diet (Du et al., 1996), which is in accordance with the results found in 2 nd , 3 rd and 4 th experimantal group of chickens.
Chicken thigh muscles are considered oxidative with more mitochondria and a higher content of myoglobin compared to glycolytic breast muscles.Oxidative muscles use fatty acids as energy substrates and have lower activities of phosphorylases than the glycolytic muscles which use glycogen as a source of energy.The higher levels of lipid peroxidation in the thigh muscle compared to the breast muscle confirmed the presence of higher amount of fatty acids in this part of chicken meat.
It is hypothesized, that in slaughtered animals the Fe containing haem groups of haemoglobin and myoglobin play an important role in the biochemical processes that turn the muscle into meat and thus influence the resistance of meat during its storage.The latter fact is of importance as the poultry meat contains a significantly higher concentration of haemoglobin as compared to other tissues and organs (Kranen et al., 1999).Moreover, the addition of manganese into the broiler diet decreased MDA content in the leg muscle by increasing the MnSOD activity (Lu et al., 2006) and might reduce the fat deposition by decreasing the activities of lipoprotein lipase and malate dehydrogenase activities or increasing the activity of hormone-sensitive lipase in the adipose tissue (Lu et al., 2007).The reduced amount of supplemented trace elements in the organic form in the 4 th group of broilers had more beneficial effect on the lipid peroxidation than the supplementation of large portion of trace minerals in the inorganic form in the 1 st group.This could be explained probably due to a higher bioavailability of nutrients presented in a feed in the proteinated form.

CONCLUSIONS
In conclusion, the feeding of diet supplemented with the organic form of trace elements, which were reduced to 50% (Cu), 20% (Fe, Zn and Mn) and on its regular level (Se) have the same effect on the body weight, weight of breast and thigh parts and the concentration of supplemented minerals (except of Se) in the muscles as the feeding of diet with recommended dose of nutrients in the inorganic form.The selenized yeast showed to be more effective in the formation of Se deposit in the muscle of broilers than sodium selenite.On the other hand, the feeding of diet supplemented only with inorganic forms of Cu, Fe, Zn, Mn and Se influenced the quality of broiler meat assessed after the refrigerated and frozen storage.The peroxide value and malondialdehyde value increased significantly in breast and thigh muscle of chickens in this group, what could be possibly explained by the negative interactions between sodium selenite and transition metal ions when supplemented in the form of inorganic salts.

Figure 1 .
Figure1.The effects of supplementation of the diet for broiler chickens with different form and amount of Cu, Fe, Zn, Mn and Se on the peroxide values in breast and thigh muscle assessed after 24 h storage at + 4°C.Values are means ± SD, n=10 in each group.Distinct letters above columns mean significant differences between treatments ( XY P<0.001)

Table 1 .
The composition of diets fed to the broiler chickens Ross 308 during the entire experimental period

Table 2 .
The effects of supplementation of the diet for broiler chickens with different form and amount of Cu, Fe, Zn, Mn and Se on the concentration of these trace elements (mg/kg -1 DM) in breast and thigh muscle after slaughter

Table 3 .
The effects of supplementation of the diet for broiler chickens with different form and amount of Cu, Fe, Zn, Mn and Se on the peroxide values (μg/g -1 ) in breast and thigh muscle assessed after the refrigerated and frozen storage A 7.8 b 9.21 c 11.2 D 0.78 69.3A 30.9 B 25.2 C 44.7 D 2.4 distinct superscripts within row -significant difference ( xY P<0.05; xY P<0.01; XY P<0.001).Values are means; n=10

Table 4 .
The effects of supplementation of the diet for broiler chickens with different forms and amounts of Cu, Fe, Zn, Mn and Se on the thiobarbituric acid reactive substances (μg MDA .g -1 ) in breast and thigh muscle assessed after the refrigerated and frozen storage A 0.218 b 0.169 B 0.192 b 0.056 2 months -18°C 0.057 A 0.052 A 0.048 a 0.037 B 0.008 0.228 A 0.155 B 0.182 b 0.177 b 0.026 6 months, -18°C 0.081 A 0.064 B 0.06 B 0.054 C 0.006 0.32 A 0.198 b 0.221 b 0.196 b 0.015 distinct superscripts within row -significant difference ( xy P<0.01; XY P<0.001).Values are means; n=10 (Table