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The effect of dietary supplementation with the natural carotenoids curcumin and lutein on pigmentation, oxidative stability and quality of meat from broiler chickens affected by a coccidiosis challenge a
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N. Rajput , S. Ali , M. Naeem , M.A. Khan & T. Wang a
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, P. R. China b
Key Laboratory of Meat Processing and Quality Control, Nanjing Agricultural University, Nanjing, P. R. China Accepted author version posted online: 23 May 2014.Published online: 22 Aug 2014.
To cite this article: N. Rajput, S. Ali, M. Naeem, M.A. Khan & T. Wang (2014): The effect of dietary supplementation with the natural carotenoids curcumin and lutein on pigmentation, oxidative stability and quality of meat from broiler chickens affected by a coccidiosis challenge, British Poultry Science, DOI: 10.1080/00071668.2014.925537 To link to this article: http://dx.doi.org/10.1080/00071668.2014.925537
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British Poultry Science, 2014 http://dx.doi.org/10.1080/00071668.2014.925537
The effect of dietary supplementation with the natural carotenoids curcumin and lutein on pigmentation, oxidative stability and quality of meat from broiler chickens affected by a coccidiosis challenge N. RAJPUT, S. ALI1, M. NAEEM, M.A. KHAN1
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T. WANG1
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College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, P. R. China, and 1 Key Laboratory of Meat Processing and Quality Control, Nanjing Agricultural University, Nanjing, P. R. China
Abstract 1. An experiment was performed to evaluate the effectiveness of the antioxidants curcumin (CRM) and lutein (LTN) on the quality of meat from coccidiosis-infected broilers. A total of 200 one-dayold Arbor Acre chicks were randomly assigned to a treatment group with 5 replicates. The treatments included a basal diet without carotenoid supplementation (control), with 300 mg/kg CRM, with 300 mg/kg LTN or with a combination (C + L) of 150 mg/kg CRM and 150 mg/kg LTN. All chickens were challenged with Eimeria maxima at 21 d old. 2. The results revealed that the coccidiosis reduced redness of meat, while supplementation with carotenoids improved the fresh meat’s redness (a*) and yellowness (b*) and contributed to colour stability maintenance after storage (1 month at −18°C and 3 d at 4°C). 3. Coccidiosis did not produce lipid and protein oxidation in fresh meat, but after storage for one month, the malondialdehyde levels and carbonyl contents were lower in the CRM and C + L birds and the sulfhydryl contents were higher in C + L birds. 4. The sodium dodecyl sulphate–polyacrylamide gel electrophoresis banding pattern showed equivalent myosin chain fragmentations in all treatment groups, whereas lower intensity actin bands were observed in the control group (CONT). Moreover, myofibril protein denaturation (differential scanning calorimetry) profiles showed a reduction in the CONT myosin and actin peaks. Coccidiosis reduced the meat’s water holding capacity in non-supplemented chicken meat and was improved by natural carotenoid. 5. These results emphasise that coccidiosis did not decrease the eating quality of fresh meat, that natural carotenoids are efficient antioxidants and that CRM (300 mg/kg) fed individually or combined with LTN was the most effective supplemented antioxidant compound.
INTRODUCTION Coccidiosis caused by various species of Eimeria is considered to be the most commonly occurring disease in chickens and may produce health and welfare problems and economical loses (Velkers et al., 2010; Shaw et al., 2012). Structural changes and decomposition in muscle tissue have been reported due to Eimeria acervulina infection (Fetterer and Allen, 2000). Coccidiosis severely increases blood loss in the muscles, which results in decreased tissue pigmentation and pale meat
(Fetterer and Allen, 2001). Immune cells are activated by the pathogenic organism and produce various mediators (i.e. reactive oxygen and nitrogen species, singlet oxygen, peroxides and cytokines) to remove and destroy the organisms that can produce oxidative stress (Shanmugasundaram and Selvaraj, 2011). The excessive production of immune cell mediators against oxidative stress may also cause oxidative damage (Smet et al., 2008). Oxidation is a common process that affects pigments, fat and proteins and reduces the shelf life of meat (Morrissey et al., 1998).
Correspondence to: Tian Wang, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, P. R. China. E-mail: [email protected] Accepted for publication 29 March 2014.
© 2014 British Poultry Science Ltd
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The oxygenated free-radicals damage cellular structures such as lipids, proteins and myoglobin, which, in turn, stimulates pigment oxidation (Faustman and Cassens, 1990; Mercier et al., 1998). Lipid oxidation can also be initiated by protein-free radicals (Stadtman and Berlett, 1997; Mercier et al., 1998). Cellular damage by free radicals has not been properly addressed in chicken meat. Anti-coccidial drugs and vaccinations are the most commonly applied methods of controlling coccidiosis. However, certain laws limit the use of antimicrobial drugs, and satisfactory cost-effective vaccines have not yet been developed. Furthermore, the residual effect of drugs in meat and increasing consumer demand for organic food (Velkers et al., 2010; Anosa and Okoro, 2011) have highlighted the need for studies in this area and prompted searches for alternate feed additives to prevent coccidiosis. Carotenoids are natural colouring agents synthesised by plants that produce skin pigments in birds and have strong antioxidant, anti-inflammatory and antimicrobial properties (Lokaewmanee et al., 2011). Lutein (LTN) is a carotenoid that is naturally found in marigold flowers and dark green vegetables. Curcumin (CRM) is not only a food colouring agent but also an important compound in Ayurveda medicine, known as a “cleanser of the body” (Gandhi et al., 2011). In China, the United States and many other countries, the colour of chicken skin and meat plays an important role in consumer preference, as meat quality is often assessed on the basis of pigmentation (Liu et al., 2008; Valeria and Williams, 2011). From a commercial point of view, the major drawback of coccidiosis is a decrease in the quality and colour stability of the meat. Previous studies have shown that carotenoids (especially CRM at a dose of 200 mg/ kg feed) have positive effects on the gut morphology, digestion and immunomodulation of broilers and on the pigmentation and oxidation of broiler meat (Rajput et al., 2012, 2013a, 2013b). In the present trial, broilers were challenged with Eimeria maxima parasites, as it is well established that coccidiosis weakens the immunity of broilers and decreases the pigmentation and quality of their meat. We hypothesised that carotenoids can decrease the activity of free radicals produced by the oxidative stress caused by coccidiosis. Many studies on the effect of coccidiosis in broiler production have been conducted; however, few studies have examined the effect of coccidiosis on the oxidative stability and the shelf life of meat. To the best of our knowledge, no studies have been conducted on the effects of natural carotenoids on the quality and oxidative stability of the meat of coccidiainfected chickens.
MATERIALS AND METHODS Feeding management A total of 200, one-day-old Arbor Acre broiler chicks were randomly assigned into 4 treatment groups with 5 replicates. The antibiotic and coccidiostat-free basal diet was a rice-soybean-based diet (Table 1), formulated on the recommendation of National Research Council (NRC, 1994), to fulfil the nutrient requirements of broiler chicken. The birds were fed starter feed (1–21 d) and grower feed (22–42 d). The basal diet was supplemented with 300 mg/kg CRM, 300 mg/kg LTN or a combination (C + L) of CRM 150 mg and LTN 150 mg/kg diet. The birds in the control group (CONT) were fed on the basal diet without supplementation of any pigment. On d 21, all the birds were orally infected with purified Emeria maxima sporulated oocysts (5000 oocysts per broiler) diluted in 0.5 ml of distilled water. A suspension containing a specific amount of infective E. maxima sporulated oocysts (Figure 1) was kindly supplied by Key lab of Veterinary Parasitology, Nanjing Agricultural University, Nanjing, People’s Republic of China. CRM and LTN were obtained from a commercial company (Guangzhou Leader Biotechnology Co Ltd, China) with the purity of 98%. The Institutional Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, People’s Republic of China) approved all of the procedures. Table 1. Feed ingredients and nutrient composition of basal diets for broiler chickens Item Ingredients (g/kg) Rice Soybean meal Maize gluten meal Limestone Dicalcium phosphate Sodium chloride Feed premix1 L-Lysine DL-Methionine
Starter (1–21 d)
Finisher (22–42 d)
602.5 325 30 12 20 3 5 1 1.5
654.7 280 25 12 18 3 5 1 1.3
Calculated chemical composition of diet (g/kg) ME (MJ/kg) 12.54 Crude protein 211.9 Lysine 12.1 Methionine 4.96 Methionine + cystine 8.0 Ca 9.88 Available P 5.31 1
12.65 192.0 10.9 4.40 7.2 9.49 4.85
Mineral premix provided the following per kilogram of diet: transretinyl acetate, 25 mg; cholecalciferol, 6 mg; menadione, 1.2 mg; thiamine, 2.3 mg; riboflavin, 8 mg; nicotinamide, 42 mg; choline chloride, 400 mg; calcium pantothenate, 10 mg; pyridoxine HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; cobalamin, 0.012 mg; Fe (from ferrous sulphate), 82 mg; Cu (from copper sulphate), 7.5 mg; Mn (from manganese sulphate), 110 mg; Zn (from zinc oxide), 64 mg; I (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.28 mg.
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Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE)
Figure 1. Sporulated oocysts of Eimeria maxima orally given to chickens.
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Sample collection At 42 d of age (marketing age), blood samples were taken from one randomly selected bird per replicate; plasma was separated by centrifugation at 300 × g for 10 min at 4°C and stored at −20°C for further analysis. After blood collection, all birds were killed by exsanguination, de-feathered, and after 45 min of post-mortem, the colour and pH of the meat was measured. The meat samples were cut into 4 × 3 × 1 inch cubes and all of the visible fat and connective tissues were removed. The antioxidant parameters were determined 3 h after slaughtering; these data were classified as “fresh meat” data. The remaining samples were immediately packed in moisture-impermeable polyethylene bags and stored at −20°C for one month. After one month, all of the frozen samples were thawed in a refrigerator at 4°C for 3 d and antioxidant activities were measured; these data were classified as “stored meat” data. Muscle colour and pH One randomly selected bird per replicate was used for the determination of breast and thigh muscle pH and colour. The breast muscle pH was measured by inserting the probe of a pH meter (Thermo Scientific Orion 3 Star, Singapore) into the muscles. The L* value (lightness), a* value (redness) and b* value (yellowness) of the breast and thigh muscles were measured with a Konica Minolta chroma meter CR-400 (Minolta Camera Co., Osaka, Japan) 45 min after air exposure to allow for blooming.
The meat was treated in Mini-PROTEAN Tetra Cell (Bio-Rad sodium dodecyl sulphate-polyacrylamide gel electrophoresis) (Bertram et al., 2006). The sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, in which a sample of minced meat was dissolved to obtain a 1 µg/µl protein concentration, was composed of 4% SDS, 20% glycerol, 0.125 M tris HCl and 10% 2-mercaptoethanol with a pH of 6.8. Ten microliter aliquots of the samples were loaded in the sample wells of the stacking gel. A molecular weight (MW) standard, consisting of a cocktail of proteins of known molecular weight (TaKaRa, Dalian Co. Ltd., Beijing, China), was also run, and protein bands in meat samples were tentatively identified by comparing their electrophoretic patterns with standard markers.
Protein denaturation: differential scanning calorimetry Differential scanning calorimetry (DSC) was carried out using a Micro DSC-II Setaram Calorimeter (Setaram, Caluire, France); 0.2 g of minced whole muscle was loaded into three 1 cm stainless steel cuvettes. The samples were scanned over the temperature range 10–90°C at a rate of 0.5°C/min against a water reference (Bertram et al., 2006).
Water holding capacity The water holding capacity of the meat was measured according to the method of Omana et al. (2010). Raw meat (approximately 2 g) was placed between a pre-weighed Whatman (No. 1) filter paper and between two glass plates. Using the texture profile analyser (TA-XT Express, Stable Micro Systems Ltd., Surrey, England) in adhesive test mode, the samples were tested with a target force of 111 kg for 5 min. After the test, the filter paper with the absorbed water was immediately weighed. Expressible moisture was measured as the quantity of water released per gram of meat and expressed as a percentage. Expressible waterð%Þ ¼ ðwet paper drypaper=meat weightÞ 100
Carbonyl contents and sulfhydryl level
Statistical analysis
Carbonyl groups were detected by the reactivity with 2, 4-dinitrophenylhydrazine to form protein hydrazones, and sulphydryl (SH) groups (thiol content) were determined according to previously described methods (Xia et al., 2009).
Statistical analyses were performed using SPSS software (SPSS 15.0 K for Windows, Chicago, IL, USA), and all data were expressed as the means ± SD values. Comparisons of the mean values were performed by one-way analysis of
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antioxidants can attenuate these effects (Anosa and Okoro, 2011). Carotenoid supplementation did not produce any effect on muscle pH before or after storage of meat (Table 2).
variance. Significant differences among means were evaluated by Tukey’s comparison test at P < 0.05.
RESULTS AND DISCUSSION
Lipid oxidation The extreme oxidation of lipids caused by high levels of free radicals is the main cause of oxidative stress; this oxidation, in turn, enhances the levels of malondialdehyde (MDA), which is the end product of lipid peroxidation (Kim et al., 2012). In the current study, lipid oxidation (as measured by MDA) was significantly lower in the meat of animals from the C + L and CRM group (Figure 2). In addition, lipid oxidation tends to increase with longer frozen storage times and higher meat fat contents (Wood and Enser, 1997). We previously reported that CRM decreased the cholesterol and fat content of meat in a dose-dependent manner (Rajput et al., 2013a). Lipid and pigment oxidation in meat are closely associated, and supplementation with antioxidants can increase the colour stability of the meat (Faustman and Cassens, 1990). Moreover, a review of the literature revealed that supplementation with vitamin E improved the colour and lipid stability of meat by delaying the oxidation of phospholipids (Mercier et al., 1998); interestingly, CRM has been reported to be a better antioxidant than vitamin E (Reddy and Lokesh, 1992).
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Pigmentation Meat colour is considered the most important factor for consumers when assessing meat quality in the market. The results of the breast (Table 2) meat pigmentation analysis showed that the L* value (lightness) of fresh breast meat was significantly lower in supplemented broilers than in non-supplemented (control) broilers (P < 0.05). The decreased L* value (lightness) of the meat of supplemented broilers might be due to the lower intramuscular fat content of this meat (Priolo et al., 2001). Indeed, our previous results showed that CRM supplementation decreased fat and cholesterol in broilers (Rajput et al., 2013a). The b* values of meat were higher in the breast muscle of LTN-supplemented broilers; similar results were found in our previous study (Rajput et al., 2012). However, after storage, the b* value was higher in the chickens of the C + L group than in the chickens of each of the other groups (P < 0.05). The a* values (redness) of breast meat were not affected by supplementation; however, the a* value of thigh (fresh) meat was higher in chickens of the CRM group (P < 0.05), which may be due to higher levels of haemoglobin in the tissue. Fresh meat contains residual blood after slaughtering, which may affect the a* value (redness) of meat (Faustman and Cassens, 1990). A decreased a* value, on the other hand, may be linked to decreased mineral availability in muscle tissue (Mancini and Hunt, 2005). Coccidiosis increases blood loss and decreases mineral absorption in muscle tissue (Shaw et al., 2012) and natural Table 2.
Protein oxidation Carbonyl contents and SH level
The concentrations of carbonyl and SH are indicators of biochemical changes or protein oxidation in meat. Lipid oxidation can initiate and/or potentiate protein oxidation and vice-versa (Park
The effects of dietary supplementation with the natural carotenoids CRM and LTN on fresh and stored breast muscle colour and pH of coccidiosis challenged broiler chickens (mean ± SD, n = 5) Dietary treatment
Parameters
CONT
CRM
LTN
C+L
Fresh meat L* value a* value b* value pH
49.15 3.73 9.13 6.24
± ± ± ±
3.01a 0.92 1.42b 0.22
46.81 4.06 9.883 6.24
± ± ± ±
2.40ab 1.29 2.01ab 0.17
47.32 3.13 12.25 6.23
± 2.95ab ± 0.83 ±1.30a ± 0.31
44.28 4.45 10.36 6.39
± ± ± ±
3.11b 0.66 1.14ab 0.12
Stored meat L* value a* value b* value pH
53.48 2.98 4.90 6.01
± ± ± ±
3.07 0.91 1.79b 0.22
49.57 3.39 6.67 6.02
± ± ± ±
3.01 0.63 1.62ab 0.29
50.06 2.23 7.86 6.21
± ± ± ±
49.15 2.75 8.03 5.85
± ± ± ±
4.52 0.91 1.73a 0.20
2.10 0.47 1.40a 0.32
CONT = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with LTN supplementation at 300 mg/kg; C + L = basal diet with the combination of CRM + LTN (150 + 150 mg/kg) supplementation; L* = lightness; a* = redness; b* = yellowness. abc Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05.
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Figure 2. Effects of dietary supplementation with the natural carotenoids CRM and LTN on MDA levels of fresh and stored breast muscle of coccidiosis challenged broiler chickens. Control = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with lutein supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; measuring units = nmol/mg protein. abcMean values within the same row sharing a common superscript letter are not statistically different at P < 0.05, n = 5.
et al., 2006). In the present study, carbonyl contents were lowest in the meat of broilers of the CRM group, followed by those of the C + L group (Figure 3). SH levels were highest in the meat of broilers of the C + L group (Figure 4), indicating that broilers supplemented with a combination of CRM and LTN experienced lower levels of protein oxidation in their breast muscle. Protein oxidation was higher in the meat of LTNsupplemented and non-supplemented chickens. The increased levels of lipid and protein oxidation might be due to the secretion of pro-oxidant and oxidative enzymes from various ruptured cellular organelles (Xia et al., 2009). Such ruptured organelles may reduce the function of muscle proteins and promote water losses in muscle tissue (Xiong, 2000). Natural carotenoids can mitigate these effects, as found in the present study; however, LTN appeared to produce adverse effects on the liver, which might indicate that the amount provided (300 mg/kg) was toxic. In the present
study, protein oxidation was not observed in the fresh meat but was observed after frozen storage and suggests that coccidiosis did not produce biochemical changes in the muscle protein. Instead, coccidial organisms may act as pro-oxidant agents, which initiate oxidation with the passage of time. Protein denaturation: DSC The protein denaturation results were further confirmed with the use of DSC. The three steps of muscle protein denaturation have been described earlier: briefly, actin is denatured at ~80°C, sarcoplasmic/collagen protein denaturation occurs at ~60–70°C and myosin denaturation occurs at ~40–60°C (Omana et al., 2010). The DSC profile of the frozen stored breast meat demonstrated that myosin denaturation had taken place (Figure 5); in particular, a decrease in the myosin peaks in the meat of control chickens and chickens in the LTN group suggested that protein
Figure 3. Effects of dietary supplementation with the natural carotenoids CRM and LTN on carbonyl contents of fresh and stored breast muscle of coccidiosis challenged broiler chickens. Control = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with lutein supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; measuring unist = nmol/mg protein. abcMean values within the same row sharing a common superscript letter are not statistically different at P < 0.05, n = 5.
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Figure 4. Effects of dietary supplementation with the natural carotenoids CRM and LTN on SH contents of fresh and stored breast muscle of coccidiosis challenged broiler chickens. Control = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with LTN supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; measuring unit = nmol/mg protein. abcMean values within the same row sharing a common superscript letter are not statistically different at P < 0.05, n = 5.
Figure 5. DSC thermogram of frozen stored chicken breast mince of control sample (CONT), sample supplemented with CRM, sample supplemented with LTN, sample supplemented with combination of CRM and LTN (C + L). All meat samples were taken from coccidiosischallenged broiler chickens.
denaturation may have occurred. The DSC profile of fresh meat was similar for chickens of all groups. Similarly, a previous study also reported a decrease in the two myosin peaks of meat during frozen storage (Careche et al., 2002). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis A literature review revealed that oxidation can cause cross-linking, polymerisation and aggregate formation in myosin heavy chains (MHC) via the formation of disulphide and carbonyl bonds (Careche et al., 2002). Muscle oxidation was further investigated by SDS-PAGE. The
SDS–PAGE gel (Figure 6) indicated that the band intensity of MHC was similar in all treatment groups. However, new peptide bands in the range of 210–98 kDa appeared, showing that protein oxidation may have caused fragmentation or structural modification of the myosin heavy chain. The SDS-PAGE gel also indicated that a decrease in the relative size and intensity of bands of actin occurred in the samples from chickens in the non-supplemented (control), LTN, C + L and CRM groups. The gel pattern further indicated that some new bands between 60 and 30 kDa may have resulted from the denaturation of actin. Martinaud et al. (1997) also reported the appearance of myosin degradation products in oxidised
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Figure 6. Effects of dietary supplementation with the natural carotenoids CRM and LTN on the myofibrillar proteins of stored breast muscle of coccidiosis-challenged broiler chickens. CON = basal diet without any carotenoid supplementation; CRM = basal diet with curcumin supplementation at 300 mg/kg; LTN = basal diet with lutein supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; MW = molecular weight of protein standard; MHC, myosin heavy chain.
myofibrillar proteins. The fragmentation, caused by OH attack, most likely contributed to the formation of carbonyl bonds and the increase in total amines in oxidised myofibrils. Oxidation also caused a slight reduction in the band intensity of actin (45 kDa), which might have been caused by the cleavage of actin into small peptides or by the cross-linking of actin through non-disulphide covalent bonds to form polymers. Water holding capacity Lipid oxidation is believed to stimulate changes in protein structures and thus alter the water holding capacity of the muscle (Stadtman and Berlett, 1997); loss of water can also lead to the loss of
protein. The majority of the water in meat is trapped between the myofibrils and the cell membrane, within the myofibrils and between the myofibrils. Proteins, especially the myofibrillar protein that holds and stores water, play a vital role in promoting the water holding capacity of muscle; pH and protein oxidation also have a direct effect on the water holding capacity of the muscle tissue (Huff-Lonergan and Lonergan, 2005). In the present study, the water holding capacity of muscle tissue was higher in CRM- and LTN-supplemented chickens and lower in the control chickens (Figure 7). The lower water holding capacity might be due to the significantly higher oxidation of protein (as illustrated by the carbonyl content and SH) and relatively lower pH
Figure 7. Effects of dietary supplementation with the natural carotenoids CRM and LTN on water holding capacity of fresh and stored breast muscle of coccidiosis-challenged broiler chickens. Control = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with LTN supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; measuring unit = percentage; WHC = water holding capacity. abcMean values within the same row sharing a common superscript letter are not statistically different at P < 0.05, n = 5.
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of non-supplemented chicken breast muscle. In addition, the variation in the water holding capacity is to some extent due to differences in proteolysis and cell shrinkage and to the mobilisation of water to the extra cellular space (HuffLonergan and Lonergan, 2005). In conclusion, coccidiosis decreased the pigmentation and water holding capacity of fresh breast meat but did not produce adverse effects on lipid and protein oxidation. After storage (one month at −18°C and 3 d at 4°C), MDA, carbonyl contents and myosin denaturation increased and SH contents, the water holding capacity and the colour stability of the meat decreased. Plant-origin carotenoids reduced the lipid and protein oxidation and improved the colour stability and quality of breast meat in coccidiosis-infected chickens.
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British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
The effect of dietary supplementation with the natural carotenoids curcumin and lutein on pigmentation, oxidative stability and quality of meat from broiler chickens affected by a coccidiosis challenge a
b
a
b
b
N. Rajput , S. Ali , M. Naeem , M.A. Khan & T. Wang a
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, P. R. China b
Key Laboratory of Meat Processing and Quality Control, Nanjing Agricultural University, Nanjing, P. R. China Accepted author version posted online: 23 May 2014.Published online: 22 Aug 2014.
To cite this article: N. Rajput, S. Ali, M. Naeem, M.A. Khan & T. Wang (2014): The effect of dietary supplementation with the natural carotenoids curcumin and lutein on pigmentation, oxidative stability and quality of meat from broiler chickens affected by a coccidiosis challenge, British Poultry Science, DOI: 10.1080/00071668.2014.925537 To link to this article: http://dx.doi.org/10.1080/00071668.2014.925537
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British Poultry Science, 2014 http://dx.doi.org/10.1080/00071668.2014.925537
The effect of dietary supplementation with the natural carotenoids curcumin and lutein on pigmentation, oxidative stability and quality of meat from broiler chickens affected by a coccidiosis challenge N. RAJPUT, S. ALI1, M. NAEEM, M.A. KHAN1
AND
T. WANG1
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College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, P. R. China, and 1 Key Laboratory of Meat Processing and Quality Control, Nanjing Agricultural University, Nanjing, P. R. China
Abstract 1. An experiment was performed to evaluate the effectiveness of the antioxidants curcumin (CRM) and lutein (LTN) on the quality of meat from coccidiosis-infected broilers. A total of 200 one-dayold Arbor Acre chicks were randomly assigned to a treatment group with 5 replicates. The treatments included a basal diet without carotenoid supplementation (control), with 300 mg/kg CRM, with 300 mg/kg LTN or with a combination (C + L) of 150 mg/kg CRM and 150 mg/kg LTN. All chickens were challenged with Eimeria maxima at 21 d old. 2. The results revealed that the coccidiosis reduced redness of meat, while supplementation with carotenoids improved the fresh meat’s redness (a*) and yellowness (b*) and contributed to colour stability maintenance after storage (1 month at −18°C and 3 d at 4°C). 3. Coccidiosis did not produce lipid and protein oxidation in fresh meat, but after storage for one month, the malondialdehyde levels and carbonyl contents were lower in the CRM and C + L birds and the sulfhydryl contents were higher in C + L birds. 4. The sodium dodecyl sulphate–polyacrylamide gel electrophoresis banding pattern showed equivalent myosin chain fragmentations in all treatment groups, whereas lower intensity actin bands were observed in the control group (CONT). Moreover, myofibril protein denaturation (differential scanning calorimetry) profiles showed a reduction in the CONT myosin and actin peaks. Coccidiosis reduced the meat’s water holding capacity in non-supplemented chicken meat and was improved by natural carotenoid. 5. These results emphasise that coccidiosis did not decrease the eating quality of fresh meat, that natural carotenoids are efficient antioxidants and that CRM (300 mg/kg) fed individually or combined with LTN was the most effective supplemented antioxidant compound.
INTRODUCTION Coccidiosis caused by various species of Eimeria is considered to be the most commonly occurring disease in chickens and may produce health and welfare problems and economical loses (Velkers et al., 2010; Shaw et al., 2012). Structural changes and decomposition in muscle tissue have been reported due to Eimeria acervulina infection (Fetterer and Allen, 2000). Coccidiosis severely increases blood loss in the muscles, which results in decreased tissue pigmentation and pale meat
(Fetterer and Allen, 2001). Immune cells are activated by the pathogenic organism and produce various mediators (i.e. reactive oxygen and nitrogen species, singlet oxygen, peroxides and cytokines) to remove and destroy the organisms that can produce oxidative stress (Shanmugasundaram and Selvaraj, 2011). The excessive production of immune cell mediators against oxidative stress may also cause oxidative damage (Smet et al., 2008). Oxidation is a common process that affects pigments, fat and proteins and reduces the shelf life of meat (Morrissey et al., 1998).
Correspondence to: Tian Wang, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, P. R. China. E-mail: [email protected] Accepted for publication 29 March 2014.
© 2014 British Poultry Science Ltd
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The oxygenated free-radicals damage cellular structures such as lipids, proteins and myoglobin, which, in turn, stimulates pigment oxidation (Faustman and Cassens, 1990; Mercier et al., 1998). Lipid oxidation can also be initiated by protein-free radicals (Stadtman and Berlett, 1997; Mercier et al., 1998). Cellular damage by free radicals has not been properly addressed in chicken meat. Anti-coccidial drugs and vaccinations are the most commonly applied methods of controlling coccidiosis. However, certain laws limit the use of antimicrobial drugs, and satisfactory cost-effective vaccines have not yet been developed. Furthermore, the residual effect of drugs in meat and increasing consumer demand for organic food (Velkers et al., 2010; Anosa and Okoro, 2011) have highlighted the need for studies in this area and prompted searches for alternate feed additives to prevent coccidiosis. Carotenoids are natural colouring agents synthesised by plants that produce skin pigments in birds and have strong antioxidant, anti-inflammatory and antimicrobial properties (Lokaewmanee et al., 2011). Lutein (LTN) is a carotenoid that is naturally found in marigold flowers and dark green vegetables. Curcumin (CRM) is not only a food colouring agent but also an important compound in Ayurveda medicine, known as a “cleanser of the body” (Gandhi et al., 2011). In China, the United States and many other countries, the colour of chicken skin and meat plays an important role in consumer preference, as meat quality is often assessed on the basis of pigmentation (Liu et al., 2008; Valeria and Williams, 2011). From a commercial point of view, the major drawback of coccidiosis is a decrease in the quality and colour stability of the meat. Previous studies have shown that carotenoids (especially CRM at a dose of 200 mg/ kg feed) have positive effects on the gut morphology, digestion and immunomodulation of broilers and on the pigmentation and oxidation of broiler meat (Rajput et al., 2012, 2013a, 2013b). In the present trial, broilers were challenged with Eimeria maxima parasites, as it is well established that coccidiosis weakens the immunity of broilers and decreases the pigmentation and quality of their meat. We hypothesised that carotenoids can decrease the activity of free radicals produced by the oxidative stress caused by coccidiosis. Many studies on the effect of coccidiosis in broiler production have been conducted; however, few studies have examined the effect of coccidiosis on the oxidative stability and the shelf life of meat. To the best of our knowledge, no studies have been conducted on the effects of natural carotenoids on the quality and oxidative stability of the meat of coccidiainfected chickens.
MATERIALS AND METHODS Feeding management A total of 200, one-day-old Arbor Acre broiler chicks were randomly assigned into 4 treatment groups with 5 replicates. The antibiotic and coccidiostat-free basal diet was a rice-soybean-based diet (Table 1), formulated on the recommendation of National Research Council (NRC, 1994), to fulfil the nutrient requirements of broiler chicken. The birds were fed starter feed (1–21 d) and grower feed (22–42 d). The basal diet was supplemented with 300 mg/kg CRM, 300 mg/kg LTN or a combination (C + L) of CRM 150 mg and LTN 150 mg/kg diet. The birds in the control group (CONT) were fed on the basal diet without supplementation of any pigment. On d 21, all the birds were orally infected with purified Emeria maxima sporulated oocysts (5000 oocysts per broiler) diluted in 0.5 ml of distilled water. A suspension containing a specific amount of infective E. maxima sporulated oocysts (Figure 1) was kindly supplied by Key lab of Veterinary Parasitology, Nanjing Agricultural University, Nanjing, People’s Republic of China. CRM and LTN were obtained from a commercial company (Guangzhou Leader Biotechnology Co Ltd, China) with the purity of 98%. The Institutional Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, People’s Republic of China) approved all of the procedures. Table 1. Feed ingredients and nutrient composition of basal diets for broiler chickens Item Ingredients (g/kg) Rice Soybean meal Maize gluten meal Limestone Dicalcium phosphate Sodium chloride Feed premix1 L-Lysine DL-Methionine
Starter (1–21 d)
Finisher (22–42 d)
602.5 325 30 12 20 3 5 1 1.5
654.7 280 25 12 18 3 5 1 1.3
Calculated chemical composition of diet (g/kg) ME (MJ/kg) 12.54 Crude protein 211.9 Lysine 12.1 Methionine 4.96 Methionine + cystine 8.0 Ca 9.88 Available P 5.31 1
12.65 192.0 10.9 4.40 7.2 9.49 4.85
Mineral premix provided the following per kilogram of diet: transretinyl acetate, 25 mg; cholecalciferol, 6 mg; menadione, 1.2 mg; thiamine, 2.3 mg; riboflavin, 8 mg; nicotinamide, 42 mg; choline chloride, 400 mg; calcium pantothenate, 10 mg; pyridoxine HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; cobalamin, 0.012 mg; Fe (from ferrous sulphate), 82 mg; Cu (from copper sulphate), 7.5 mg; Mn (from manganese sulphate), 110 mg; Zn (from zinc oxide), 64 mg; I (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.28 mg.
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Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE)
Figure 1. Sporulated oocysts of Eimeria maxima orally given to chickens.
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Sample collection At 42 d of age (marketing age), blood samples were taken from one randomly selected bird per replicate; plasma was separated by centrifugation at 300 × g for 10 min at 4°C and stored at −20°C for further analysis. After blood collection, all birds were killed by exsanguination, de-feathered, and after 45 min of post-mortem, the colour and pH of the meat was measured. The meat samples were cut into 4 × 3 × 1 inch cubes and all of the visible fat and connective tissues were removed. The antioxidant parameters were determined 3 h after slaughtering; these data were classified as “fresh meat” data. The remaining samples were immediately packed in moisture-impermeable polyethylene bags and stored at −20°C for one month. After one month, all of the frozen samples were thawed in a refrigerator at 4°C for 3 d and antioxidant activities were measured; these data were classified as “stored meat” data. Muscle colour and pH One randomly selected bird per replicate was used for the determination of breast and thigh muscle pH and colour. The breast muscle pH was measured by inserting the probe of a pH meter (Thermo Scientific Orion 3 Star, Singapore) into the muscles. The L* value (lightness), a* value (redness) and b* value (yellowness) of the breast and thigh muscles were measured with a Konica Minolta chroma meter CR-400 (Minolta Camera Co., Osaka, Japan) 45 min after air exposure to allow for blooming.
The meat was treated in Mini-PROTEAN Tetra Cell (Bio-Rad sodium dodecyl sulphate-polyacrylamide gel electrophoresis) (Bertram et al., 2006). The sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, in which a sample of minced meat was dissolved to obtain a 1 µg/µl protein concentration, was composed of 4% SDS, 20% glycerol, 0.125 M tris HCl and 10% 2-mercaptoethanol with a pH of 6.8. Ten microliter aliquots of the samples were loaded in the sample wells of the stacking gel. A molecular weight (MW) standard, consisting of a cocktail of proteins of known molecular weight (TaKaRa, Dalian Co. Ltd., Beijing, China), was also run, and protein bands in meat samples were tentatively identified by comparing their electrophoretic patterns with standard markers.
Protein denaturation: differential scanning calorimetry Differential scanning calorimetry (DSC) was carried out using a Micro DSC-II Setaram Calorimeter (Setaram, Caluire, France); 0.2 g of minced whole muscle was loaded into three 1 cm stainless steel cuvettes. The samples were scanned over the temperature range 10–90°C at a rate of 0.5°C/min against a water reference (Bertram et al., 2006).
Water holding capacity The water holding capacity of the meat was measured according to the method of Omana et al. (2010). Raw meat (approximately 2 g) was placed between a pre-weighed Whatman (No. 1) filter paper and between two glass plates. Using the texture profile analyser (TA-XT Express, Stable Micro Systems Ltd., Surrey, England) in adhesive test mode, the samples were tested with a target force of 111 kg for 5 min. After the test, the filter paper with the absorbed water was immediately weighed. Expressible moisture was measured as the quantity of water released per gram of meat and expressed as a percentage. Expressible waterð%Þ ¼ ðwet paper drypaper=meat weightÞ 100
Carbonyl contents and sulfhydryl level
Statistical analysis
Carbonyl groups were detected by the reactivity with 2, 4-dinitrophenylhydrazine to form protein hydrazones, and sulphydryl (SH) groups (thiol content) were determined according to previously described methods (Xia et al., 2009).
Statistical analyses were performed using SPSS software (SPSS 15.0 K for Windows, Chicago, IL, USA), and all data were expressed as the means ± SD values. Comparisons of the mean values were performed by one-way analysis of
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antioxidants can attenuate these effects (Anosa and Okoro, 2011). Carotenoid supplementation did not produce any effect on muscle pH before or after storage of meat (Table 2).
variance. Significant differences among means were evaluated by Tukey’s comparison test at P < 0.05.
RESULTS AND DISCUSSION
Lipid oxidation The extreme oxidation of lipids caused by high levels of free radicals is the main cause of oxidative stress; this oxidation, in turn, enhances the levels of malondialdehyde (MDA), which is the end product of lipid peroxidation (Kim et al., 2012). In the current study, lipid oxidation (as measured by MDA) was significantly lower in the meat of animals from the C + L and CRM group (Figure 2). In addition, lipid oxidation tends to increase with longer frozen storage times and higher meat fat contents (Wood and Enser, 1997). We previously reported that CRM decreased the cholesterol and fat content of meat in a dose-dependent manner (Rajput et al., 2013a). Lipid and pigment oxidation in meat are closely associated, and supplementation with antioxidants can increase the colour stability of the meat (Faustman and Cassens, 1990). Moreover, a review of the literature revealed that supplementation with vitamin E improved the colour and lipid stability of meat by delaying the oxidation of phospholipids (Mercier et al., 1998); interestingly, CRM has been reported to be a better antioxidant than vitamin E (Reddy and Lokesh, 1992).
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Pigmentation Meat colour is considered the most important factor for consumers when assessing meat quality in the market. The results of the breast (Table 2) meat pigmentation analysis showed that the L* value (lightness) of fresh breast meat was significantly lower in supplemented broilers than in non-supplemented (control) broilers (P < 0.05). The decreased L* value (lightness) of the meat of supplemented broilers might be due to the lower intramuscular fat content of this meat (Priolo et al., 2001). Indeed, our previous results showed that CRM supplementation decreased fat and cholesterol in broilers (Rajput et al., 2013a). The b* values of meat were higher in the breast muscle of LTN-supplemented broilers; similar results were found in our previous study (Rajput et al., 2012). However, after storage, the b* value was higher in the chickens of the C + L group than in the chickens of each of the other groups (P < 0.05). The a* values (redness) of breast meat were not affected by supplementation; however, the a* value of thigh (fresh) meat was higher in chickens of the CRM group (P < 0.05), which may be due to higher levels of haemoglobin in the tissue. Fresh meat contains residual blood after slaughtering, which may affect the a* value (redness) of meat (Faustman and Cassens, 1990). A decreased a* value, on the other hand, may be linked to decreased mineral availability in muscle tissue (Mancini and Hunt, 2005). Coccidiosis increases blood loss and decreases mineral absorption in muscle tissue (Shaw et al., 2012) and natural Table 2.
Protein oxidation Carbonyl contents and SH level
The concentrations of carbonyl and SH are indicators of biochemical changes or protein oxidation in meat. Lipid oxidation can initiate and/or potentiate protein oxidation and vice-versa (Park
The effects of dietary supplementation with the natural carotenoids CRM and LTN on fresh and stored breast muscle colour and pH of coccidiosis challenged broiler chickens (mean ± SD, n = 5) Dietary treatment
Parameters
CONT
CRM
LTN
C+L
Fresh meat L* value a* value b* value pH
49.15 3.73 9.13 6.24
± ± ± ±
3.01a 0.92 1.42b 0.22
46.81 4.06 9.883 6.24
± ± ± ±
2.40ab 1.29 2.01ab 0.17
47.32 3.13 12.25 6.23
± 2.95ab ± 0.83 ±1.30a ± 0.31
44.28 4.45 10.36 6.39
± ± ± ±
3.11b 0.66 1.14ab 0.12
Stored meat L* value a* value b* value pH
53.48 2.98 4.90 6.01
± ± ± ±
3.07 0.91 1.79b 0.22
49.57 3.39 6.67 6.02
± ± ± ±
3.01 0.63 1.62ab 0.29
50.06 2.23 7.86 6.21
± ± ± ±
49.15 2.75 8.03 5.85
± ± ± ±
4.52 0.91 1.73a 0.20
2.10 0.47 1.40a 0.32
CONT = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with LTN supplementation at 300 mg/kg; C + L = basal diet with the combination of CRM + LTN (150 + 150 mg/kg) supplementation; L* = lightness; a* = redness; b* = yellowness. abc Mean values within the same row sharing a common superscript letter are not statistically different at P < 0.05.
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Figure 2. Effects of dietary supplementation with the natural carotenoids CRM and LTN on MDA levels of fresh and stored breast muscle of coccidiosis challenged broiler chickens. Control = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with lutein supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; measuring units = nmol/mg protein. abcMean values within the same row sharing a common superscript letter are not statistically different at P < 0.05, n = 5.
et al., 2006). In the present study, carbonyl contents were lowest in the meat of broilers of the CRM group, followed by those of the C + L group (Figure 3). SH levels were highest in the meat of broilers of the C + L group (Figure 4), indicating that broilers supplemented with a combination of CRM and LTN experienced lower levels of protein oxidation in their breast muscle. Protein oxidation was higher in the meat of LTNsupplemented and non-supplemented chickens. The increased levels of lipid and protein oxidation might be due to the secretion of pro-oxidant and oxidative enzymes from various ruptured cellular organelles (Xia et al., 2009). Such ruptured organelles may reduce the function of muscle proteins and promote water losses in muscle tissue (Xiong, 2000). Natural carotenoids can mitigate these effects, as found in the present study; however, LTN appeared to produce adverse effects on the liver, which might indicate that the amount provided (300 mg/kg) was toxic. In the present
study, protein oxidation was not observed in the fresh meat but was observed after frozen storage and suggests that coccidiosis did not produce biochemical changes in the muscle protein. Instead, coccidial organisms may act as pro-oxidant agents, which initiate oxidation with the passage of time. Protein denaturation: DSC The protein denaturation results were further confirmed with the use of DSC. The three steps of muscle protein denaturation have been described earlier: briefly, actin is denatured at ~80°C, sarcoplasmic/collagen protein denaturation occurs at ~60–70°C and myosin denaturation occurs at ~40–60°C (Omana et al., 2010). The DSC profile of the frozen stored breast meat demonstrated that myosin denaturation had taken place (Figure 5); in particular, a decrease in the myosin peaks in the meat of control chickens and chickens in the LTN group suggested that protein
Figure 3. Effects of dietary supplementation with the natural carotenoids CRM and LTN on carbonyl contents of fresh and stored breast muscle of coccidiosis challenged broiler chickens. Control = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with lutein supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; measuring unist = nmol/mg protein. abcMean values within the same row sharing a common superscript letter are not statistically different at P < 0.05, n = 5.
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Figure 4. Effects of dietary supplementation with the natural carotenoids CRM and LTN on SH contents of fresh and stored breast muscle of coccidiosis challenged broiler chickens. Control = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with LTN supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; measuring unit = nmol/mg protein. abcMean values within the same row sharing a common superscript letter are not statistically different at P < 0.05, n = 5.
Figure 5. DSC thermogram of frozen stored chicken breast mince of control sample (CONT), sample supplemented with CRM, sample supplemented with LTN, sample supplemented with combination of CRM and LTN (C + L). All meat samples were taken from coccidiosischallenged broiler chickens.
denaturation may have occurred. The DSC profile of fresh meat was similar for chickens of all groups. Similarly, a previous study also reported a decrease in the two myosin peaks of meat during frozen storage (Careche et al., 2002). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis A literature review revealed that oxidation can cause cross-linking, polymerisation and aggregate formation in myosin heavy chains (MHC) via the formation of disulphide and carbonyl bonds (Careche et al., 2002). Muscle oxidation was further investigated by SDS-PAGE. The
SDS–PAGE gel (Figure 6) indicated that the band intensity of MHC was similar in all treatment groups. However, new peptide bands in the range of 210–98 kDa appeared, showing that protein oxidation may have caused fragmentation or structural modification of the myosin heavy chain. The SDS-PAGE gel also indicated that a decrease in the relative size and intensity of bands of actin occurred in the samples from chickens in the non-supplemented (control), LTN, C + L and CRM groups. The gel pattern further indicated that some new bands between 60 and 30 kDa may have resulted from the denaturation of actin. Martinaud et al. (1997) also reported the appearance of myosin degradation products in oxidised
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Figure 6. Effects of dietary supplementation with the natural carotenoids CRM and LTN on the myofibrillar proteins of stored breast muscle of coccidiosis-challenged broiler chickens. CON = basal diet without any carotenoid supplementation; CRM = basal diet with curcumin supplementation at 300 mg/kg; LTN = basal diet with lutein supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; MW = molecular weight of protein standard; MHC, myosin heavy chain.
myofibrillar proteins. The fragmentation, caused by OH attack, most likely contributed to the formation of carbonyl bonds and the increase in total amines in oxidised myofibrils. Oxidation also caused a slight reduction in the band intensity of actin (45 kDa), which might have been caused by the cleavage of actin into small peptides or by the cross-linking of actin through non-disulphide covalent bonds to form polymers. Water holding capacity Lipid oxidation is believed to stimulate changes in protein structures and thus alter the water holding capacity of the muscle (Stadtman and Berlett, 1997); loss of water can also lead to the loss of
protein. The majority of the water in meat is trapped between the myofibrils and the cell membrane, within the myofibrils and between the myofibrils. Proteins, especially the myofibrillar protein that holds and stores water, play a vital role in promoting the water holding capacity of muscle; pH and protein oxidation also have a direct effect on the water holding capacity of the muscle tissue (Huff-Lonergan and Lonergan, 2005). In the present study, the water holding capacity of muscle tissue was higher in CRM- and LTN-supplemented chickens and lower in the control chickens (Figure 7). The lower water holding capacity might be due to the significantly higher oxidation of protein (as illustrated by the carbonyl content and SH) and relatively lower pH
Figure 7. Effects of dietary supplementation with the natural carotenoids CRM and LTN on water holding capacity of fresh and stored breast muscle of coccidiosis-challenged broiler chickens. Control = basal diet without any carotenoid supplementation; CRM = basal diet with CRM supplementation at 300 mg/kg; LTN = basal diet with LTN supplementation at 300 mg/kg; C + L = basal diet with combination of CRM + LTN supplementation at 150 + 150 mg/kg; measuring unit = percentage; WHC = water holding capacity. abcMean values within the same row sharing a common superscript letter are not statistically different at P < 0.05, n = 5.
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of non-supplemented chicken breast muscle. In addition, the variation in the water holding capacity is to some extent due to differences in proteolysis and cell shrinkage and to the mobilisation of water to the extra cellular space (HuffLonergan and Lonergan, 2005). In conclusion, coccidiosis decreased the pigmentation and water holding capacity of fresh breast meat but did not produce adverse effects on lipid and protein oxidation. After storage (one month at −18°C and 3 d at 4°C), MDA, carbonyl contents and myosin denaturation increased and SH contents, the water holding capacity and the colour stability of the meat decreased. Plant-origin carotenoids reduced the lipid and protein oxidation and improved the colour stability and quality of breast meat in coccidiosis-infected chickens.
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