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Effect of hesperidin dietary supplementation on hen performance, egg quality and yolk oxidative stability a
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M. Goliomytis , H. Orfanou , E. Petrou , M. A. Charismiadou , P. E. Simitzis & S. G. Deligeorgis
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Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece Accepted author version posted online: 08 Jan 2014.Published online: 16 Apr 2014.
To cite this article: M. Goliomytis, H. Orfanou, E. Petrou, M. A. Charismiadou, P. E. Simitzis & S. G. Deligeorgis (2014) Effect of hesperidin dietary supplementation on hen performance, egg quality and yolk oxidative stability, British Poultry Science, 55:1, 98-104, DOI: 10.1080/00071668.2013.870328 To link to this article: http://dx.doi.org/10.1080/00071668.2013.870328
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British Poultry Science, 2014 Vol. 55, No. 1, 98–104, http://dx.doi.org/10.1080/00071668.2013.870328
Effect of hesperidin dietary supplementation on hen performance, egg quality and yolk oxidative stability M. GOLIOMYTIS, H. ORFANOU, E. PETROU, M. A. CHARISMIADOU, P. E. SIMITZIS AND S. G. DELIGEORGIS
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Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece
Abstract 1. The purpose of this study was to evaluate the effects of dietary supplementation with hesperidin (one or 3 g/kg of feed) for 31 d on the performance, egg quality and yolk oxidative stability of brown and white laying hens (26-wk old). 2. Supplementation with hesperidin did not affect egg production, egg weight and egg quality traits. 3. No hesperidin effect on yolk and plasma cholesterol was observed. A strain effect was found with lower total and per g yolk cholesterol of brown hens in comparison to the white ones. 4. Oxidative stability of egg yolk, expressed as ng MDA/g yolk, was significantly improved in the hesperidin groups even from the first week of supplementation. At the same time, a significant improvement in the oxidative stability of egg yolk due to the incorporation of hesperidin in hens’ diet was observed after 30 and 90 d of storage at 20°C and 4°C, respectively. 5. No hesperidin by strain interaction was detected for any of the traits measured. 6. In conclusion, incorporation of hesperidin to laying hens’ feed did not affect productive and egg qualitative traits. On the other hand, dietary hesperidin supplementation significantly improved oxidative stability of both fresh and stored eggs. Antioxidant properties of hesperidin seem to make it a promising natural agent for improving the shelf life of eggs.
INTRODUCTION Eggs have a high nutritional value and contain a variety of necessary components for the maintenance and the normal function of the human organism. Feeding strategies have been developed in order to increase the n-3 fatty acid content of eggs by enriching poultry diets with polyunsaturated fatty acids (Huang et al., 1990). However, these constituents increase the degree of unsaturation and the susceptibility of eggs to oxidative deterioration. Lipid oxidation by free radicals is one of the primary mechanisms of quality deterioration in eggs. It is initiated in the highly unsaturated fatty acid fraction of membrane phospholipids, leading to the production of hydroperoxides, which are susceptible to further oxidation or decomposition
to secondary reaction products such as short-chain aldehydes, ketones and other oxygenated compounds that may adversely affect lipids, pigments, proteins, carbohydrates, vitamins and the overall quality by causing loss of flavour, colour and nutritive value and limiting shelf-life (Angelo et al., 1996; Cherian et al., 1996). In the past, synthetic antioxidants were used with the intention of preventing lipid oxidation by scavenging chain-carrying peroxyl radicals or diminishing the formation of lipid radicals. In the last decade, there has been a strong tendency towards using organic antioxidants from natural sources for the protection of animal health and their products against oxidation (Wenk, 2003). As a result, considerable interest has arisen in the use of natural antioxidants derived from aromatic
Correspondence to: Michael Goliomytis, Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece. E-mail: [email protected] Accepted for publication 9 October 2013.
© 2014 British Poultry Science Ltd
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plants that could serve as alternatives to synthetic supplements on purpose to improve egg quality, without leaving residues in the product or the environment (Galobart et al., 2001; Florou-paneri et al., 2005, 2006; Botsoglou et al., 2005a, 2005b). The increased awareness of consumers towards a diet rich in natural, safe and healthpromoting ingredients has led to the search of promising sources of materials that may be used in the food and feed industry because of their valuable nutritional properties. Dried citrus pulp appears to be a promising candidate and is the main by-product from the citrus-processing industry. It is produced after extraction of the juice from citrus fruits and drying of the residues and is a mixture of peel, inside portions and culled fruits of the citrus family, rich in energy, fibre and calcium. Fibres from citrus fruits have an additional advantage over dietary fibres from other sources due to the presence of associated bioactive compounds (i.e. flavonoids) (Gorinstein et al., 2001). These compounds usually contain one or more aromatic hydroxyl groups, which actively scavenge free radicals and are responsible for the respective antioxidant properties (Pietta, 2000). Hesperidin, a bioflavonoid, is an abundant and inexpensive by-product of citrus cultivation. It is a naturally occurring polyphenolic compound widely distributed in the plant kingdom as a secondary metabolite. The traditional extraction method, with a very low recovery, treats triturated peel with alkaline compounds to dissolve the flavonoids, which, after separation of solid residue, are precipitated by acidification. An innovative method to recover hesperidin from orange peel and waste water utilises styrene-divinylbenzene as the absorbing phase, removing the flavonoid with alcoholic NaOH. The results may be a useful starting point for industrial applications (Lanza, 2003). Hesperidin is a flavanone glycoside comprising of an aglycone, hesperetin or methyl eriodictyol and an attached disaccharide, rutinose. Pure hesperidin occurs as long hair-like needles, tan or pale yellow in colour. It is tasteless and odourless (Garg et al., 2001). A deficiency of this substance in human diet has been linked with abnormal capillary leakiness as well as pain in the extremities causing aches, weakness and leg cramps. Moreover, hesperidin has been reported to possess antioxidant activity and radical scavenging properties (Garg et al., 2001). The combination of increased disposal costs in many parts of the world with the antioxidant properties of citrus by-products have increased interest in their utilisation as alternative feeds in animal production. The objective of the present study was therefore the evaluation of the effects of different concentrations of hesperidin dietary
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supplementation on hen performance and egg quality characteristics.
MATERIALS AND METHODS Animals and diets A total of 24 white (LSL-classic) and 24 brown (Brown-Classic) Lohmann individually caged laying hens (26 wk old) were randomly assigned into three equal treatment groups (16 hens each). One of the groups served as a control (C) and was given a commercial basal diet, whereas the other two groups were given the same diet further supplemented with hesperidin (MP Biomedicals, LLC, France) at 1 g/kg (HE1), and 3 g/kg (HE2) for 31 d. Water was provided ad libitum throughout the experimental period and the light regimen was 16 h of continuous light per d. Each hen of the white and the brown strain consumed approximately 105 and 110 g feed/d, respectively. Table 1 presents the ingredients and the composition of the control diet. Egg production was daily recorded throughout the experimental study. Egg weight, laboratory evaluations of egg quality and measurements of yolk cholesterol were performed on eggs collected from 8 hens per dietary group, at d 28 of the experiment. Yolk oxidative stability was assessed by using the malondialdehyde (MDA) assay on eggs collected from 8 hens per Table 1.
Ingredients and chemical composition of the diet
Ingredients Maize Soybean meal, 47% CP Wheat Wheat bran Soybean oil Limestone Monocalcium phosphate Sodium chloride Sodium bicarbonate Methionine 99% Choline chloride 70% Phytase, Natuphos® Antioxidant Pigment (Carophyl® Red) Vitamin and Mineral premix1 Chemical composition, calculated Metabolisable energy (MJ/kg) Crude protein Lysine Methionine + cysteine Calcium Available phosphorus 1
g/kg 540 246 50 46 12 89 9 2.4 2.7 1.1 0.6 0.06 0.1 0.04 1 11.48 175 9 6.8 37.5 3.6
The vitamin and mineral premix provided per kg of diet: 3 mg of retinol (vitamin A), 62.5 μg of cholecalciferol (vitamin D3), 30 mg of tocopherol (vitamin E), 5 mg of menadione (vitamin K3), one milligram of thiamine (vitamin B1), 5 mg of riboflavin (vitamin B2), 3 mg of pyridoxin (vitamin B6), 20 μg of cobalamin (vitamin B12), 30 mg of nicotinic acid, 10 mg of pantothenic acid, 0.8 mg of folic acid, 100 μg of biotin, 10 mg of ascorbic acid (Vitamin C), 450 mg of choline chloride, 0.2 mg of Co, 0.5 mg of I, 0.3 mg of Se, 25 mg of Fe, 120 mg of Mn, 10 mg of Cu and 100 mg of Zn.
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dietary group at 0, 7, 14, 21 and 28 d after hesperidin dietary supplementation. MDA concentration was also measured in eggs collected at d 29, 30 and the 31 of the experiment and consequently stored for 15 and 30 d at room temperature (~20° C) and for 90 d at 4°C (refrigerated storage). At d 28 of the experiment, blood samples from 8 hens per dietary group were also collected from the brachial wing vein for the plasma cholesterol determination. The methods used in the present experiment were in accordance with the national legislation and the guidelines of the Research Ethics Committee of the Department of Animal Science and Aquaculture of the Agricultural University of Athens.
anticoagulant (2 mg EDTA/ml of blood), were centrifuged at 800 × g for 20 min and supernatant was collected. Plasma cholesterol was measured photometrically at 540 nm in a spectrophotometer (Hitachi U3010 Spectrophotometer) by using a commercial cholesterol reagent kit (Biosis commercial kits; Athens, Greece). Yolk cholesterol was determined following the method described by Pasin et al. (1998). In detail, 3 g of egg yolk was diluted with 27 ml NaCl-solution (20 g/kg), stirred for 2 h using a magnetic stirrer, and 1 ml of the above solution was further diluted with 9 ml NaCl (20 g/kg). The rest of the determination procedure was the same to the one described above for plasma cholesterol. Determination of lipid oxidation (MDA analysis)
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Quality measurements Egg quality measurements were based on determination of external and internal indices, and egg components. The external indices included eggshape index and eggshell strength; those of interior quality were Haugh unit score, albumen pH, yolk index, yolk weight and yolk percentage expressed as yolk weight/egg weight. In detail, the collected eggs were individually weighed and their width and length were measured. Eggshell breaking strength was assessed by a Zwick Testing Machine (Model Z2.5/TN1S, Zwick GmbH & Co, Germany). The measurement was carried out on eggs with the major axis parallel to the compression surfaces (force applied at equator). Crosshead speed was set at 60 mm/min, with a programmed automatic stop set when a 40% reduction of the maximum load occurred (no loss of egg contents occurred, since the inner shell membrane remained intact (Guardbouldin and Buhr, 2006). Eggs were then broken and Haugh units were determined on each egg albumen with a Haugh meter (Model S-8400, B.C. Ames Inc., USA). Yolk height was determined using a tripod micrometer, while yolk diameter was measured using a steel vernier calliper. Yolk index was calculated as yolk height × 100 divided by yolk diameter. After the separation of albumen and yolk, 5 g of the albumen was placed in a separate 100-ml beaker for the measurement of albumen pH. Distilled water (45 ml) was added to each beaker and mixed thoroughly using a blender (Edmund Buehler 7400 Tuebingen/H04, Germany). The pH measurements were obtained by inserting the electrode of the pH meter (pHM120, MeterLab) in the derived mixture. Determination of plasma and yolk cholesterol The blood samples (2 ml) that were collected from the hens in tubes containing EDTA
Lipid oxidation was assessed on the basis of the malondialdehyde (MDA) formed during storage, a secondary lipid oxidation product formed by hydrolysis of lipid hydroperoxides. In the present study, yolk MDA concentration was determined by using a selective third-order derivative spectrophotometric method (Botsoglou et al., 1994). Derivative versus conventional spectrophotometry was adopted because it offers improved sensitivity, specificity and reliability of the measurements, since it eliminates potential interferences from other reactive compounds. In brief, 2 g of each sample (two samples per egg) were homogenised (Edmund Buehler, 7400 Tuebingen/H04, Germany) in the presence of 8 ml aqueous trichloroacetic acid (TCA) (50 g/l) and 5 ml butylated hydroxytoluene (BHT) in hexane (8 g/l), and the mixture was centrifuged for 3 min at 3000 g. The top hexane layer was discarded and a 2.5 ml aliquot from the bottom layer was mixed with 1.5 ml aqueous 2-thiobarbituric acid (TBA) (8 g/l) to be further incubated at 70ºC for 30 min. Following incubation, the mixture was cooled under tap water and submitted to thirdorder derivative (3D) spectrophotometry (Hitachi U3010 Spectrophotometer) in the range of 500– 550 nm. The concentration of MDA (ng/g yolk) in analysed samples was calculated on the basis of the height of the third-order derivative peak at 521.5 nm by referring to the slope and intercept data of the computed least-squares fit of standard calibration curve prepared using 1,1,3,3tetraethoxypropane (TEP), the malondialdehyde precursor. Statistical analysis Data were subjected to analysis of variance with hesperidin supplementation, strain of the hens and their interaction as fixed effects using the general linear models of SAS software (SAS Institute, Inc., 2005). An arcsine transformation
DIETARY HESPERIDIN FOR LAYERS
was applied to egg production data prior to statistical analysis. Mean differences were tested at 0.05 significance level with Bonferroni adjustment. Results are presented as mean ± standard error.
Table 3. Effect of 28-d dietary hesperidin supplementation on yolk and plasma cholesterol (mean ± SE, n=8) Hesperidin Variable
RESULTS Egg production and quality measurements
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The effects on egg production and egg quality characteristics after 28 d of hesperidin dietary supplementation are shown in Table 2. Neither egg production nor egg quality measurements appeared to be influenced by the incorporation of hesperidin in hens’ diet (P > 0.05). On the other hand, the genetic origin of hens affected egg index, yolk weight and yolk/egg weight ratio (P < 0.05). However, no interaction between hesperidin and strain of hens was detected (P > 0.05). Yolk and plasma cholesterol Results presented in Table 3 showed that hesperidin dietary supplementation for 28 d did not influence either plasma or yolk cholesterol (P > 0.05). A strain effect (P < 0.05) was detected for cholesterol (mg) per g yolk (8.0 ± 0.4 and 6.7 ± 0.5 for white and brown hens, respectively) and total cholesterol (mg) per egg (114.5 ± 6.9 and 84.0 ±
Table 2. Effect of 28-d dietary hesperidin supplementation on egg production and quality traits in laying hens (mean ± se, n = 8*) Hesperidin C Egg production (%) Egg weight (g) Eggshell strength (N) Egg index Yolk index Haugh units Albumen pH Yolk weight (g) Yolk weight/egg weight
86.1 ± 4.63 57.0 ± 1.56 27.4 ± 1.33 75.6 ± 0.73 46.5 ± 0.75 94.9 ± 1.01 8.4 ± 0.08 13.6 ± 0.54 0.25 ± 0.010
HE1 93.6 57.1 28.2 75.5 47.1 95.6 8.2 13.4 0.24
± ± ± ± ± ± ± ± ±
HE2
0.90 1.21 1.07 0.60 0.94 1.78 0.09 0.73 0.013
89.8 56.9 27.8 75.8 45.1 93.5 8.2 13.5 0.24
± ± ± ± ± ± ± ± ±
C, 0 g/kg feed; HE1, 1 g/kg feed; HE2, 3 g/kg feed. *n for egg production = 16.
Table 4.
1.79 1.03 0.99 0.39 0.84 2.04 0.16 0.32 0.010
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C
HE1
HE2
Yolk cholesterol (mg/g) 8.4 ± 0.85 7.3 ± 0.33 6.8 ± 0.49 Total cholesterol/egg 114.7 ± 14.82 97.1 ± 4.98 92.4 ± 8.24 (mg) Plasma cholesterol 98.7 ± 23.62 89.18 ± 9.12 80.5 ± 7.42 (mg/dl) C, 0 g/kg feed; HE1, 1 g/kg feed; HE2, 3 g/kg feed.
4.3 for white and brown hens, respectively) but hesperidin by strain interaction appeared not to be significant for the measured cholesterol parameters (P > 0.05). Egg yolk oxidative stability Table 4 presents egg yolk oxidative stability throughout the experimental period, expressed as ng MDA/g yolk. Smaller MDA values indicate improved oxidative stability compared to greater ones. As it was found, dietary hesperidin supplementation improved yolk oxidative stability even from the first week of the experiment (P < 0.05). Egg yolk of HE1 and HE2 groups showed improved antioxidant capacity compared to the C group, from the beginning until the end of the experiment (P < 0.05). No significant effects of strain and the interaction of hesperidin with strain were observed (P > 0.05). Yolk oxidative stability from eggs collected between the 29th and the 31st day of the experiment, which were then stored either at room temperature (~20°C) for 15 and 30 d or at 4°C for 90 d, is presented in Table 5. Regardless of the duration of the storage period, MDA values for hesperidin treatment groups were smaller compared to the control one. However, a significant improvement in oxidative stability in the HE2 group compared to the other treatment groups was detected for eggs stored at 4°C for 90 d (P < 0.05). The lack of significant mean differences for eggs stored at room temperature for 30 d, even though a significant hesperidin effect was detected, is attributed to the conservative
Effect of dietary hesperidin on egg yolk oxidative stability (ng MDA/g yolk) (mean ± SE, n = 8) Duration of supplementation (d)
Effect Hesperidin C HE1 HE2 P values
0
7
14
21
28
13.1 ± 1.09 13.8 ± 1.52 11.0 ± 1.23 P > 0.05
12.1 ± 1.36a 7.7 ± 1.12b 6.7 ± 0.33b P < 0.01
11.7 ± 1.19a 6.8 ± 0.87b 7.1 ± 0.98b P < 0.05
10.2 ± 1.05a 5.9 ± 0.70b 6.0 ± 0.32b P < 0.01
8.1 ± 0.51a 5.3 ± 0.33b 4.6 ± 0.22b P < 0.001
C, 0 g/kg feed; HE1, 1 g/kg feed; HE2, 3 g/kg feed. a,b Within a column, values not sharing a common superscript letter are significantly different (P < 0.05).
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Table 5. Effect of dietary hesperidin on egg yolk oxidative stability (ng MDA/g yolk) during storage (mean ± SE, n = 8) Storage1 (d) Effect Hesperidin C HE1 HE2 P values
15
30
90
31.3 ± 4.84 25.5 ± 3.90 22.6 ± 2.55 P > 0.05
82.3 ± 6.35 57.7 ± 6.11 56.6 ± 7.78 P < 0.05
138.9 ± 3.28a 126.5 ± 5.25ab 114.8 ± 6.11b P < 0.01
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C, 0 g/kg feed; HE1, 1 g/kg feed; HE2, 3 g/kg feed. a,b Within a column, values not sharing a common superscript letter are significantly different (P < 0.05). 1 Eggs were stored for 15 and 30 d at room temperature (15–20°C) and for 90 d at 4°C.
Bonferroni adjustment method applied for multiple comparisons. Strain and interaction of hesperidin by strain did not influence antioxidant capacity of egg yolks stored either at room temperature (~20°C) for 15 and 30 d or at 4°C for 90 d (P > 0.05).
DISCUSSION Egg production and quality measurements No effects of the different concentrations of hesperidin dietary supplementation on egg production, weight and quality measurements were found. Ting et al. (2011) and Lien et al. (2008) reached the same conclusions for eggshell strength and egg weight after the incorporation of hesperetin (the aglycone of hesperidin) in hens’ diet at a variety of concentrations (0.5-4 g/kg feed). However, yolk weight and the ratio of yolk weight/egg weight were increased after the incorporation of hesperetin in the diet at a concentration of 0.5 g/kg feed (Lien et al., 2008). On the other hand, egg production (%) appeared to decrease after the hesperetin dietary supplementation for 10 weeks at the concentrations of 1 and 4 g/kg feed (Ting et al., 2011). Dietary supplementation of the flavonoid daidzein at a concentration of 10 mg/kg diet to post-peak laying hens improved egg production, and the eggshell quality with no effect on egg weight (Ni et al., 2007). The authors attributed daidzein’s positive effects to its oestrogen-like action (phytoestrogen). According to previous studies (Orban et al., 1993; Zapata and Gernat, 1995; Keshavarz, 1996), ascorbic acid (vitamin C) dietary supplementation (2–3 g/kg or 250–500 mg/kg or 250 mg/kg feed, respectively) appeared to enhance egg weight. However, Kassim and Norziha (1995) reported that the incorporation of ascorbic acid (400– 600 mg/kg feed) in hens’ diet had a significant negative influence on egg weight but not on egg production. Previous studies with α-tocopherol acetate (up to 200 mg/kg feed) have also shown to have no effect on egg production, egg weight
and shape, yolk shape, Haugh units and shell thickness (Jiang et al., 1994; Qi and Sim, 1998). No effect on egg production and weight was observed by Cabuk et al. (2006) when a mixture of essential oils, consisting of oregano (Origanum sp.), laurel leaf (Laurus nobilis L.), sage leaf (Salvia triloba L.), myrtle leaf (Myrtus communis), fennel seed (Foeniculum vulgare) and citrus peel (Citrus sp.) was incorporated in layer hens’ diet during the summer season. Moreover, feeding hens with rosemary or oregano spices that contain a wide range of different phenolic compounds (carnosic acid, carnosol and carvacrol, and thymol, respectively) did not have an effect on egg production, egg weight and shape, yolk shape, Haugh units and shell thickness (Botsoglou et al., 2005b). The exact reasons for the discrepancies between the above studies are speculative but they could have been due to different antioxidant type, different concentrations of dietary supplementation, the period of supplementation and the hen genotype. Moreover, the variability in the effects of antioxidants on hens’ performance and egg quality could also be attributed to, among others, the composition of the basal diet, hygienic standards and environmental conditions. Wellnourished healthy hens may not respond to a feed supplement when they are housed under clean, disinfected conditions and a moderate stocking density (Brenes and Roura, 2010). Yolk and plasma cholesterol The results of the present study indicate no significant effect of hesperidin dietary supplementation on the plasma and yolk cholesterol concentration, although values for the above parameters in HE1 and HE2 group of hens were smaller compared to the C group. Although, a strain effect was detected for yolk cholesterol (total and per g of yolk), no hesperidin by strain interaction was observed. In agreement with the findings of the present study, differences in yolk cholesterol concentration among hens from different populations, i.e. breeds or strains, have been also previously reported by Campo (1995), Sheridan et al. (1982) and Kovacs et al. (1998). Apart from the species, breed or strain, age and rate of egg production, dietary manipulation of poultry may alter yolk cholesterol content (Elkin, 2006). According to previously implemented studies, many plants and phytochemicals may alter cholesterol content of yolk and blood in laying hens. Serum and yolk cholesterol content decreases after hesperetin or naringenin supplementation in laying hens (Lien et al., 2008; Ting et al., 2011), probably as a result of the inhibition of the key enzyme in the cholesterol synthesis, HMG-CoA reductase (Lee et al., 2003; Choi et al., 2004). Chowdhury et al. (2002) and Mottaghitalab and Taraz (2004) observed a
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significant reduction in yolk and plasma cholesterol due to the incorporation of garlic at concentrations ranging from 0.5% to 10% in layer diets. Uuganbayar et al. (2005) reported that dietary green tea reduced yolk cholesterol in laying hens while, on the contrary, Park et al. (2010) found that dietary supplementation with Epimedium koreanum, a plant known for its antioxidant properties, improved egg weight and yolk colour but at the same time had the adverse effect of increasing yolk cholesterol. As it was previously pointed out, discrepancies among previously implemented studies may be attributed to the different mode of action of the various substances and their dosages. However, the observed differences between the present study and the studies of Lien et al. (2008) and Ting et al. (2011), which are attributed to hesperetin administration, may be attributed to the longer period of hesperetin supplementation, 8 and 10 weeks, respectively. Undoubtedly, cholesterol measurements during smaller time intervals, such as weeks, would be an optimal alternative for future research so as to predict the duration of diet enrichment with hesperidin required for detecting the reduction in yolk and plasma cholesterol. Egg yolk oxidative stability In the present study, the extent of egg yolk lipid oxidation was lower in the HE1 and HE2 compared to the control group between the first and the fourth week after the hesperidin supplementation of the diet. A significant improvement in yolk antioxidative capacity was also observed in eggs that were laid from hens fed with hesperidin for approximately 30 d and then stored for 30 and 90 d. A possible explanation of MDA values reduction is the transfer of hesperidin and/or its metabolites into the hen metabolism through feeding and the further inhibition of the chain reactions involved in oxidation of the lipids leading to a reduction of oxidation products in the yolk, thus increasing the egg antioxidative potential. Marshall et al. (1994) and Cherian et al. (1996) demonstrated that the incorporation of α-tocopherol in diet increased the oxidative stability of fresh eggs. During the last decade, considerable interest has arisen in the use of natural antioxidants derived from aromatic plants for improving egg quality by increasing its antioxidative potential. In several studies, a positive effect on oxidation status of conserved eggs was demonstrated after supplementing hens’ diet with oregano, rosemary, thyme or saffron (Botsoglou et al., 1997, 2005a, 2005b; Florou-paneri et al., 2005, 2006). Lien et al. (2008) and Ting et al. (2011) investigated the antioxidant activity on hens’ blood samples and found that serum superoxide dismutase (SOD) concentration was relatively high after hesperetin
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and naringenin supplementation (0.5-4 g/kg), resulting in a reduced superoxide anion concentration. The above results indicate that hesperetin and naringenin can terminate chain radical reaction by donating hydrogen atoms to the free radicals; an action comparable to the action of vitamin E (Cook and Samman, 1996; Van Acker et al., 2000) Consumer concerns about the nutritive value of foods have triggered the interest in improving the quality and the antioxidant properties of eggs with the intention to produce a high-quality functional food. Results of the present study reveal that when hesperidin is incorporated in hens’ diet, at concentrations of 1 and 3 g/kg feed, it can pass into egg yolk its antioxidant properties, efficiently prevent lipid oxidation and increase shelf-life of eggs. However, further experimentation is warranted to elucidate its exact action in hen’s metabolism.
REFERENCES ANGELO, A.J.S., VERCELLOTTI, J., JACKS, T. & LEGENDRE, M. (1996) Lipid oxidation in foods. Critical Reviews in Food Science and Nutrition, 36: 175–224. BOTSOGLOU, N.A., FLOROU-PANERI, P., NIKOLAKAKIS, I., GIANNENAS, I., DOTAS, V., BOTSOGLOU, E.N. & AGGELOPOULOS, S. (2005a) Effect of dietary saffron (Crocus sativus L.) on the oxidative stability of egg yolk. British Poultry Science, 46: 701–707. BOTSOGLOU, N., FLOROU-PANERI, P., BOTSOGLOU, E., DOTAS, V., GIANNENAS, I., KOIDIS, A. & MITRAKOS, P. (2005b) The effect of feeding rosemary, oregano, saffron and α-tocopheryl acetate on hen performance and oxidative stability of eggs. South African Journal of Animal Science, 35: 143–151. BOTSOGLOU, N.A., FLETOURIS, D.J., PAPAGEORGIOU, G.E., VASSILOPOULOS, V.N., MANTIS, A.J. & TRAKATELLIS, A.G. (1994) A rapid, sensitive, and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissues, food, and feedstuff samples. Journal of Agriculture and Food Chemistry, 42: 1931–1937. BOTSOGLOU, N.A., YANNAKOPOULOS, A.L., FLETOURIS, D.J., TSERVENI-GOUSSI, A.S. & FORTOMARIS, P.D. (1997) Effect of dietary thyme on the oxidative stability of egg yolk. Journal of Agricultural and Food Chemistry, 45: 3711–3716. BRENES, A. & ROURA, E. (2010) Essential oils in poultry nutrition: main effects and modes of action. Animal Feed Science and Technology, 158: 1–14. CABUK, M., BOZKURT, M., ALCICEK, A., CATLI, A.U. & BASER, K.H. C. (2006) Effect of dietary essential oil mixture on performance of laying hens in summer season. South African Journal of Animal Science, 36: 215–221. CAMPO, J.L. (1995) Comparative yolk cholesterol content in four Spanish breeds of hens, an F2 cross, and a white Leghorn population. Poultry Science, 74: 1061–1066. CHERIAN, G., WOLFE, F.W. & SIM, J.S. (1996) Feeding dietary oils with tocopherols: effects on internal qualities of eggs during storage. Journal of Food Science, 61: 15–18. CHOI, G.S., LEE, S., JEONG, T.S., LEE, M.K., LEE, J.S., JUNG, U.J., KIM, H.J., PARK, Y.B., BOK, S.H. & CHOI, M.S. (2004) Evaluation of hesperetin 7-O-lauryl ether as lipid-lowering agent in high-cholesterol-fed rats. Bioorganic and Medicinal Chemistry, 12: 3599–3605. CHOWDHURY, S.R., CHOWDHURY, S.D.& SMITH, T.K. (2002) Effects of dietary garlic on cholesterol metabolism in laying hens. Poultry Science, 81: 1856–1862. COOK, N.C. & SAMMAN, S. (1996) Flavonoids – chemistry, metabolism, cardioprotective effects, and dietary sources. The Journal of Nutritional Biochemistry, 7: 66–67.
Downloaded by [UQ Library] at 10:57 16 June 2014
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ELKIN, R.G. (2006) Reducing shell egg cholesterol content. I. Overview, genetic approaches, and nutritional strategies. World’s Poultry Science Journal, 62: 665–687. FLOROU-PANERI, P., NIKOLAKAKIS, I., GIANNENAS, I., KOIDIS, A., BOTSOGLOU, E., DOTAS, V. & MITSOPOULOS, I. (2005) Hen performance and egg quality as affected by dietary oregano essential oil and α-tocopheryl acetate supplementation. International Journal of Poultry Science, 4: 449–454. FLOROU-PANERI, P., DOTAS, D., MITSOPOULOS, I., DOTAS, V., BOTSOGLOU, E., NIKOLAKAKIS, I. & BOTSOGLOU, N. (2006) Effect of feeding rosemary and α-tocopheryl acetate on hen performance and egg quality. The Journal of Poultry Science, 43: 143–149. GALOBART, J., BARROETA, A.C., BAUCELLS, M. D., CODONY, R. & TERNES, W. (2001) Effect of dietary supplementation with rosemary extract and α-tocopheryl acetate on lipid oxidation in eggs enriched with omega-3 fatty acids. Poultry Science, 80: 460–467. GARG, A., GARG, S., ZANEVELD, L.J.D. & SINGLA, A.K. (2001) Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytotherapy Research, 15: 655–669. GORINSTEIN, S., MARTIN-BELLOSO, O., PARK, Y.S., HARUENKIT, R., LOJEK, A., CIZ, M., CASPI, A., LIBMAN, I. & TRAKHTENBERG, S. (2001) Comparison of some biochemical characteristics of different citrus fruits. Food Chemistry, 74: 309–315. GUARD-BOULDIN, J. & BUHR, R.J. (2006) Evaluation of eggshell quality of hens infected with Salmonella enteritidis by application of compression. Poultry Science, 85: 129–135. HUANG, Z., LEIBOVITZ, H., LEE, C.M. & MILLAR, R. (1990) Effect of dietary fish oil on omega-3 fatty acid levels in chicken eggs and thigh flesh. Journal of Agricultural and Food Chemistry, 38: 743–747. JIANG, Y.H., MCGEACHIN, R.B. & BAILEY, C.A. (1994) Alpha-tocopherol, beta-carotene, and retinal enrichment of chicken eggs. Poultry Science, 73: 1137–1143. KASSIM, H. & NORZIHA, I. (1995) Effects of ascorbic acid (vitamin C) supplementation in layer and broiler diets in the tropics. Asian-Australian Journal of Animal Science, 8: 607–610. KESHAVARZ, K. (1996) The effect of different levels of vitamin C and cholecalciferol with adequate or marginal levels of dietary calcium on performance and eggshell quality of laying hens. Poultry Science, 75: 1227–1235. KOVACS, G., DUBLECZ, K., HUSVETH, F., WAGNER, L., GERENDAI, D., ORBAN, J. & MANILLA, H. (1998) Effects of different hybrids, strains and age of laying hens on the cholesterol content of the table egg. Acta Veterinaria Hungarica, 46: 285–294. LANZA, M. (2003) Processed and derived products of oranges. in: CABALLERO, B., TRUGO, L. & FINGLAS, P.M. (Eds) Encyclopedia of Food Sciences and Nutrition, 2nd edn, pp.1346–1354 (Oxford, Elsevier Science Ltd). LEE, M.K., MOON, S.S., LEE, S.E., BOK, S.H., JEONG, T.S., PARK, Y. B.& CHOI, M.S. (2003) Naringenin 7-O-cetyl ether as inhibitor of HMG-CoA reductase and modulator of plasma and hepatic lipids in high cholesterol-fed rats. Bioorganic and Medicinal Chemistry, 11: 393–398.
LIEN, T.F., YEH, H.S.& SU, W.T. (2008) Effect of adding extracted hesperetin, naringenin and pectin on egg cholesterol, serum traits and antioxidant activity in laying hens. Archives of Animal Nutrition, 62: 33–43. MARSHALL, A.C., SAMS, A.R. & VAN ELSWYK, M.E. (1994) Oxidative stability and sensory quality of stored eggs from hens fed 1.5% menhaden oil. Journal of Food Science, 59: 561–563. MOTTAGHITALAB, M. & TARAZ, Z. (2004) Garlic powder as blood serum and egg yolk cholesterol lowering agent. Journal of Poultry Science, 41: 50–57. NI, Y.D., ZHU, Q., ZHOU, Z.L., GROSSMANN, R., CHEN, J. & ZHAO, R.Q. (2007) Effect of dietary daidzein on egg production, shell quality, and gene expression of ER-r, GH-R, and IGFIR in shell glands of laying hens. Agricultural and Food Chemistry, 55: 6997–7001. ORBAN, J.I., ROLAND, D.A., CUMMINS, K. & LOVELL, R.T. (1993) Influence of large doses of ascorbic acid on performance, plasma calcium, bone characteristics, and eggshell quality in broilers and Leghorn hens. Poultry Science, 72: 691–700. PARK, K.M., JIN, Y.H., LEE, K.T., LEE, W.I., NAM, S.W. & HAN, Y. K. (2010) Influence of Epimedium koreanum on the performance of laying hens, egg quality, and fat soluble vitamin and cholesterol contents in the yolk. Journal of Medicinal Plant Research, 4: 1971–1976. PASIN, G., SMITH, G.M. & MAHONY, M.O. (1998) Rapid determination of total cholesterol in egg yolk using commercial diagnostic cholesterol reagent. Food Chemistry, 61: 255–259. PIETTA, P.G. (2000) Flavonoids as antioxidants. The Journal of Natural Products, 63: 1035–1042. QI, G.H. & SIM, J.S. (1998) Natural tocopherol enrichment and its effect in n-3 fatty acid modified chicken eggs. Journal of Agriculture and Food Chemistry, 46: 920–926. SAS INSTITUTE, INC. (2005) Statistical Analysis Systems User’s Guide. Version 9.1.3. (Cary, NC, SAS Institute). SHERIDAN, A.K., HUMPHRIS, C.S. & NICHOLLS, P.J. (1982) The cholesterol content of eggs produced by Australian egglaying strains. British Poultry Science, 23: 569–575. TING, S., YEH, H.S. & LIEN, T.F. (2011) Effects of supplemental levels of hesperetin and naringenin on egg quality, serum traits and antioxidant activity of laying hens. Animal Feed Science and Technology, 163: 59–66. UUGANBAYAR, D., BAE, I.H., CHOI, K.S., SHIN, I.S., FIRMAN, J.D. & YANG, C.J. (2005) Effects of green tea powder on laying performance and egg quality in laying hens. AsianAustralasian Journal of Animal Sciences, 18: 1769–1774. VAN ACKER, F.A.A., SCHOUTEN, O., HAENEN, G.R.M.M., VAN DER VIJGH, W.J.F. & BAST, A. (2000) Flavonoids can replace a-tocopheryl as an antioxidant. FEBS Letters, 473: 145–148. WENK, C. (2003) Herbs and botanicals as feed additives in monogastric animals. Asian-Australasian Journal of Animal Sciences, 16: 282–289. ZAPATA, L.F. & GERNAT, A.G. (1995) The effect of four levels of ascorbic acid and two levels of calcium on eggshell quality of forced-molted White Leghorn hens. Poultry Science, 74: 1049–1052.
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effect of hesperidin dietary supplementation on hen performance, egg quality and yolk oxidative stability a
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M. Goliomytis , H. Orfanou , E. Petrou , M. A. Charismiadou , P. E. Simitzis & S. G. Deligeorgis
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Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece Accepted author version posted online: 08 Jan 2014.Published online: 16 Apr 2014.
To cite this article: M. Goliomytis, H. Orfanou, E. Petrou, M. A. Charismiadou, P. E. Simitzis & S. G. Deligeorgis (2014) Effect of hesperidin dietary supplementation on hen performance, egg quality and yolk oxidative stability, British Poultry Science, 55:1, 98-104, DOI: 10.1080/00071668.2013.870328 To link to this article: http://dx.doi.org/10.1080/00071668.2013.870328
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British Poultry Science, 2014 Vol. 55, No. 1, 98–104, http://dx.doi.org/10.1080/00071668.2013.870328
Effect of hesperidin dietary supplementation on hen performance, egg quality and yolk oxidative stability M. GOLIOMYTIS, H. ORFANOU, E. PETROU, M. A. CHARISMIADOU, P. E. SIMITZIS AND S. G. DELIGEORGIS
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Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece
Abstract 1. The purpose of this study was to evaluate the effects of dietary supplementation with hesperidin (one or 3 g/kg of feed) for 31 d on the performance, egg quality and yolk oxidative stability of brown and white laying hens (26-wk old). 2. Supplementation with hesperidin did not affect egg production, egg weight and egg quality traits. 3. No hesperidin effect on yolk and plasma cholesterol was observed. A strain effect was found with lower total and per g yolk cholesterol of brown hens in comparison to the white ones. 4. Oxidative stability of egg yolk, expressed as ng MDA/g yolk, was significantly improved in the hesperidin groups even from the first week of supplementation. At the same time, a significant improvement in the oxidative stability of egg yolk due to the incorporation of hesperidin in hens’ diet was observed after 30 and 90 d of storage at 20°C and 4°C, respectively. 5. No hesperidin by strain interaction was detected for any of the traits measured. 6. In conclusion, incorporation of hesperidin to laying hens’ feed did not affect productive and egg qualitative traits. On the other hand, dietary hesperidin supplementation significantly improved oxidative stability of both fresh and stored eggs. Antioxidant properties of hesperidin seem to make it a promising natural agent for improving the shelf life of eggs.
INTRODUCTION Eggs have a high nutritional value and contain a variety of necessary components for the maintenance and the normal function of the human organism. Feeding strategies have been developed in order to increase the n-3 fatty acid content of eggs by enriching poultry diets with polyunsaturated fatty acids (Huang et al., 1990). However, these constituents increase the degree of unsaturation and the susceptibility of eggs to oxidative deterioration. Lipid oxidation by free radicals is one of the primary mechanisms of quality deterioration in eggs. It is initiated in the highly unsaturated fatty acid fraction of membrane phospholipids, leading to the production of hydroperoxides, which are susceptible to further oxidation or decomposition
to secondary reaction products such as short-chain aldehydes, ketones and other oxygenated compounds that may adversely affect lipids, pigments, proteins, carbohydrates, vitamins and the overall quality by causing loss of flavour, colour and nutritive value and limiting shelf-life (Angelo et al., 1996; Cherian et al., 1996). In the past, synthetic antioxidants were used with the intention of preventing lipid oxidation by scavenging chain-carrying peroxyl radicals or diminishing the formation of lipid radicals. In the last decade, there has been a strong tendency towards using organic antioxidants from natural sources for the protection of animal health and their products against oxidation (Wenk, 2003). As a result, considerable interest has arisen in the use of natural antioxidants derived from aromatic
Correspondence to: Michael Goliomytis, Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece. E-mail: [email protected] Accepted for publication 9 October 2013.
© 2014 British Poultry Science Ltd
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plants that could serve as alternatives to synthetic supplements on purpose to improve egg quality, without leaving residues in the product or the environment (Galobart et al., 2001; Florou-paneri et al., 2005, 2006; Botsoglou et al., 2005a, 2005b). The increased awareness of consumers towards a diet rich in natural, safe and healthpromoting ingredients has led to the search of promising sources of materials that may be used in the food and feed industry because of their valuable nutritional properties. Dried citrus pulp appears to be a promising candidate and is the main by-product from the citrus-processing industry. It is produced after extraction of the juice from citrus fruits and drying of the residues and is a mixture of peel, inside portions and culled fruits of the citrus family, rich in energy, fibre and calcium. Fibres from citrus fruits have an additional advantage over dietary fibres from other sources due to the presence of associated bioactive compounds (i.e. flavonoids) (Gorinstein et al., 2001). These compounds usually contain one or more aromatic hydroxyl groups, which actively scavenge free radicals and are responsible for the respective antioxidant properties (Pietta, 2000). Hesperidin, a bioflavonoid, is an abundant and inexpensive by-product of citrus cultivation. It is a naturally occurring polyphenolic compound widely distributed in the plant kingdom as a secondary metabolite. The traditional extraction method, with a very low recovery, treats triturated peel with alkaline compounds to dissolve the flavonoids, which, after separation of solid residue, are precipitated by acidification. An innovative method to recover hesperidin from orange peel and waste water utilises styrene-divinylbenzene as the absorbing phase, removing the flavonoid with alcoholic NaOH. The results may be a useful starting point for industrial applications (Lanza, 2003). Hesperidin is a flavanone glycoside comprising of an aglycone, hesperetin or methyl eriodictyol and an attached disaccharide, rutinose. Pure hesperidin occurs as long hair-like needles, tan or pale yellow in colour. It is tasteless and odourless (Garg et al., 2001). A deficiency of this substance in human diet has been linked with abnormal capillary leakiness as well as pain in the extremities causing aches, weakness and leg cramps. Moreover, hesperidin has been reported to possess antioxidant activity and radical scavenging properties (Garg et al., 2001). The combination of increased disposal costs in many parts of the world with the antioxidant properties of citrus by-products have increased interest in their utilisation as alternative feeds in animal production. The objective of the present study was therefore the evaluation of the effects of different concentrations of hesperidin dietary
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supplementation on hen performance and egg quality characteristics.
MATERIALS AND METHODS Animals and diets A total of 24 white (LSL-classic) and 24 brown (Brown-Classic) Lohmann individually caged laying hens (26 wk old) were randomly assigned into three equal treatment groups (16 hens each). One of the groups served as a control (C) and was given a commercial basal diet, whereas the other two groups were given the same diet further supplemented with hesperidin (MP Biomedicals, LLC, France) at 1 g/kg (HE1), and 3 g/kg (HE2) for 31 d. Water was provided ad libitum throughout the experimental period and the light regimen was 16 h of continuous light per d. Each hen of the white and the brown strain consumed approximately 105 and 110 g feed/d, respectively. Table 1 presents the ingredients and the composition of the control diet. Egg production was daily recorded throughout the experimental study. Egg weight, laboratory evaluations of egg quality and measurements of yolk cholesterol were performed on eggs collected from 8 hens per dietary group, at d 28 of the experiment. Yolk oxidative stability was assessed by using the malondialdehyde (MDA) assay on eggs collected from 8 hens per Table 1.
Ingredients and chemical composition of the diet
Ingredients Maize Soybean meal, 47% CP Wheat Wheat bran Soybean oil Limestone Monocalcium phosphate Sodium chloride Sodium bicarbonate Methionine 99% Choline chloride 70% Phytase, Natuphos® Antioxidant Pigment (Carophyl® Red) Vitamin and Mineral premix1 Chemical composition, calculated Metabolisable energy (MJ/kg) Crude protein Lysine Methionine + cysteine Calcium Available phosphorus 1
g/kg 540 246 50 46 12 89 9 2.4 2.7 1.1 0.6 0.06 0.1 0.04 1 11.48 175 9 6.8 37.5 3.6
The vitamin and mineral premix provided per kg of diet: 3 mg of retinol (vitamin A), 62.5 μg of cholecalciferol (vitamin D3), 30 mg of tocopherol (vitamin E), 5 mg of menadione (vitamin K3), one milligram of thiamine (vitamin B1), 5 mg of riboflavin (vitamin B2), 3 mg of pyridoxin (vitamin B6), 20 μg of cobalamin (vitamin B12), 30 mg of nicotinic acid, 10 mg of pantothenic acid, 0.8 mg of folic acid, 100 μg of biotin, 10 mg of ascorbic acid (Vitamin C), 450 mg of choline chloride, 0.2 mg of Co, 0.5 mg of I, 0.3 mg of Se, 25 mg of Fe, 120 mg of Mn, 10 mg of Cu and 100 mg of Zn.
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dietary group at 0, 7, 14, 21 and 28 d after hesperidin dietary supplementation. MDA concentration was also measured in eggs collected at d 29, 30 and the 31 of the experiment and consequently stored for 15 and 30 d at room temperature (~20° C) and for 90 d at 4°C (refrigerated storage). At d 28 of the experiment, blood samples from 8 hens per dietary group were also collected from the brachial wing vein for the plasma cholesterol determination. The methods used in the present experiment were in accordance with the national legislation and the guidelines of the Research Ethics Committee of the Department of Animal Science and Aquaculture of the Agricultural University of Athens.
anticoagulant (2 mg EDTA/ml of blood), were centrifuged at 800 × g for 20 min and supernatant was collected. Plasma cholesterol was measured photometrically at 540 nm in a spectrophotometer (Hitachi U3010 Spectrophotometer) by using a commercial cholesterol reagent kit (Biosis commercial kits; Athens, Greece). Yolk cholesterol was determined following the method described by Pasin et al. (1998). In detail, 3 g of egg yolk was diluted with 27 ml NaCl-solution (20 g/kg), stirred for 2 h using a magnetic stirrer, and 1 ml of the above solution was further diluted with 9 ml NaCl (20 g/kg). The rest of the determination procedure was the same to the one described above for plasma cholesterol. Determination of lipid oxidation (MDA analysis)
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Quality measurements Egg quality measurements were based on determination of external and internal indices, and egg components. The external indices included eggshape index and eggshell strength; those of interior quality were Haugh unit score, albumen pH, yolk index, yolk weight and yolk percentage expressed as yolk weight/egg weight. In detail, the collected eggs were individually weighed and their width and length were measured. Eggshell breaking strength was assessed by a Zwick Testing Machine (Model Z2.5/TN1S, Zwick GmbH & Co, Germany). The measurement was carried out on eggs with the major axis parallel to the compression surfaces (force applied at equator). Crosshead speed was set at 60 mm/min, with a programmed automatic stop set when a 40% reduction of the maximum load occurred (no loss of egg contents occurred, since the inner shell membrane remained intact (Guardbouldin and Buhr, 2006). Eggs were then broken and Haugh units were determined on each egg albumen with a Haugh meter (Model S-8400, B.C. Ames Inc., USA). Yolk height was determined using a tripod micrometer, while yolk diameter was measured using a steel vernier calliper. Yolk index was calculated as yolk height × 100 divided by yolk diameter. After the separation of albumen and yolk, 5 g of the albumen was placed in a separate 100-ml beaker for the measurement of albumen pH. Distilled water (45 ml) was added to each beaker and mixed thoroughly using a blender (Edmund Buehler 7400 Tuebingen/H04, Germany). The pH measurements were obtained by inserting the electrode of the pH meter (pHM120, MeterLab) in the derived mixture. Determination of plasma and yolk cholesterol The blood samples (2 ml) that were collected from the hens in tubes containing EDTA
Lipid oxidation was assessed on the basis of the malondialdehyde (MDA) formed during storage, a secondary lipid oxidation product formed by hydrolysis of lipid hydroperoxides. In the present study, yolk MDA concentration was determined by using a selective third-order derivative spectrophotometric method (Botsoglou et al., 1994). Derivative versus conventional spectrophotometry was adopted because it offers improved sensitivity, specificity and reliability of the measurements, since it eliminates potential interferences from other reactive compounds. In brief, 2 g of each sample (two samples per egg) were homogenised (Edmund Buehler, 7400 Tuebingen/H04, Germany) in the presence of 8 ml aqueous trichloroacetic acid (TCA) (50 g/l) and 5 ml butylated hydroxytoluene (BHT) in hexane (8 g/l), and the mixture was centrifuged for 3 min at 3000 g. The top hexane layer was discarded and a 2.5 ml aliquot from the bottom layer was mixed with 1.5 ml aqueous 2-thiobarbituric acid (TBA) (8 g/l) to be further incubated at 70ºC for 30 min. Following incubation, the mixture was cooled under tap water and submitted to thirdorder derivative (3D) spectrophotometry (Hitachi U3010 Spectrophotometer) in the range of 500– 550 nm. The concentration of MDA (ng/g yolk) in analysed samples was calculated on the basis of the height of the third-order derivative peak at 521.5 nm by referring to the slope and intercept data of the computed least-squares fit of standard calibration curve prepared using 1,1,3,3tetraethoxypropane (TEP), the malondialdehyde precursor. Statistical analysis Data were subjected to analysis of variance with hesperidin supplementation, strain of the hens and their interaction as fixed effects using the general linear models of SAS software (SAS Institute, Inc., 2005). An arcsine transformation
DIETARY HESPERIDIN FOR LAYERS
was applied to egg production data prior to statistical analysis. Mean differences were tested at 0.05 significance level with Bonferroni adjustment. Results are presented as mean ± standard error.
Table 3. Effect of 28-d dietary hesperidin supplementation on yolk and plasma cholesterol (mean ± SE, n=8) Hesperidin Variable
RESULTS Egg production and quality measurements
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The effects on egg production and egg quality characteristics after 28 d of hesperidin dietary supplementation are shown in Table 2. Neither egg production nor egg quality measurements appeared to be influenced by the incorporation of hesperidin in hens’ diet (P > 0.05). On the other hand, the genetic origin of hens affected egg index, yolk weight and yolk/egg weight ratio (P < 0.05). However, no interaction between hesperidin and strain of hens was detected (P > 0.05). Yolk and plasma cholesterol Results presented in Table 3 showed that hesperidin dietary supplementation for 28 d did not influence either plasma or yolk cholesterol (P > 0.05). A strain effect (P < 0.05) was detected for cholesterol (mg) per g yolk (8.0 ± 0.4 and 6.7 ± 0.5 for white and brown hens, respectively) and total cholesterol (mg) per egg (114.5 ± 6.9 and 84.0 ±
Table 2. Effect of 28-d dietary hesperidin supplementation on egg production and quality traits in laying hens (mean ± se, n = 8*) Hesperidin C Egg production (%) Egg weight (g) Eggshell strength (N) Egg index Yolk index Haugh units Albumen pH Yolk weight (g) Yolk weight/egg weight
86.1 ± 4.63 57.0 ± 1.56 27.4 ± 1.33 75.6 ± 0.73 46.5 ± 0.75 94.9 ± 1.01 8.4 ± 0.08 13.6 ± 0.54 0.25 ± 0.010
HE1 93.6 57.1 28.2 75.5 47.1 95.6 8.2 13.4 0.24
± ± ± ± ± ± ± ± ±
HE2
0.90 1.21 1.07 0.60 0.94 1.78 0.09 0.73 0.013
89.8 56.9 27.8 75.8 45.1 93.5 8.2 13.5 0.24
± ± ± ± ± ± ± ± ±
C, 0 g/kg feed; HE1, 1 g/kg feed; HE2, 3 g/kg feed. *n for egg production = 16.
Table 4.
1.79 1.03 0.99 0.39 0.84 2.04 0.16 0.32 0.010
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C
HE1
HE2
Yolk cholesterol (mg/g) 8.4 ± 0.85 7.3 ± 0.33 6.8 ± 0.49 Total cholesterol/egg 114.7 ± 14.82 97.1 ± 4.98 92.4 ± 8.24 (mg) Plasma cholesterol 98.7 ± 23.62 89.18 ± 9.12 80.5 ± 7.42 (mg/dl) C, 0 g/kg feed; HE1, 1 g/kg feed; HE2, 3 g/kg feed.
4.3 for white and brown hens, respectively) but hesperidin by strain interaction appeared not to be significant for the measured cholesterol parameters (P > 0.05). Egg yolk oxidative stability Table 4 presents egg yolk oxidative stability throughout the experimental period, expressed as ng MDA/g yolk. Smaller MDA values indicate improved oxidative stability compared to greater ones. As it was found, dietary hesperidin supplementation improved yolk oxidative stability even from the first week of the experiment (P < 0.05). Egg yolk of HE1 and HE2 groups showed improved antioxidant capacity compared to the C group, from the beginning until the end of the experiment (P < 0.05). No significant effects of strain and the interaction of hesperidin with strain were observed (P > 0.05). Yolk oxidative stability from eggs collected between the 29th and the 31st day of the experiment, which were then stored either at room temperature (~20°C) for 15 and 30 d or at 4°C for 90 d, is presented in Table 5. Regardless of the duration of the storage period, MDA values for hesperidin treatment groups were smaller compared to the control one. However, a significant improvement in oxidative stability in the HE2 group compared to the other treatment groups was detected for eggs stored at 4°C for 90 d (P < 0.05). The lack of significant mean differences for eggs stored at room temperature for 30 d, even though a significant hesperidin effect was detected, is attributed to the conservative
Effect of dietary hesperidin on egg yolk oxidative stability (ng MDA/g yolk) (mean ± SE, n = 8) Duration of supplementation (d)
Effect Hesperidin C HE1 HE2 P values
0
7
14
21
28
13.1 ± 1.09 13.8 ± 1.52 11.0 ± 1.23 P > 0.05
12.1 ± 1.36a 7.7 ± 1.12b 6.7 ± 0.33b P < 0.01
11.7 ± 1.19a 6.8 ± 0.87b 7.1 ± 0.98b P < 0.05
10.2 ± 1.05a 5.9 ± 0.70b 6.0 ± 0.32b P < 0.01
8.1 ± 0.51a 5.3 ± 0.33b 4.6 ± 0.22b P < 0.001
C, 0 g/kg feed; HE1, 1 g/kg feed; HE2, 3 g/kg feed. a,b Within a column, values not sharing a common superscript letter are significantly different (P < 0.05).
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Table 5. Effect of dietary hesperidin on egg yolk oxidative stability (ng MDA/g yolk) during storage (mean ± SE, n = 8) Storage1 (d) Effect Hesperidin C HE1 HE2 P values
15
30
90
31.3 ± 4.84 25.5 ± 3.90 22.6 ± 2.55 P > 0.05
82.3 ± 6.35 57.7 ± 6.11 56.6 ± 7.78 P < 0.05
138.9 ± 3.28a 126.5 ± 5.25ab 114.8 ± 6.11b P < 0.01
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C, 0 g/kg feed; HE1, 1 g/kg feed; HE2, 3 g/kg feed. a,b Within a column, values not sharing a common superscript letter are significantly different (P < 0.05). 1 Eggs were stored for 15 and 30 d at room temperature (15–20°C) and for 90 d at 4°C.
Bonferroni adjustment method applied for multiple comparisons. Strain and interaction of hesperidin by strain did not influence antioxidant capacity of egg yolks stored either at room temperature (~20°C) for 15 and 30 d or at 4°C for 90 d (P > 0.05).
DISCUSSION Egg production and quality measurements No effects of the different concentrations of hesperidin dietary supplementation on egg production, weight and quality measurements were found. Ting et al. (2011) and Lien et al. (2008) reached the same conclusions for eggshell strength and egg weight after the incorporation of hesperetin (the aglycone of hesperidin) in hens’ diet at a variety of concentrations (0.5-4 g/kg feed). However, yolk weight and the ratio of yolk weight/egg weight were increased after the incorporation of hesperetin in the diet at a concentration of 0.5 g/kg feed (Lien et al., 2008). On the other hand, egg production (%) appeared to decrease after the hesperetin dietary supplementation for 10 weeks at the concentrations of 1 and 4 g/kg feed (Ting et al., 2011). Dietary supplementation of the flavonoid daidzein at a concentration of 10 mg/kg diet to post-peak laying hens improved egg production, and the eggshell quality with no effect on egg weight (Ni et al., 2007). The authors attributed daidzein’s positive effects to its oestrogen-like action (phytoestrogen). According to previous studies (Orban et al., 1993; Zapata and Gernat, 1995; Keshavarz, 1996), ascorbic acid (vitamin C) dietary supplementation (2–3 g/kg or 250–500 mg/kg or 250 mg/kg feed, respectively) appeared to enhance egg weight. However, Kassim and Norziha (1995) reported that the incorporation of ascorbic acid (400– 600 mg/kg feed) in hens’ diet had a significant negative influence on egg weight but not on egg production. Previous studies with α-tocopherol acetate (up to 200 mg/kg feed) have also shown to have no effect on egg production, egg weight
and shape, yolk shape, Haugh units and shell thickness (Jiang et al., 1994; Qi and Sim, 1998). No effect on egg production and weight was observed by Cabuk et al. (2006) when a mixture of essential oils, consisting of oregano (Origanum sp.), laurel leaf (Laurus nobilis L.), sage leaf (Salvia triloba L.), myrtle leaf (Myrtus communis), fennel seed (Foeniculum vulgare) and citrus peel (Citrus sp.) was incorporated in layer hens’ diet during the summer season. Moreover, feeding hens with rosemary or oregano spices that contain a wide range of different phenolic compounds (carnosic acid, carnosol and carvacrol, and thymol, respectively) did not have an effect on egg production, egg weight and shape, yolk shape, Haugh units and shell thickness (Botsoglou et al., 2005b). The exact reasons for the discrepancies between the above studies are speculative but they could have been due to different antioxidant type, different concentrations of dietary supplementation, the period of supplementation and the hen genotype. Moreover, the variability in the effects of antioxidants on hens’ performance and egg quality could also be attributed to, among others, the composition of the basal diet, hygienic standards and environmental conditions. Wellnourished healthy hens may not respond to a feed supplement when they are housed under clean, disinfected conditions and a moderate stocking density (Brenes and Roura, 2010). Yolk and plasma cholesterol The results of the present study indicate no significant effect of hesperidin dietary supplementation on the plasma and yolk cholesterol concentration, although values for the above parameters in HE1 and HE2 group of hens were smaller compared to the C group. Although, a strain effect was detected for yolk cholesterol (total and per g of yolk), no hesperidin by strain interaction was observed. In agreement with the findings of the present study, differences in yolk cholesterol concentration among hens from different populations, i.e. breeds or strains, have been also previously reported by Campo (1995), Sheridan et al. (1982) and Kovacs et al. (1998). Apart from the species, breed or strain, age and rate of egg production, dietary manipulation of poultry may alter yolk cholesterol content (Elkin, 2006). According to previously implemented studies, many plants and phytochemicals may alter cholesterol content of yolk and blood in laying hens. Serum and yolk cholesterol content decreases after hesperetin or naringenin supplementation in laying hens (Lien et al., 2008; Ting et al., 2011), probably as a result of the inhibition of the key enzyme in the cholesterol synthesis, HMG-CoA reductase (Lee et al., 2003; Choi et al., 2004). Chowdhury et al. (2002) and Mottaghitalab and Taraz (2004) observed a
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DIETARY HESPERIDIN FOR LAYERS
significant reduction in yolk and plasma cholesterol due to the incorporation of garlic at concentrations ranging from 0.5% to 10% in layer diets. Uuganbayar et al. (2005) reported that dietary green tea reduced yolk cholesterol in laying hens while, on the contrary, Park et al. (2010) found that dietary supplementation with Epimedium koreanum, a plant known for its antioxidant properties, improved egg weight and yolk colour but at the same time had the adverse effect of increasing yolk cholesterol. As it was previously pointed out, discrepancies among previously implemented studies may be attributed to the different mode of action of the various substances and their dosages. However, the observed differences between the present study and the studies of Lien et al. (2008) and Ting et al. (2011), which are attributed to hesperetin administration, may be attributed to the longer period of hesperetin supplementation, 8 and 10 weeks, respectively. Undoubtedly, cholesterol measurements during smaller time intervals, such as weeks, would be an optimal alternative for future research so as to predict the duration of diet enrichment with hesperidin required for detecting the reduction in yolk and plasma cholesterol. Egg yolk oxidative stability In the present study, the extent of egg yolk lipid oxidation was lower in the HE1 and HE2 compared to the control group between the first and the fourth week after the hesperidin supplementation of the diet. A significant improvement in yolk antioxidative capacity was also observed in eggs that were laid from hens fed with hesperidin for approximately 30 d and then stored for 30 and 90 d. A possible explanation of MDA values reduction is the transfer of hesperidin and/or its metabolites into the hen metabolism through feeding and the further inhibition of the chain reactions involved in oxidation of the lipids leading to a reduction of oxidation products in the yolk, thus increasing the egg antioxidative potential. Marshall et al. (1994) and Cherian et al. (1996) demonstrated that the incorporation of α-tocopherol in diet increased the oxidative stability of fresh eggs. During the last decade, considerable interest has arisen in the use of natural antioxidants derived from aromatic plants for improving egg quality by increasing its antioxidative potential. In several studies, a positive effect on oxidation status of conserved eggs was demonstrated after supplementing hens’ diet with oregano, rosemary, thyme or saffron (Botsoglou et al., 1997, 2005a, 2005b; Florou-paneri et al., 2005, 2006). Lien et al. (2008) and Ting et al. (2011) investigated the antioxidant activity on hens’ blood samples and found that serum superoxide dismutase (SOD) concentration was relatively high after hesperetin
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and naringenin supplementation (0.5-4 g/kg), resulting in a reduced superoxide anion concentration. The above results indicate that hesperetin and naringenin can terminate chain radical reaction by donating hydrogen atoms to the free radicals; an action comparable to the action of vitamin E (Cook and Samman, 1996; Van Acker et al., 2000) Consumer concerns about the nutritive value of foods have triggered the interest in improving the quality and the antioxidant properties of eggs with the intention to produce a high-quality functional food. Results of the present study reveal that when hesperidin is incorporated in hens’ diet, at concentrations of 1 and 3 g/kg feed, it can pass into egg yolk its antioxidant properties, efficiently prevent lipid oxidation and increase shelf-life of eggs. However, further experimentation is warranted to elucidate its exact action in hen’s metabolism.
REFERENCES ANGELO, A.J.S., VERCELLOTTI, J., JACKS, T. & LEGENDRE, M. (1996) Lipid oxidation in foods. Critical Reviews in Food Science and Nutrition, 36: 175–224. BOTSOGLOU, N.A., FLOROU-PANERI, P., NIKOLAKAKIS, I., GIANNENAS, I., DOTAS, V., BOTSOGLOU, E.N. & AGGELOPOULOS, S. (2005a) Effect of dietary saffron (Crocus sativus L.) on the oxidative stability of egg yolk. British Poultry Science, 46: 701–707. BOTSOGLOU, N., FLOROU-PANERI, P., BOTSOGLOU, E., DOTAS, V., GIANNENAS, I., KOIDIS, A. & MITRAKOS, P. (2005b) The effect of feeding rosemary, oregano, saffron and α-tocopheryl acetate on hen performance and oxidative stability of eggs. South African Journal of Animal Science, 35: 143–151. BOTSOGLOU, N.A., FLETOURIS, D.J., PAPAGEORGIOU, G.E., VASSILOPOULOS, V.N., MANTIS, A.J. & TRAKATELLIS, A.G. (1994) A rapid, sensitive, and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissues, food, and feedstuff samples. Journal of Agriculture and Food Chemistry, 42: 1931–1937. BOTSOGLOU, N.A., YANNAKOPOULOS, A.L., FLETOURIS, D.J., TSERVENI-GOUSSI, A.S. & FORTOMARIS, P.D. (1997) Effect of dietary thyme on the oxidative stability of egg yolk. Journal of Agricultural and Food Chemistry, 45: 3711–3716. BRENES, A. & ROURA, E. (2010) Essential oils in poultry nutrition: main effects and modes of action. Animal Feed Science and Technology, 158: 1–14. CABUK, M., BOZKURT, M., ALCICEK, A., CATLI, A.U. & BASER, K.H. C. (2006) Effect of dietary essential oil mixture on performance of laying hens in summer season. South African Journal of Animal Science, 36: 215–221. CAMPO, J.L. (1995) Comparative yolk cholesterol content in four Spanish breeds of hens, an F2 cross, and a white Leghorn population. Poultry Science, 74: 1061–1066. CHERIAN, G., WOLFE, F.W. & SIM, J.S. (1996) Feeding dietary oils with tocopherols: effects on internal qualities of eggs during storage. Journal of Food Science, 61: 15–18. CHOI, G.S., LEE, S., JEONG, T.S., LEE, M.K., LEE, J.S., JUNG, U.J., KIM, H.J., PARK, Y.B., BOK, S.H. & CHOI, M.S. (2004) Evaluation of hesperetin 7-O-lauryl ether as lipid-lowering agent in high-cholesterol-fed rats. Bioorganic and Medicinal Chemistry, 12: 3599–3605. CHOWDHURY, S.R., CHOWDHURY, S.D.& SMITH, T.K. (2002) Effects of dietary garlic on cholesterol metabolism in laying hens. Poultry Science, 81: 1856–1862. COOK, N.C. & SAMMAN, S. (1996) Flavonoids – chemistry, metabolism, cardioprotective effects, and dietary sources. The Journal of Nutritional Biochemistry, 7: 66–67.
Downloaded by [UQ Library] at 10:57 16 June 2014
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ELKIN, R.G. (2006) Reducing shell egg cholesterol content. I. Overview, genetic approaches, and nutritional strategies. World’s Poultry Science Journal, 62: 665–687. FLOROU-PANERI, P., NIKOLAKAKIS, I., GIANNENAS, I., KOIDIS, A., BOTSOGLOU, E., DOTAS, V. & MITSOPOULOS, I. (2005) Hen performance and egg quality as affected by dietary oregano essential oil and α-tocopheryl acetate supplementation. International Journal of Poultry Science, 4: 449–454. FLOROU-PANERI, P., DOTAS, D., MITSOPOULOS, I., DOTAS, V., BOTSOGLOU, E., NIKOLAKAKIS, I. & BOTSOGLOU, N. (2006) Effect of feeding rosemary and α-tocopheryl acetate on hen performance and egg quality. The Journal of Poultry Science, 43: 143–149. GALOBART, J., BARROETA, A.C., BAUCELLS, M. D., CODONY, R. & TERNES, W. (2001) Effect of dietary supplementation with rosemary extract and α-tocopheryl acetate on lipid oxidation in eggs enriched with omega-3 fatty acids. Poultry Science, 80: 460–467. GARG, A., GARG, S., ZANEVELD, L.J.D. & SINGLA, A.K. (2001) Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytotherapy Research, 15: 655–669. GORINSTEIN, S., MARTIN-BELLOSO, O., PARK, Y.S., HARUENKIT, R., LOJEK, A., CIZ, M., CASPI, A., LIBMAN, I. & TRAKHTENBERG, S. (2001) Comparison of some biochemical characteristics of different citrus fruits. Food Chemistry, 74: 309–315. GUARD-BOULDIN, J. & BUHR, R.J. (2006) Evaluation of eggshell quality of hens infected with Salmonella enteritidis by application of compression. Poultry Science, 85: 129–135. HUANG, Z., LEIBOVITZ, H., LEE, C.M. & MILLAR, R. (1990) Effect of dietary fish oil on omega-3 fatty acid levels in chicken eggs and thigh flesh. Journal of Agricultural and Food Chemistry, 38: 743–747. JIANG, Y.H., MCGEACHIN, R.B. & BAILEY, C.A. (1994) Alpha-tocopherol, beta-carotene, and retinal enrichment of chicken eggs. Poultry Science, 73: 1137–1143. KASSIM, H. & NORZIHA, I. (1995) Effects of ascorbic acid (vitamin C) supplementation in layer and broiler diets in the tropics. Asian-Australian Journal of Animal Science, 8: 607–610. KESHAVARZ, K. (1996) The effect of different levels of vitamin C and cholecalciferol with adequate or marginal levels of dietary calcium on performance and eggshell quality of laying hens. Poultry Science, 75: 1227–1235. KOVACS, G., DUBLECZ, K., HUSVETH, F., WAGNER, L., GERENDAI, D., ORBAN, J. & MANILLA, H. (1998) Effects of different hybrids, strains and age of laying hens on the cholesterol content of the table egg. Acta Veterinaria Hungarica, 46: 285–294. LANZA, M. (2003) Processed and derived products of oranges. in: CABALLERO, B., TRUGO, L. & FINGLAS, P.M. (Eds) Encyclopedia of Food Sciences and Nutrition, 2nd edn, pp.1346–1354 (Oxford, Elsevier Science Ltd). LEE, M.K., MOON, S.S., LEE, S.E., BOK, S.H., JEONG, T.S., PARK, Y. B.& CHOI, M.S. (2003) Naringenin 7-O-cetyl ether as inhibitor of HMG-CoA reductase and modulator of plasma and hepatic lipids in high cholesterol-fed rats. Bioorganic and Medicinal Chemistry, 11: 393–398.
LIEN, T.F., YEH, H.S.& SU, W.T. (2008) Effect of adding extracted hesperetin, naringenin and pectin on egg cholesterol, serum traits and antioxidant activity in laying hens. Archives of Animal Nutrition, 62: 33–43. MARSHALL, A.C., SAMS, A.R. & VAN ELSWYK, M.E. (1994) Oxidative stability and sensory quality of stored eggs from hens fed 1.5% menhaden oil. Journal of Food Science, 59: 561–563. MOTTAGHITALAB, M. & TARAZ, Z. (2004) Garlic powder as blood serum and egg yolk cholesterol lowering agent. Journal of Poultry Science, 41: 50–57. NI, Y.D., ZHU, Q., ZHOU, Z.L., GROSSMANN, R., CHEN, J. & ZHAO, R.Q. (2007) Effect of dietary daidzein on egg production, shell quality, and gene expression of ER-r, GH-R, and IGFIR in shell glands of laying hens. Agricultural and Food Chemistry, 55: 6997–7001. ORBAN, J.I., ROLAND, D.A., CUMMINS, K. & LOVELL, R.T. (1993) Influence of large doses of ascorbic acid on performance, plasma calcium, bone characteristics, and eggshell quality in broilers and Leghorn hens. Poultry Science, 72: 691–700. PARK, K.M., JIN, Y.H., LEE, K.T., LEE, W.I., NAM, S.W. & HAN, Y. K. (2010) Influence of Epimedium koreanum on the performance of laying hens, egg quality, and fat soluble vitamin and cholesterol contents in the yolk. Journal of Medicinal Plant Research, 4: 1971–1976. PASIN, G., SMITH, G.M. & MAHONY, M.O. (1998) Rapid determination of total cholesterol in egg yolk using commercial diagnostic cholesterol reagent. Food Chemistry, 61: 255–259. PIETTA, P.G. (2000) Flavonoids as antioxidants. The Journal of Natural Products, 63: 1035–1042. QI, G.H. & SIM, J.S. (1998) Natural tocopherol enrichment and its effect in n-3 fatty acid modified chicken eggs. Journal of Agriculture and Food Chemistry, 46: 920–926. SAS INSTITUTE, INC. (2005) Statistical Analysis Systems User’s Guide. Version 9.1.3. (Cary, NC, SAS Institute). SHERIDAN, A.K., HUMPHRIS, C.S. & NICHOLLS, P.J. (1982) The cholesterol content of eggs produced by Australian egglaying strains. British Poultry Science, 23: 569–575. TING, S., YEH, H.S. & LIEN, T.F. (2011) Effects of supplemental levels of hesperetin and naringenin on egg quality, serum traits and antioxidant activity of laying hens. Animal Feed Science and Technology, 163: 59–66. UUGANBAYAR, D., BAE, I.H., CHOI, K.S., SHIN, I.S., FIRMAN, J.D. & YANG, C.J. (2005) Effects of green tea powder on laying performance and egg quality in laying hens. AsianAustralasian Journal of Animal Sciences, 18: 1769–1774. VAN ACKER, F.A.A., SCHOUTEN, O., HAENEN, G.R.M.M., VAN DER VIJGH, W.J.F. & BAST, A. (2000) Flavonoids can replace a-tocopheryl as an antioxidant. FEBS Letters, 473: 145–148. WENK, C. (2003) Herbs and botanicals as feed additives in monogastric animals. Asian-Australasian Journal of Animal Sciences, 16: 282–289. ZAPATA, L.F. & GERNAT, A.G. (1995) The effect of four levels of ascorbic acid and two levels of calcium on eggshell quality of forced-molted White Leghorn hens. Poultry Science, 74: 1049–1052.
