Research Article Received: 24 February 2013
Revised: 19 July 2014
Accepted article published: 25 July 2014
Published online in Wiley Online Library: 28 August 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6844
Dietary supplementation of Gracilariopsis persica is associated with some quality related sera and egg yolk parameters in laying quails Behnam Abbaspour, Sharifi S Davood* and Abdollah Mohammadi-Sangcheshmeh Abstract BACKGROUND: Gracilariopsis persica (Gp) is one of the most abundant red algae distributed in the Persian Gulf, containing various bioactive components with hypolipedimic, hypoglycemic and antioxidant properties. Therefore using laying quails as a model we aimed to investigate the effect of dietary Gp on body weight, feed conversion, estradiol, progesterone, calcium and lipid levels in serum, as well as the high-density:low-density lipoprotein (HDL:LDL) ratio. Yolk cholesterol and yolk lipid oxidation were also evaluated. To accomplish this, diets containing 0, 10, 30 and 50 g kg−1 Gp were fed to 5-week-old laying quails for 12 weeks. RESULTS: Our data revealed that Gp had no effect on body weight, feed conversion, triglycerides and estradiol levels of serum. Dietary Gp decreased the serum and yolk cholesterol in a dose-dependent manner. In addition, the sera progesterone and calcium levels and HDL:LDL ratios were increased by feeding diets containing 50 g kg−1 Gp. Our results relating to yolk lipid oxidation showed that malondialdehyde content was decreased in Gp-fed laying quails. CONCLUSIONS: The results of the present study demonstrate that not only serum and egg yolk cholesterol levels, but also susceptibility of yolk lipids to oxidation, can be decreased by feeding Gp to laying quails. © 2014 Society of Chemical Industry Keywords: quail; cholesterol; malondialdehyde; progesterone; Gracilariopsis persica
INTRODUCTION
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study Gp was demonstrated to be valuable for inclusion in the diet of laying chickens up to a level of 100 g kg−1 .14 In this regard it has been reported that Gp contains various bioactive sterols such as fucosterol, stigmasterol and 𝛽-sitosterol, which can effectively decrease plasma cholesterol, glucose and inflammation.15 Little data are available on the nutritional value of Gp or its effects on the metabolism of poultry. Therefore, a prospective study has been performed to determine possible effects of using dietary Gp on metabolism of laying quails, especially on their serum lipids, calcium, estrogen and progesterone levels, as well as on egg lipid oxidation.
MATERIAL AND METHODS Experimental diets and bird husbandry Before diet formulation, whole Gp seaweeds were ground in a hammer mill using a 1 mm mesh. A GP sample (1 kg) was prepared and after grinding finely the sample was subjected to chemical analysis (Table 1). Moisture, ash, crude fiber, ether extract, crude
∗
Correspondence to: Sharifi S Davood, Department of Animal and Poultry Science, College of Abouraihan, University of Tehran, Pakdasht, 3391-653755 Tehran, Iran. E-mail: sdsharifi@ut.ac.ir Department of Animal and Poultry Science, College of Abouraihan, University of Tehran, Pakdasht, 3391-653755 Tehran, Iran
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Egg is an excellent source of high-quality protein as well as many vitamins and minerals.1 It is well known that egg yolk is rich in fat and fat-soluble vitamins such as vitamin A, D and E, which are essential for human health. Despite the nutritional value of eggs, there are some potential health issues arising from egg quality. Egg is a major source of cholesterol, which can contribute to high blood cholesterol levels.2 It has been reported that foods with high cholesterol cause artery stenosis, which is one of the major causes of coronary heart disease.3 The cholesterol and fat content in egg yolk highly depend on the birds’ diet. Several studies have been conducted in laying hens to reduce the cholesterol content of egg through diet manipulation strategies.4 – 7 Seaweeds have been used directly as a source of health food for human consumption since very early times.8 It is evident that the seaweeds are rich sources of bioactive components as well as micro and macro nutrients.9 Thus the nutritional value of seaweeds as a food for humans10 or feedstuff for animals11 and poultry12 has been widely investigated. Gracilaria is a genus of red algae (Rhodophyceae) and a member of the Gracilariaceae family. This seaweed is well known for its economic importance as a source of agar, as well as its use as a food for humans. Gracilariopsis persica (Gp) is one of the most abundant red algae in the Persian Gulf, which introduced based on morphology and DNA sequence data.13 Recently, the nutritional value of Gp was evaluated as a feedstuff for laying hens. In that
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B Abbaspour, SS Davood, A Mohammadi-Sangcheshmeh
Table 1. Metabolizable energy (MJ kg−1 dry matter) and chemical composition (g kg−1 dry matter) of Gracilariopsis persica11 Nutrient
Amount
Dry matter Metabolizable energy Crude protein Crude fiber Ether extract Calcium Phosphorous Sodium Ash
964.0 9.2 230.5 72.0 1.0 9.0 3.4 12.1 255.0
protein, Ca, P and Na content of were determined using standard AOAC methods.16 A total of 96 laying Japanese quail (Coturnix coturnix japonica) 5 weeks of age with an average weight of 200 ± 20 g were randomly assigned to 16 cages (n = 4 cages per treatment; six birds per cage). The feeding experiment was conducted for 12 weeks. However, all quails were fed with the control diet for 2 weeks before the feeding experiment began. The dietary inclusion rates of Gp seaweed was as follows: 0 (control group), 10, 30 and 50 g kg−1 . The diets were isocaloric and isonitrogenous and were formulated to meet or exceed the nutritional recommendations of laying quails (Table 2).17 Birds were raised under environmentally controlled conditions following a standard temperature regimen (20 ± 5 ∘ C) and a 16 L:8D lighting program. Feed and water were provided for ad libitum intake. During the experimental period, egg numbers were recorded daily and feed consumption was determined weekly. The body weights of birds were measured at the beginning and end of the study to determine body weight changes during the experiment. Analysis of yellow follicles After weighing the ovaries, follicles were dissected using the method described by Gilbert et al.18 The follicles were classified into three groups according to their size: large yellow follicles (>8 mm), small yellow follicles (2–8 mm) and white follicles (2–5 mm). In this study, the diameter of the largest )F1) and the second largest (F2) ovarian follicles of the hens were measured using a digital Vernier caliper and then calculated based on the total ovarian follicles (%). The eggs laid during the last 3 days of every 4 weeks were weighed individually. Then, eggs from the same replicate (20 eggs per treatment) were broken out carefully and the yolks separated from albumen, weighed, pooled and blended. Three-gram samples of egg yolks were dissolved in a 2% NaCl solution to measure egg yolk cholesterol. Remaining egg yolk was frozen at −20 ∘ C pending antioxidant activity assessment. At the end of the experiment, two birds per cage (n = 8 per treatment) were randomly selected and their blood samples were taken from the wing vein using a sterile syringe. The blood samples were centrifuged at 1000 × g for 10 min and serum samples were stored at −20 ∘ C for later analysis.
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Analysis of egg yolk cholesterol Cholesterol concentration in yolk samples was determined enzymatically using commercial kits (Pars Azmoun, Karaj, Iran,
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Table 2. Ingredient and nutrient composition of experimental diets (as fed) Dietary Gp (g kg−1 ) Composition (g kg−1 )
0
Yellow corn 653.2 Soybean meal- 48% 189.9 Soybean oil 9.9 Gluten meal 50.0 Seaweed – Dicalcium phosphate 14.0 Oyster shell 71.7 Sodium chloride 2.0 DL-Methionine 3.1 5.0 Vitamin–mineral premixa Calculated analysis (g kg−1 as fed) 12.39 AMEn (MJ kg−1 ) CP 180 CF 19.1 Ca 31 Available P 4.50 Met + Cys 8.20 Lys 8.50
10
30
50
645.3 186.9 11.0 50.0 10.0 14.0 71.5 2.0 3.1 5.0
629.4 180.9 13.2 50.0 30.0 14.1 71.1 2.0 3.2 5.0
613.5 174.9 15.3 50.0 50.0 14.1 70.7 2.0 3.2 5.0
12.39 180 19.6 31 4.50 8.20 8.50
12.39 180 20.5 31 4.50 8.20 8.50
12.39 180 23.1 31 4.50 8.20 8.50
a Providing, per kilogram of diet: vitamin A, 12 000 IU; vitamin D3, 2000 IU; vitamin E, 35 mg; vitamin K3,4 mg; vitamin B1, 3 mg; vitamin B2, 7 mg; niacin, 20 mg; vitamin B6, 5 mg; vitamin B12, 0.015 mg; folic acid, 1 mg; biotin, 0.045 mg; ascorbic acid, 50 mg; canthaxanthine, 1.5 mg; apocarotenoic acid ester, 0.5 mg; choline chloride, 125 mg; manganese, 80 mg; iron, 60 mg; zinc, 60 mg; copper, 5 mg; cobalt, 0.2 mg; iodine, 1 mg; selenium, 0.15 mg.
CHAD-PAP 2012) according to the manufacturer’s recommendations. Briefly, 3 g yolk was dissolved in 27 mL of 2% (w/v) NaCl. The solution was then shaken slowly for 2 h. Subsequently, 1 mL of these solutions was diluted tenfold with 2% NaCl solution and then used as test sample. Iron-induced lipid oxidation Iron-induced lipid oxidation (induced TBA) was carried out as a modification of the method of Kornsbrust and Mavis,19 described by Botsoglou et al.20 Briefly, four 1 g subsamples from each egg yolk were weighed into 50 mL centrifuge tubes. Then, 1.5 mL of a solution containing 1.138 mmol L−1 ferrous sulfate and 0.368 mmol L−1 ascorbic acid were added to three of the subsamples and incubated at 37 ∘ C for 50, 100 or 150 min. Following incubation, all three iron-induced subsamples, along with the fourth non-induced subsample, were immediately submitted to malondialdehyde assay for assessing the extent of lipid oxidation. Malondialdehyde assay Malondialdehyde (secondary oxidation product) values were determined by a spectrophotometric method described by Botsoglou et al.21 For this, 4 mL aqueous trichloroacetic acid (50 g L−1 ) and 2.5 mL butyrate hydroxytoluene in hexane (8 g L−1 ) were added to samples and then vortexed for 60 s. Then, 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 3 mL aqueous 2-thiobarbituricacid (8 g L−1 ). TBA-containing tubes were incubated for 30 min at 70 ∘ C in a water bath. Following incubation, the mixture was cooled on ice and submitted
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 643–648
Gracilariopsis persica has been found to reduce serum and egg yolk cholesterol levels
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Table 3. Laying quails’ performance and egg quality parameters during an 18-week study trial Dietary Gp (g kg−1 ) Parameters
0
Body weight (g) Egg production (%) Daily feed intake (g per hen d−1 ) Feed conversion (g g−1 ) Whole egg weight (g) Egg yolk weight (g) Egg mass (g d−1 )
10
288.8 ± 14.8 89 ± 1.3ab 32.5 ± 0.5 3.2 ± 0.08 10.8 ± 0.1 3.5 ± 0.05 9.6 ± 0.2ab
30
271.3 ± 11.6 89.5 ± 1.09ab 33.6 ± 0.9 3.3 ± 0.13 10.9 ± 0.2 3.6 ± 0.04 9.8 ± 0.3a
50
266.3 ± 16.8 91.6 ± 0.9a 33.6 ± 0.6 3.2 ± 0.07 11.0 ± 0.1 3.8 ± 0.25 10.1 ± 0.2a
262.5 ± 11.8 86.8 ± 1.3b 33.33 ± 0.6 3.4 ± 0.1 10.5 ± 0.2 3.4 ± 0.04 9.12 ± 0.2b
Mean values in the same row with different letters are significantly different (P < 0.05).
Table 4. Effect of dietary Gp on some sera biochemical parameters and frequency of yellow ovarian follicles in laying quails (mean ± SD) Dietary Gp (g kg−1 ) Parameter Triglyceride (mg dL−1 ) Cholesterol (mg dL−1 ) HDL:LDL (mg dL−1 )a Yellow folliclesb (%) Estradiol (pg mL-1 ) Progesterone (ng mL−1 ) Calcium (mg dL−1 )
0 1184 ± 4.5 294.3a ± 39.71 0.48b ± 0.11 16b ± 1.1 166.3 ± 63.1 0.16b ± 0.004 24.00b ± 2.7
10
30
50
1123 ± 71.5 208.8b ± 29.69 0.71b ± 0.01 25.75ab ± 2.7 193.9 ± 29.7 0.22 b ± 0.02 29.25ab ± 1.3
1057 ± 112.5 199.8bc ± 15.37 0.76b ± 0.10 29a ± 2.5 209.5 ± 24.8 0.24 b ± 0.05 31.75ab ± 1.3
1038 ± 19.1 143.7c ±15.5 1.75a ± 0.31 31.50a ± 6.2 212.9 ± 36.1 0.38a ± 0.05 35.57a ± 1.1
Mean values in the same row with different letters (a, b) are significantly different (P < 0.05). a High-density lipoproteins:low-density lipoproteins. b Yellow follicles are expressed as percentages of total number of ovarian follicles.
to conventional spectrophotometry (UV–visible S2100, Scinco, Seoul, South Korea) at a wavelength of 521.5 nm. Analysis of blood biochemical traits Lipids (cholesterol, triglycerides and HDL:LDL ratios) and calcium of serum were measured by enzyme colorimetric methods, using a commercial kit (Pars Azmoun, Karaj, Iran). All analyses were performed on a multiple reader (Roche Cobas Mira, Elisa Reader and Dana-3200). Serum estradiol and progesterone levels were determined using radioimmunoassay, and commercial kits (DKO003 and DKO006, respectively, Delaware Biotech, Wilmington, DE, USA). Levels were determined using Tosoh AIA-360 Immunoassay Analyzer (WS-AIA360). Statistical analysis The data were analyzed using one-way analysis of variance.22 Differences among treatment means were tested for significance using Duncan’s multiple-range test.
RESULTS
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Effect of Gp on cholesterol and blood biochemical parameters There were no differences in triglycerides and estradiol of serum between treatments (Table 4). However, the concentration of triglycerides and estradiol of serum tended to be lower and higher, respectively, by increasing the level of seaweed in diets. The HDL:LDL ratio was higher (by 31%; P < 0.05) in birds receiving the diet containing 50 g kg−1 Gp as compared to the control diet (Table 4). The levels of cholesterol, progesterone and calcium concentrations in serum were significantly influenced by dietary treatments. Serum cholesterol level was lower in birds fed on diets containing Gp (P < 0.05). In contrast, in birds receiving the 50 g kg−1 Gp diet, concentrations of progesterone and calcium in serum were higher when compared to birds fed the control diet (P < 0.05). Effect of Gp on ovarian follicles, egg cholesterol content and oxidative stability of the yolk Table 4 shows our data regarding the proportion of yellow follicles (as a percentage of total ovarian follicles) in the birds. As can be seen, birds that received a diet containing 50 and 30 g kg−1 Gp represent a higher level of follicles than that of control group (P < 0.05). The effect of the dietary Gp on the cholesterol content and oxidative stability of the yolk is illustrated in Figs 1 and 2, respectively. No significant effect of dietary treatments was
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Effect of Gp on live body weight and egg production Dietary treatments had no effect on the weight of egg and yolk. Also, there were no live body weight changes, daily feed intake or feed conversion due to the dietary treatments (Table 3). No differences (P > 0.05) in egg production and egg mass were detected between birds on Gp diets and control. However, egg production
and egg mass produced by birds receiving the diet containing 50 g kg−1 Gp were lower (P < 0.05) when compared to birds fed 10 or 30 g kg−1 Gp diets.
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B Abbaspour, SS Davood, A Mohammadi-Sangcheshmeh
Figure 1. Effect of dietary Gp on yolk cholesterol levels (mean ± SD). Columns with different letters (a, b) are significantly different (P < 0.05).
observed on the yolk cholesterol at week 10. However, the cholesterol level in the egg yolks, as determined in eggs laid at weeks 14 and 18, were lower in the Gp-fed quail as compared to the respective value of the control group (P < 0.05). In egg yolk, the concentrations of malondialdehyde for birds that fed Gp diets were lower than those for birds fed the control (Fig. 2). However, significant differences were observed after 50, 100 and 150 min incubation for iron-induced lipid oxidation (P < 0.05).
DISCUSSION
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According to our findings there were no differences in body weight, egg mass, egg or yolk weight following feeding Gp. This is in great agreement with previous findings where red algae have been found to have no effect on the aforementioned parameters.6,7,23,24 The birds fed on diets containing Gp tended to show a lower body weight; however, the differences were not significant. The lower body weight, egg mass, egg and yolk weight observed in this study, especially in the 50 g kg−1 group, can be explained to some extent by the fiber- and ash-rich properties of the Gp. In the present study, we speculated that usage of Gp in the diet could be a potential to modify egg and serum cholesterol as well as some blood biochemical parameters. According to our finds, feeding Gp caused a significant reduction in the cholesterol concentration in serum, with a concomitant decrease in serum triglycerides (numerically) and yolk cholesterol content. The increased levels of serum HDL:LDL in birds receiving the diet containing 50 g kg−1 Gp may be due to hypertriglyceridemia and hypocholesterolemia induced by reactive metabolite formed during biotransformation. Similarly, Dvir et al.25 reported that the addition of biomass red micro alga (Porphyridium sp.) to rats’
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diets increased levels of serum HDL:LDL and decreased levels of plasma cholesterol, whereas no changes were observed in plasma triglyceride levels. These results were also consistent with previous findings.5 – 7 It appears from these results that feeding laying quails with Gp led to a significant reduction in yolk cholesterol content in response to decreased sera cholesterol concentration. In this study, however, the yolk cholesterol was not different among dietary groups during the first 4 weeks of Gp feeding. This finding is in agreement with a previous report by Ginzberg et al.6 This indicates that prolonged feeding of laying quails with Gp is needed to decrease the concentration of cholesterol in quail’s eggs. It has been proven that, unlike fatty acids, the cholesterol level in egg yolk is extremely resistant to any change.26 Previous studies have shown that red marine algae, including Gracilariopsis spp., are a rich source of dietary fiber (338–700 g kg−1 dry weight),7 unsaturated fatty acids27 and sterols.4 It is known that the dietary fiber, plant sterols28 and polyunsaturated fatty acids5 reduce blood cholesterol in human and animals. It is reported that dietary fiber increases intestinal viscosity and modifies the gastrointestinal transit time,28 increases fecal bile acid and neutral steroid excretion,29 inhibits lipase activity30 and consequently reduces the absorption of lipids in the small intestine. Also, in some studies, it has been reported that dietary fiber may possess hypocholesterolemic activity by inhibiting hydroxymethylglutaryl-CoA reductase (the key enzyme in cholesterol synthesis) and hepatic sterol synthesis.25 In another study, it has been shown that algal polysaccharide have no direct effect on the inhibition of hepatic cholesterol synthesis.5 Therefore, all the above findings may well explain the results of this study. Increasing serum estradiol and progesterone concentration in Gp-fed birds compared to the control group indicates that there is
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 643–648
Gracilariopsis persica has been found to reduce serum and egg yolk cholesterol levels
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Figure 2. Effect of dietary Gp on yolk malondialdehyde concentration (mean ± SD). Columns with different letters (a, c) are significantly different (P < 0.05).
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activity which can be transferred to the egg. It has been reported that Gp is a rich source of antioxidants.41 Takamatsu et al.42 reported that chlorophylls, carotenoids, tocopherol derivatives such as vitamin E, and related isoprenoids that are structurally related to plant-derived antioxidants, are found in seaweeds. Thus it appears that antioxidant constituents of Gp had passed into the developing yolk through feeding, and protected yolk lipids against iron-induced oxidation. This observation is consistent with previous work demonstrating the antioxidant activity of seaweed and marine algae.43 In conclusion, our data imply that the cholesterol levels in serum and egg yolk can be decreased by feeding Gp to laying quails. Moreover, susceptibility of yolk lipids to oxidation was found to be reduced by supplying Gp in the quail diet. It is important, however, to note that such an improvement had no detrimental effect on performance of laying quails until Gp was included up to 30 g kg−1 in diets.
ACKNOWLEDGEMENTS The authors would like to thank the Office of Research Affairs of the University of Tehran, grant number 26567-06-13, for their support. We acknowledge the support of the Research Center of Jihad-E-Agriculture of Hormozgan Province (Bandar Abbas- Iran) for provision of Gracilariopsis persica seaweed. We are also grateful to MA Nourozian from the Department of Animal and poultry Science, College of Abouraihan, University of Tehran, Iran, for their review of this paper.
REFERENCES 1 Cook R and Bird FH, Duodenal villus area and epithelial cellular migration in conventional and germ free chicks. Poult Sci 52:2276–2280 (1973).
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probably some phytoestrogenic components in Gp. Accelerated serum estradiol concentration has been demonstrated in layer hens given exogenous estrogens.31 In humans, nutrition studies of seaweed have reported increased plasma progesterone and decreased estrogen.32 Also, it has been proposed that seaweed has potent antiestrogenic effects that may be associated with high fiber content as well as low cholesterol absorption in the intestine.33 The proportions of yellow follicles were higher for birds fed Gp diets than control birds, which might be a result of the better development of the ovaries in these birds. The fact that the steroid hormones such as estrogen and progesterone originate from cholesterol may to some extent explain the observed increased steroid hormone and reduced plasma cholesterol in this study.34 The observed increase in the calcium content of serum in 50 g kg−1 Gp treatment could be related to the higher concentration of estradiol and progesterone of serum in comparison with the other treatments. Increase in plasma calcium as a consequence of estrogen administration agrees with several previous reports.35,36 Bar and Hurwitz37 reported that estrogen hormones increased intestinal absorption of calcium in laying birds. Also, Elkomy et al.38 reported that estrogen treatment increased calcium absorption and blood calcium concentration in young and old Japanese quail hens. On the other hand, the increases in quail’s serum calcium concentration may be due to the high level of iodine in Gp diets. Seaweeds have the unique ability to concentrate minerals from the ocean; thus they are rich in many minerals and trace elements such as calcium, iron and iodine.39 Accordingly, Harms et al.40 reported that feeding iodine resulted in increased blood calcium levels in laying hens. The Gp used as a dietary constituent in the current research decreased malondialdehyde concentrations of egg yolk after 50, 100 and 150 min incubation for iron-induced lipid oxidation. This indicates that Gp contains chemical compounds with antioxidant
www.soci.org 2 Kim JH, Hong ST, Lee HS and Kim HJ, Oral administration of pravastatin reduces egg cholesterol but not plasma cholesterol in laying hens. Poult Sci 83:1539–1543 (2004). 3 Weggemans RM, Zock PL and Katan MB, Dietary cholesterol from eggs increases the ratio of total cholesterol to high-density lipoprotein cholesterol in human: a meta-analysis. Am J Clin Nutr 73:885–891 (2001). 4 Jimenez-Escrig A and Goni Cambrodon I, Nutritional evaluation and physiological effects of edible seaweeds. Arch Latinoam Nutr 49:114–120 (1999). 5 Dvir I, Chayoth R, Sod-Moriah U, Shany S, Nyska A, Stark AH et al., Soluble polysaccharide and biomass of red microalga Porphyridium spp. alter intestinal morphology and reduce serum cholesterol in rats. Br J Nutr 84:469–476 (2000). 6 Ginzberg A, Cohen M, Sod-Moriah UA, Shany S, Rosenshtrauch A and Arad SM, Chickens fed with biomass of the red microalga Porphyridium spp. have reduced blood cholesterol level and modified fatty acid composition in egg yolk. J Appl Phycol 12:324–330 (2000). 7 Bocanegra A, Nieto A, Bastida S, Benedi J and Sánchez-Muniz FJ, A Nori but not a Konbu, dietary supplement decreases the cholesterolaemia, liver fat infiltration and mineral bioavailability in hypocholesterolaemic growing Wister rats. Br J Nutr 99:272–280 (2008). 8 Rimber I, Why is seaweed so important? MSc thesis, Faculty of Fisheries and Marine Sciences, Sam Ratulangi University. Campus Bahu Manado Indonesia 95115 (2007). 9 Becker W, Microalgae in human and animal nutrition, in Handbook of Macro Alga Culture, ed. by Richmond A. Blackwell, Oxford, pp. 312–351 (2004). 10 Fleurence J, Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends Food Sci Technol 10:25–28 (1999). 11 Vosough-Sharifi O, Yaghoubfar A, Sharifi SD, Mirzadeh G and Askari F, Study on the possibility of Gracilariopsis persica utilization in layer diets. J Anim Prod 14:1–10 (2011). 12 Zalabak V, Kejmar J, Rosa M and Stros F, Algae, in: Nonconventional Feedstuffs in the Nutrition of Farm Animals, ed. by Boda K. Elsevier, Amsterdam, The Netherlands (1990). 13 Rendón LU, Carrillo DS, Arellano ML, Casas G, Pérez-Gil RF and Avila GE, Chemical composition of the residue of alginates (Macrocystis pyrifera) extraction: its utilization in laying hens feeding. Cuban J Agric Sci 37:287–293 (2003). 14 Bellorin AM, Buriyo A, Sohrabipour J, Oliveria M and Oliveria EC, Gracilariopsis mclachlanii sp. nov. and Gracilariopsis persica sp. nov. of the Gracilariaceae (Gracilaria, Rhodophyceae) from the Indian Ocean. J Phycol 44:1022–1032 (2008). 15 Saeidniaa S, Permeh P, Gohari AR and Mashinchian-Moradi A, Gracilariopsis persica from Persian Gulf contains bioactive sterols. Iranian J Pharm Res 11:845–849 (2012). 16 AOAC, Official methods of Analysis of AOAC International, Vol. 41. AOAC International, Rockville, MD, pp. 19–24 (2000). 17 Leeson S and Summers JD. Commercial Poultry Nutrition (4th edn). Nottingham University Press, Nottingham, UK (2008). 18 Gilbert AB, Perry MM, Waddington D and Hardie MA, Role of atresia in establishing the follicular hierarchy in the ovary of the domestic hen (Gallus domesticus). J Reprod Fertil 69:221–227 (1983). 19 Kornsbrust DJ and Mavis RD, Relative susceptibility of microsomes from lung, heart, liver, brain and testes to lipid peroxidation: correlation with vitamin E content. Lipids 15:315–322 (1980). 20 Botsoglou NA, Florou-Paneri P, Christaki E, Fletouris DG and Spais AB, Effect of dietary oregano essential oil on performance of chickens and on iron-induced lipid oxidation of breast, thigh and abdominal fat tissues. Br Poult Sci 43:223–230 (2002). 21 Botsoglou NA, Fletouris DJ, Papageorgiou GE, Vassilopoulos VN, Mantis AJ and Trakatellis AG, Rapid sensitive and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food and feed-stuff samples. J Agric Food Chem 42:1931–1937 (1994).
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© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 643–648
Revised: 19 July 2014
Accepted article published: 25 July 2014
Published online in Wiley Online Library: 28 August 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6844
Dietary supplementation of Gracilariopsis persica is associated with some quality related sera and egg yolk parameters in laying quails Behnam Abbaspour, Sharifi S Davood* and Abdollah Mohammadi-Sangcheshmeh Abstract BACKGROUND: Gracilariopsis persica (Gp) is one of the most abundant red algae distributed in the Persian Gulf, containing various bioactive components with hypolipedimic, hypoglycemic and antioxidant properties. Therefore using laying quails as a model we aimed to investigate the effect of dietary Gp on body weight, feed conversion, estradiol, progesterone, calcium and lipid levels in serum, as well as the high-density:low-density lipoprotein (HDL:LDL) ratio. Yolk cholesterol and yolk lipid oxidation were also evaluated. To accomplish this, diets containing 0, 10, 30 and 50 g kg−1 Gp were fed to 5-week-old laying quails for 12 weeks. RESULTS: Our data revealed that Gp had no effect on body weight, feed conversion, triglycerides and estradiol levels of serum. Dietary Gp decreased the serum and yolk cholesterol in a dose-dependent manner. In addition, the sera progesterone and calcium levels and HDL:LDL ratios were increased by feeding diets containing 50 g kg−1 Gp. Our results relating to yolk lipid oxidation showed that malondialdehyde content was decreased in Gp-fed laying quails. CONCLUSIONS: The results of the present study demonstrate that not only serum and egg yolk cholesterol levels, but also susceptibility of yolk lipids to oxidation, can be decreased by feeding Gp to laying quails. © 2014 Society of Chemical Industry Keywords: quail; cholesterol; malondialdehyde; progesterone; Gracilariopsis persica
INTRODUCTION
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study Gp was demonstrated to be valuable for inclusion in the diet of laying chickens up to a level of 100 g kg−1 .14 In this regard it has been reported that Gp contains various bioactive sterols such as fucosterol, stigmasterol and 𝛽-sitosterol, which can effectively decrease plasma cholesterol, glucose and inflammation.15 Little data are available on the nutritional value of Gp or its effects on the metabolism of poultry. Therefore, a prospective study has been performed to determine possible effects of using dietary Gp on metabolism of laying quails, especially on their serum lipids, calcium, estrogen and progesterone levels, as well as on egg lipid oxidation.
MATERIAL AND METHODS Experimental diets and bird husbandry Before diet formulation, whole Gp seaweeds were ground in a hammer mill using a 1 mm mesh. A GP sample (1 kg) was prepared and after grinding finely the sample was subjected to chemical analysis (Table 1). Moisture, ash, crude fiber, ether extract, crude
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Correspondence to: Sharifi S Davood, Department of Animal and Poultry Science, College of Abouraihan, University of Tehran, Pakdasht, 3391-653755 Tehran, Iran. E-mail: sdsharifi@ut.ac.ir Department of Animal and Poultry Science, College of Abouraihan, University of Tehran, Pakdasht, 3391-653755 Tehran, Iran
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Egg is an excellent source of high-quality protein as well as many vitamins and minerals.1 It is well known that egg yolk is rich in fat and fat-soluble vitamins such as vitamin A, D and E, which are essential for human health. Despite the nutritional value of eggs, there are some potential health issues arising from egg quality. Egg is a major source of cholesterol, which can contribute to high blood cholesterol levels.2 It has been reported that foods with high cholesterol cause artery stenosis, which is one of the major causes of coronary heart disease.3 The cholesterol and fat content in egg yolk highly depend on the birds’ diet. Several studies have been conducted in laying hens to reduce the cholesterol content of egg through diet manipulation strategies.4 – 7 Seaweeds have been used directly as a source of health food for human consumption since very early times.8 It is evident that the seaweeds are rich sources of bioactive components as well as micro and macro nutrients.9 Thus the nutritional value of seaweeds as a food for humans10 or feedstuff for animals11 and poultry12 has been widely investigated. Gracilaria is a genus of red algae (Rhodophyceae) and a member of the Gracilariaceae family. This seaweed is well known for its economic importance as a source of agar, as well as its use as a food for humans. Gracilariopsis persica (Gp) is one of the most abundant red algae in the Persian Gulf, which introduced based on morphology and DNA sequence data.13 Recently, the nutritional value of Gp was evaluated as a feedstuff for laying hens. In that
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B Abbaspour, SS Davood, A Mohammadi-Sangcheshmeh
Table 1. Metabolizable energy (MJ kg−1 dry matter) and chemical composition (g kg−1 dry matter) of Gracilariopsis persica11 Nutrient
Amount
Dry matter Metabolizable energy Crude protein Crude fiber Ether extract Calcium Phosphorous Sodium Ash
964.0 9.2 230.5 72.0 1.0 9.0 3.4 12.1 255.0
protein, Ca, P and Na content of were determined using standard AOAC methods.16 A total of 96 laying Japanese quail (Coturnix coturnix japonica) 5 weeks of age with an average weight of 200 ± 20 g were randomly assigned to 16 cages (n = 4 cages per treatment; six birds per cage). The feeding experiment was conducted for 12 weeks. However, all quails were fed with the control diet for 2 weeks before the feeding experiment began. The dietary inclusion rates of Gp seaweed was as follows: 0 (control group), 10, 30 and 50 g kg−1 . The diets were isocaloric and isonitrogenous and were formulated to meet or exceed the nutritional recommendations of laying quails (Table 2).17 Birds were raised under environmentally controlled conditions following a standard temperature regimen (20 ± 5 ∘ C) and a 16 L:8D lighting program. Feed and water were provided for ad libitum intake. During the experimental period, egg numbers were recorded daily and feed consumption was determined weekly. The body weights of birds were measured at the beginning and end of the study to determine body weight changes during the experiment. Analysis of yellow follicles After weighing the ovaries, follicles were dissected using the method described by Gilbert et al.18 The follicles were classified into three groups according to their size: large yellow follicles (>8 mm), small yellow follicles (2–8 mm) and white follicles (2–5 mm). In this study, the diameter of the largest )F1) and the second largest (F2) ovarian follicles of the hens were measured using a digital Vernier caliper and then calculated based on the total ovarian follicles (%). The eggs laid during the last 3 days of every 4 weeks were weighed individually. Then, eggs from the same replicate (20 eggs per treatment) were broken out carefully and the yolks separated from albumen, weighed, pooled and blended. Three-gram samples of egg yolks were dissolved in a 2% NaCl solution to measure egg yolk cholesterol. Remaining egg yolk was frozen at −20 ∘ C pending antioxidant activity assessment. At the end of the experiment, two birds per cage (n = 8 per treatment) were randomly selected and their blood samples were taken from the wing vein using a sterile syringe. The blood samples were centrifuged at 1000 × g for 10 min and serum samples were stored at −20 ∘ C for later analysis.
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Analysis of egg yolk cholesterol Cholesterol concentration in yolk samples was determined enzymatically using commercial kits (Pars Azmoun, Karaj, Iran,
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Table 2. Ingredient and nutrient composition of experimental diets (as fed) Dietary Gp (g kg−1 ) Composition (g kg−1 )
0
Yellow corn 653.2 Soybean meal- 48% 189.9 Soybean oil 9.9 Gluten meal 50.0 Seaweed – Dicalcium phosphate 14.0 Oyster shell 71.7 Sodium chloride 2.0 DL-Methionine 3.1 5.0 Vitamin–mineral premixa Calculated analysis (g kg−1 as fed) 12.39 AMEn (MJ kg−1 ) CP 180 CF 19.1 Ca 31 Available P 4.50 Met + Cys 8.20 Lys 8.50
10
30
50
645.3 186.9 11.0 50.0 10.0 14.0 71.5 2.0 3.1 5.0
629.4 180.9 13.2 50.0 30.0 14.1 71.1 2.0 3.2 5.0
613.5 174.9 15.3 50.0 50.0 14.1 70.7 2.0 3.2 5.0
12.39 180 19.6 31 4.50 8.20 8.50
12.39 180 20.5 31 4.50 8.20 8.50
12.39 180 23.1 31 4.50 8.20 8.50
a Providing, per kilogram of diet: vitamin A, 12 000 IU; vitamin D3, 2000 IU; vitamin E, 35 mg; vitamin K3,4 mg; vitamin B1, 3 mg; vitamin B2, 7 mg; niacin, 20 mg; vitamin B6, 5 mg; vitamin B12, 0.015 mg; folic acid, 1 mg; biotin, 0.045 mg; ascorbic acid, 50 mg; canthaxanthine, 1.5 mg; apocarotenoic acid ester, 0.5 mg; choline chloride, 125 mg; manganese, 80 mg; iron, 60 mg; zinc, 60 mg; copper, 5 mg; cobalt, 0.2 mg; iodine, 1 mg; selenium, 0.15 mg.
CHAD-PAP 2012) according to the manufacturer’s recommendations. Briefly, 3 g yolk was dissolved in 27 mL of 2% (w/v) NaCl. The solution was then shaken slowly for 2 h. Subsequently, 1 mL of these solutions was diluted tenfold with 2% NaCl solution and then used as test sample. Iron-induced lipid oxidation Iron-induced lipid oxidation (induced TBA) was carried out as a modification of the method of Kornsbrust and Mavis,19 described by Botsoglou et al.20 Briefly, four 1 g subsamples from each egg yolk were weighed into 50 mL centrifuge tubes. Then, 1.5 mL of a solution containing 1.138 mmol L−1 ferrous sulfate and 0.368 mmol L−1 ascorbic acid were added to three of the subsamples and incubated at 37 ∘ C for 50, 100 or 150 min. Following incubation, all three iron-induced subsamples, along with the fourth non-induced subsample, were immediately submitted to malondialdehyde assay for assessing the extent of lipid oxidation. Malondialdehyde assay Malondialdehyde (secondary oxidation product) values were determined by a spectrophotometric method described by Botsoglou et al.21 For this, 4 mL aqueous trichloroacetic acid (50 g L−1 ) and 2.5 mL butyrate hydroxytoluene in hexane (8 g L−1 ) were added to samples and then vortexed for 60 s. Then, 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 3 mL aqueous 2-thiobarbituricacid (8 g L−1 ). TBA-containing tubes were incubated for 30 min at 70 ∘ C in a water bath. Following incubation, the mixture was cooled on ice and submitted
© 2014 Society of Chemical Industry
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Gracilariopsis persica has been found to reduce serum and egg yolk cholesterol levels
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Table 3. Laying quails’ performance and egg quality parameters during an 18-week study trial Dietary Gp (g kg−1 ) Parameters
0
Body weight (g) Egg production (%) Daily feed intake (g per hen d−1 ) Feed conversion (g g−1 ) Whole egg weight (g) Egg yolk weight (g) Egg mass (g d−1 )
10
288.8 ± 14.8 89 ± 1.3ab 32.5 ± 0.5 3.2 ± 0.08 10.8 ± 0.1 3.5 ± 0.05 9.6 ± 0.2ab
30
271.3 ± 11.6 89.5 ± 1.09ab 33.6 ± 0.9 3.3 ± 0.13 10.9 ± 0.2 3.6 ± 0.04 9.8 ± 0.3a
50
266.3 ± 16.8 91.6 ± 0.9a 33.6 ± 0.6 3.2 ± 0.07 11.0 ± 0.1 3.8 ± 0.25 10.1 ± 0.2a
262.5 ± 11.8 86.8 ± 1.3b 33.33 ± 0.6 3.4 ± 0.1 10.5 ± 0.2 3.4 ± 0.04 9.12 ± 0.2b
Mean values in the same row with different letters are significantly different (P < 0.05).
Table 4. Effect of dietary Gp on some sera biochemical parameters and frequency of yellow ovarian follicles in laying quails (mean ± SD) Dietary Gp (g kg−1 ) Parameter Triglyceride (mg dL−1 ) Cholesterol (mg dL−1 ) HDL:LDL (mg dL−1 )a Yellow folliclesb (%) Estradiol (pg mL-1 ) Progesterone (ng mL−1 ) Calcium (mg dL−1 )
0 1184 ± 4.5 294.3a ± 39.71 0.48b ± 0.11 16b ± 1.1 166.3 ± 63.1 0.16b ± 0.004 24.00b ± 2.7
10
30
50
1123 ± 71.5 208.8b ± 29.69 0.71b ± 0.01 25.75ab ± 2.7 193.9 ± 29.7 0.22 b ± 0.02 29.25ab ± 1.3
1057 ± 112.5 199.8bc ± 15.37 0.76b ± 0.10 29a ± 2.5 209.5 ± 24.8 0.24 b ± 0.05 31.75ab ± 1.3
1038 ± 19.1 143.7c ±15.5 1.75a ± 0.31 31.50a ± 6.2 212.9 ± 36.1 0.38a ± 0.05 35.57a ± 1.1
Mean values in the same row with different letters (a, b) are significantly different (P < 0.05). a High-density lipoproteins:low-density lipoproteins. b Yellow follicles are expressed as percentages of total number of ovarian follicles.
to conventional spectrophotometry (UV–visible S2100, Scinco, Seoul, South Korea) at a wavelength of 521.5 nm. Analysis of blood biochemical traits Lipids (cholesterol, triglycerides and HDL:LDL ratios) and calcium of serum were measured by enzyme colorimetric methods, using a commercial kit (Pars Azmoun, Karaj, Iran). All analyses were performed on a multiple reader (Roche Cobas Mira, Elisa Reader and Dana-3200). Serum estradiol and progesterone levels were determined using radioimmunoassay, and commercial kits (DKO003 and DKO006, respectively, Delaware Biotech, Wilmington, DE, USA). Levels were determined using Tosoh AIA-360 Immunoassay Analyzer (WS-AIA360). Statistical analysis The data were analyzed using one-way analysis of variance.22 Differences among treatment means were tested for significance using Duncan’s multiple-range test.
RESULTS
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Effect of Gp on cholesterol and blood biochemical parameters There were no differences in triglycerides and estradiol of serum between treatments (Table 4). However, the concentration of triglycerides and estradiol of serum tended to be lower and higher, respectively, by increasing the level of seaweed in diets. The HDL:LDL ratio was higher (by 31%; P < 0.05) in birds receiving the diet containing 50 g kg−1 Gp as compared to the control diet (Table 4). The levels of cholesterol, progesterone and calcium concentrations in serum were significantly influenced by dietary treatments. Serum cholesterol level was lower in birds fed on diets containing Gp (P < 0.05). In contrast, in birds receiving the 50 g kg−1 Gp diet, concentrations of progesterone and calcium in serum were higher when compared to birds fed the control diet (P < 0.05). Effect of Gp on ovarian follicles, egg cholesterol content and oxidative stability of the yolk Table 4 shows our data regarding the proportion of yellow follicles (as a percentage of total ovarian follicles) in the birds. As can be seen, birds that received a diet containing 50 and 30 g kg−1 Gp represent a higher level of follicles than that of control group (P < 0.05). The effect of the dietary Gp on the cholesterol content and oxidative stability of the yolk is illustrated in Figs 1 and 2, respectively. No significant effect of dietary treatments was
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Effect of Gp on live body weight and egg production Dietary treatments had no effect on the weight of egg and yolk. Also, there were no live body weight changes, daily feed intake or feed conversion due to the dietary treatments (Table 3). No differences (P > 0.05) in egg production and egg mass were detected between birds on Gp diets and control. However, egg production
and egg mass produced by birds receiving the diet containing 50 g kg−1 Gp were lower (P < 0.05) when compared to birds fed 10 or 30 g kg−1 Gp diets.
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B Abbaspour, SS Davood, A Mohammadi-Sangcheshmeh
Figure 1. Effect of dietary Gp on yolk cholesterol levels (mean ± SD). Columns with different letters (a, b) are significantly different (P < 0.05).
observed on the yolk cholesterol at week 10. However, the cholesterol level in the egg yolks, as determined in eggs laid at weeks 14 and 18, were lower in the Gp-fed quail as compared to the respective value of the control group (P < 0.05). In egg yolk, the concentrations of malondialdehyde for birds that fed Gp diets were lower than those for birds fed the control (Fig. 2). However, significant differences were observed after 50, 100 and 150 min incubation for iron-induced lipid oxidation (P < 0.05).
DISCUSSION
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According to our findings there were no differences in body weight, egg mass, egg or yolk weight following feeding Gp. This is in great agreement with previous findings where red algae have been found to have no effect on the aforementioned parameters.6,7,23,24 The birds fed on diets containing Gp tended to show a lower body weight; however, the differences were not significant. The lower body weight, egg mass, egg and yolk weight observed in this study, especially in the 50 g kg−1 group, can be explained to some extent by the fiber- and ash-rich properties of the Gp. In the present study, we speculated that usage of Gp in the diet could be a potential to modify egg and serum cholesterol as well as some blood biochemical parameters. According to our finds, feeding Gp caused a significant reduction in the cholesterol concentration in serum, with a concomitant decrease in serum triglycerides (numerically) and yolk cholesterol content. The increased levels of serum HDL:LDL in birds receiving the diet containing 50 g kg−1 Gp may be due to hypertriglyceridemia and hypocholesterolemia induced by reactive metabolite formed during biotransformation. Similarly, Dvir et al.25 reported that the addition of biomass red micro alga (Porphyridium sp.) to rats’
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diets increased levels of serum HDL:LDL and decreased levels of plasma cholesterol, whereas no changes were observed in plasma triglyceride levels. These results were also consistent with previous findings.5 – 7 It appears from these results that feeding laying quails with Gp led to a significant reduction in yolk cholesterol content in response to decreased sera cholesterol concentration. In this study, however, the yolk cholesterol was not different among dietary groups during the first 4 weeks of Gp feeding. This finding is in agreement with a previous report by Ginzberg et al.6 This indicates that prolonged feeding of laying quails with Gp is needed to decrease the concentration of cholesterol in quail’s eggs. It has been proven that, unlike fatty acids, the cholesterol level in egg yolk is extremely resistant to any change.26 Previous studies have shown that red marine algae, including Gracilariopsis spp., are a rich source of dietary fiber (338–700 g kg−1 dry weight),7 unsaturated fatty acids27 and sterols.4 It is known that the dietary fiber, plant sterols28 and polyunsaturated fatty acids5 reduce blood cholesterol in human and animals. It is reported that dietary fiber increases intestinal viscosity and modifies the gastrointestinal transit time,28 increases fecal bile acid and neutral steroid excretion,29 inhibits lipase activity30 and consequently reduces the absorption of lipids in the small intestine. Also, in some studies, it has been reported that dietary fiber may possess hypocholesterolemic activity by inhibiting hydroxymethylglutaryl-CoA reductase (the key enzyme in cholesterol synthesis) and hepatic sterol synthesis.25 In another study, it has been shown that algal polysaccharide have no direct effect on the inhibition of hepatic cholesterol synthesis.5 Therefore, all the above findings may well explain the results of this study. Increasing serum estradiol and progesterone concentration in Gp-fed birds compared to the control group indicates that there is
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J Sci Food Agric 2015; 95: 643–648
Gracilariopsis persica has been found to reduce serum and egg yolk cholesterol levels
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Figure 2. Effect of dietary Gp on yolk malondialdehyde concentration (mean ± SD). Columns with different letters (a, c) are significantly different (P < 0.05).
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activity which can be transferred to the egg. It has been reported that Gp is a rich source of antioxidants.41 Takamatsu et al.42 reported that chlorophylls, carotenoids, tocopherol derivatives such as vitamin E, and related isoprenoids that are structurally related to plant-derived antioxidants, are found in seaweeds. Thus it appears that antioxidant constituents of Gp had passed into the developing yolk through feeding, and protected yolk lipids against iron-induced oxidation. This observation is consistent with previous work demonstrating the antioxidant activity of seaweed and marine algae.43 In conclusion, our data imply that the cholesterol levels in serum and egg yolk can be decreased by feeding Gp to laying quails. Moreover, susceptibility of yolk lipids to oxidation was found to be reduced by supplying Gp in the quail diet. It is important, however, to note that such an improvement had no detrimental effect on performance of laying quails until Gp was included up to 30 g kg−1 in diets.
ACKNOWLEDGEMENTS The authors would like to thank the Office of Research Affairs of the University of Tehran, grant number 26567-06-13, for their support. We acknowledge the support of the Research Center of Jihad-E-Agriculture of Hormozgan Province (Bandar Abbas- Iran) for provision of Gracilariopsis persica seaweed. We are also grateful to MA Nourozian from the Department of Animal and poultry Science, College of Abouraihan, University of Tehran, Iran, for their review of this paper.
REFERENCES 1 Cook R and Bird FH, Duodenal villus area and epithelial cellular migration in conventional and germ free chicks. Poult Sci 52:2276–2280 (1973).
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probably some phytoestrogenic components in Gp. Accelerated serum estradiol concentration has been demonstrated in layer hens given exogenous estrogens.31 In humans, nutrition studies of seaweed have reported increased plasma progesterone and decreased estrogen.32 Also, it has been proposed that seaweed has potent antiestrogenic effects that may be associated with high fiber content as well as low cholesterol absorption in the intestine.33 The proportions of yellow follicles were higher for birds fed Gp diets than control birds, which might be a result of the better development of the ovaries in these birds. The fact that the steroid hormones such as estrogen and progesterone originate from cholesterol may to some extent explain the observed increased steroid hormone and reduced plasma cholesterol in this study.34 The observed increase in the calcium content of serum in 50 g kg−1 Gp treatment could be related to the higher concentration of estradiol and progesterone of serum in comparison with the other treatments. Increase in plasma calcium as a consequence of estrogen administration agrees with several previous reports.35,36 Bar and Hurwitz37 reported that estrogen hormones increased intestinal absorption of calcium in laying birds. Also, Elkomy et al.38 reported that estrogen treatment increased calcium absorption and blood calcium concentration in young and old Japanese quail hens. On the other hand, the increases in quail’s serum calcium concentration may be due to the high level of iodine in Gp diets. Seaweeds have the unique ability to concentrate minerals from the ocean; thus they are rich in many minerals and trace elements such as calcium, iron and iodine.39 Accordingly, Harms et al.40 reported that feeding iodine resulted in increased blood calcium levels in laying hens. The Gp used as a dietary constituent in the current research decreased malondialdehyde concentrations of egg yolk after 50, 100 and 150 min incubation for iron-induced lipid oxidation. This indicates that Gp contains chemical compounds with antioxidant
www.soci.org 2 Kim JH, Hong ST, Lee HS and Kim HJ, Oral administration of pravastatin reduces egg cholesterol but not plasma cholesterol in laying hens. Poult Sci 83:1539–1543 (2004). 3 Weggemans RM, Zock PL and Katan MB, Dietary cholesterol from eggs increases the ratio of total cholesterol to high-density lipoprotein cholesterol in human: a meta-analysis. Am J Clin Nutr 73:885–891 (2001). 4 Jimenez-Escrig A and Goni Cambrodon I, Nutritional evaluation and physiological effects of edible seaweeds. Arch Latinoam Nutr 49:114–120 (1999). 5 Dvir I, Chayoth R, Sod-Moriah U, Shany S, Nyska A, Stark AH et al., Soluble polysaccharide and biomass of red microalga Porphyridium spp. alter intestinal morphology and reduce serum cholesterol in rats. Br J Nutr 84:469–476 (2000). 6 Ginzberg A, Cohen M, Sod-Moriah UA, Shany S, Rosenshtrauch A and Arad SM, Chickens fed with biomass of the red microalga Porphyridium spp. have reduced blood cholesterol level and modified fatty acid composition in egg yolk. J Appl Phycol 12:324–330 (2000). 7 Bocanegra A, Nieto A, Bastida S, Benedi J and Sánchez-Muniz FJ, A Nori but not a Konbu, dietary supplement decreases the cholesterolaemia, liver fat infiltration and mineral bioavailability in hypocholesterolaemic growing Wister rats. Br J Nutr 99:272–280 (2008). 8 Rimber I, Why is seaweed so important? MSc thesis, Faculty of Fisheries and Marine Sciences, Sam Ratulangi University. Campus Bahu Manado Indonesia 95115 (2007). 9 Becker W, Microalgae in human and animal nutrition, in Handbook of Macro Alga Culture, ed. by Richmond A. Blackwell, Oxford, pp. 312–351 (2004). 10 Fleurence J, Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends Food Sci Technol 10:25–28 (1999). 11 Vosough-Sharifi O, Yaghoubfar A, Sharifi SD, Mirzadeh G and Askari F, Study on the possibility of Gracilariopsis persica utilization in layer diets. J Anim Prod 14:1–10 (2011). 12 Zalabak V, Kejmar J, Rosa M and Stros F, Algae, in: Nonconventional Feedstuffs in the Nutrition of Farm Animals, ed. by Boda K. Elsevier, Amsterdam, The Netherlands (1990). 13 Rendón LU, Carrillo DS, Arellano ML, Casas G, Pérez-Gil RF and Avila GE, Chemical composition of the residue of alginates (Macrocystis pyrifera) extraction: its utilization in laying hens feeding. Cuban J Agric Sci 37:287–293 (2003). 14 Bellorin AM, Buriyo A, Sohrabipour J, Oliveria M and Oliveria EC, Gracilariopsis mclachlanii sp. nov. and Gracilariopsis persica sp. nov. of the Gracilariaceae (Gracilaria, Rhodophyceae) from the Indian Ocean. J Phycol 44:1022–1032 (2008). 15 Saeidniaa S, Permeh P, Gohari AR and Mashinchian-Moradi A, Gracilariopsis persica from Persian Gulf contains bioactive sterols. Iranian J Pharm Res 11:845–849 (2012). 16 AOAC, Official methods of Analysis of AOAC International, Vol. 41. AOAC International, Rockville, MD, pp. 19–24 (2000). 17 Leeson S and Summers JD. Commercial Poultry Nutrition (4th edn). Nottingham University Press, Nottingham, UK (2008). 18 Gilbert AB, Perry MM, Waddington D and Hardie MA, Role of atresia in establishing the follicular hierarchy in the ovary of the domestic hen (Gallus domesticus). J Reprod Fertil 69:221–227 (1983). 19 Kornsbrust DJ and Mavis RD, Relative susceptibility of microsomes from lung, heart, liver, brain and testes to lipid peroxidation: correlation with vitamin E content. Lipids 15:315–322 (1980). 20 Botsoglou NA, Florou-Paneri P, Christaki E, Fletouris DG and Spais AB, Effect of dietary oregano essential oil on performance of chickens and on iron-induced lipid oxidation of breast, thigh and abdominal fat tissues. Br Poult Sci 43:223–230 (2002). 21 Botsoglou NA, Fletouris DJ, Papageorgiou GE, Vassilopoulos VN, Mantis AJ and Trakatellis AG, Rapid sensitive and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food and feed-stuff samples. J Agric Food Chem 42:1931–1937 (1994).
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