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Effects of dietary supplementation with Gynura procumbens (Merr.) on egg yolk cholesterol, excreta microflora and laying hen performance a
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A. Lokhande , S. L. Ingale , S. H. Lee , S. Sen , C. Khong , B. J. Chae & I. K. Kwon
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Department of Animal Products and Food Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea b
Department of Animal Resources Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea Accepted author version posted online: 10 Jul 2014.Published online: 15 Aug 2014.
Click for updates To cite this article: A. Lokhande, S. L. Ingale, S. H. Lee, S. Sen, C. Khong, B. J. Chae & I. K. Kwon (2014) Effects of dietary supplementation with Gynura procumbens (Merr.) on egg yolk cholesterol, excreta microflora and laying hen performance, British Poultry Science, 55:4, 524-531, DOI: 10.1080/00071668.2014.938020 To link to this article: http://dx.doi.org/10.1080/00071668.2014.938020
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British Poultry Science, 2014 Vol. 55, No. 4, 524–531, http://dx.doi.org/10.1080/00071668.2014.938020
Effects of dietary supplementation with Gynura procumbens (Merr.) on egg yolk cholesterol, excreta microflora and laying hen performance A. LOKHANDE, S. L. INGALE1, S. H. LEE1, S. SEN1, C. KHONG1, B. J. CHAE1 AND I. K. KWON
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Department of Animal Products and Food Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea, and 1Department of Animal Resources Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea
Abstract 1. The present study investigated the effects of dietary supplementation with Gynura procumbens on egg yolk and serum cholesterol and triglycerides, excreta microflora, laying performance and egg quality. 2. A total of 160 Hy-Line Brown layers (45 weeks old) were randomly assigned into 4 treatments on the basis of laying performance. Each treatment had 4 replicates with 10 birds each. 3. Dietary treatments were basal diet supplemented with 0 (control), 2.5, 5.0 and 7.5 g/kg diet G. procumbens during 56-d feeding period. 4. Serum (d 21, 42 and 56) and egg yolk (d 28, 42 and 56) cholesterol and triglycerides concentrations were linearly reduced with increasing dietary concentrations of G. procumbens. 5. Increasing dietary concentrations of G. procumbens linearly reduced the excreta total anaerobic bacteria (d 28), Clostridium sp. and Escherichia coli (d 28 and 56) populations. 6. Overall egg production and egg mass were linearly increased, and overall feed efficiency was linearly improved with increase in dietary G. procumbens. 7. Dietary increasing concentrations of G. procumbens linearly improved egg yolk colour (d 28 and 56) and breaking strength of eggs (d 56). 8. The results obtained in the present experiment indicate that dietary supplementation with G. procumbens could reduce the egg yolk cholesterol, suppresses harmful excreta microflora and improves layers performance.
INTRODUCTION Chicken eggs are one of the most widely consumed animal food products and generally considered as an excellent source of calcium, phosphorus, high-quality proteins, unsaturated fats, folates and other B vitamins for persons of all age group (USDA, 1991). However, chicken egg yolk is rich source of cholesterol (213 mg/ egg; USDA, 1991), and over 95% of yolk cholesterol is associated with triglyceride-rich lipoprotein (Griffin, 1992). To avoid the elevation of blood cholesterol and heart-related diseases of human, it has been recommended to restrict
dietary cholesterol intake to below 200 mg/d for individuals at high risk of cardiovascular disease, to below 300 mg/d for healthy individuals (WHO, 2003) and limit the consumption of eggs (Weggemans et al., 2001). The poultry industry has continued to seek for production of “designer” eggs with value-added traits such as decreased cholesterol, triglycerides and increased omega-3 fatty acids to meet out the demand of health conscious consumers (Hernandez et al., 2009). Previous attempts to lowering egg yolk cholesterol have centred mostly on genetic selection, pharmacological intervention or alteration of the laying hen diets with feed additives that convey
Correspondence to: I. K. Kwon, Department of Animal Products and Food Science, Kangwon National University, Chuncheon, 200-701, Republic of Korea. E-mail: [email protected] Accepted for publication 10 April 2014. Lokhande and Ingale have contributed equally to this work.
© 2014 British Poultry Science Ltd
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GYNURA PROCUMBENS IN LAYING HEN DIETS
positive eggs traits (Wang and Tong, 2002). However, genetic selection had limited cholesterol reducing effect (Ansah et al., 1985), and use of pharmaceuticals causes serious problem like drug residues in eggs (Mathur and Singh, 2005). In this scene, use of phytogenic feed additives to reduce egg cholesterol could be an attractive alternative. Previous studies have been reported that supplementation of garlic paste (Chowdhury et al., 2002), Fuzhuan tea (Xu et al., 2012), tamarind (Chowdhury et al., 2005) had potential to reduce serum and egg yolk cholesterol concentrations. Gynura procumbens (Merr.) is an annual evergreen herbaceous plant, widely found in SouthEast Asia, especially Indonesia, Malaysia and Thailand. It has been extensively used in folk medicines as remedy for eruptive fevers, rash, kidney diseases, hypertension, diabetes and cancer (Perry, 1980). It has been reported that G. procumbens is a rich source of total phenolic compounds and flavonoids and has high free radical scavenging activity (Mustafa et al., 2010). Pharmacological studies have indicated that G. procumbens has antioxidant (Rosidah et al., 2008), anti-hyperglycaemic (Akowuah et al., 2002), antiinflammatory (Iskander et al., 2002), anti-ulcerogenic (Mahmood et al., 2010) and anti-carcinogenic (Agustina et al., 2006) capabilities. Despite the various medicinal and pharmacological uses of G. procumbens over a long time period, there is no report on the effects of G. procumbens on cholesterol metabolism and performance of laying hens. Therefore, the aim of the present study is to investigate the effects of dietary supplementation with G. procumbens on egg yolk and serum cholesterol and triglycerides, excreta microflora laying performance and egg quality
MATERIALS AND METHODS The protocol for this experiment was approved, and birds were cared for according to the guidelines of the Institutional Animal Care and Use Committee of Kangwon National University, Chuncheon, Republic of Korea. Plant material Gynura procumbens seedlings were grown at Kangwon National University Farm, Chuncheon, Republic of Korea. Whole plants of G. procumbens, excluding roots, were harvested and transported to the laboratory. Plants were air-dried under shade for 3–4 d and then dried using a forced air drying oven at 60°C for 72 h, and ground in Wiley mill (Thomas Model 4 Wiley Mill, Thomas Scientific, Swedesboro, NJ, USA) using a 1-mm screen and stored at 4°C until further use.
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Preparation of the extract A total of 50 g of ground G. procumbens was extracted with 500 ml of pure methanol at 40°C for 2 h. The extract was then concentrated in vacuum at 40°C in a rotary evaporator and stored at −20°C until analysed for total phenolic compounds and flavonoids. Analytical compositions of G. procumbens Gynura procumbens were analysed in triplicate for dry matter (DM) (Method 930.15), crude protein (CP) (Method 990.03), crude fat (Method 954.02), Ca and P (Method 985.01) using AOAC (2007). Total phenolic compounds were determined using Folin and Ciocalteu’s phenol reagent and tannic acid standard (Sigma Aldrich, St. Louis, MO, USA) according to the procedure described by AOAC (2005). The total flavonoids content was measured using aluminium chloride colorimetric method with quercetin (Sigma Aldrich, USA) as a standard according to the procedure described by Zhishen et al. (1999). Chlorophyll a, b and carotenoids contents were measured according to the procedure of Yang et al. (1998). The analytical compositions of G. procumbens are shown in Table 1. Birds, diets and management A total of 160 Hy-Line Brown layers (45 weeks old) were randomly assigned to 4 treatments on the basis of laying performance. Each treatment had 4 replicates with 10 hens each (40 hens per treatment). Two hens were confined in a cage, size 35 × 35 × 40 cm, and each group of 10 birds (5 cages) shared a common feed trough between them forming one experimental unit. Dietary treatments were basal diet supplemented with 0 (control), 2.5, 5.0 and 7.5 g/kg diet G. procumbens. All the birds were fed isocaloric (Metabolisable energy, ME, 11.51 MJ/kg) and isonitrogenous (CP, 166.0 g/kg) diets in mash form for 56 d. Diets were formulated to meet or exceeded the nutrient requirements recommended by NRC (1994). Ingredients and chemical composition of basal diet and G. procumbens are shown in Table 1. The birds were provided daily ad libitum feed and clean drinking water during 56-d feeding period. Laying hens were exposed to a 16-h incandescent light period. Sample collection The laying hens were weighed at the beginning and on d 28 and 56 of experimental feeding. Feed consumption was recorded weekly, and feed conversion efficiency (feed intake/egg mass) was calculated during 56-d experimental period. Daily
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Table 1. Ingredients and chemical composition of basal diet and G. procumbens (as-fed basis)1
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Items
Basal diet
Ingredients, g/kg Maize 481.3 Wheat 50.0 Soybean meal, 450 g/kg 203.0 Rape seed meal 20.0 Maize gluten meal 59.0 Distillers dried grains with solubles 30.0 Rice bran 30.0 Animal fat 16.2 Choline chloride, 500 g/kg 0.6 0.9 L-Lysine, 240 g/kg Methionine hydroxyl analogue, 0.8 880 g/kg Limestone 95.3 Dicalcium phosphate 7.6 Sodium chloride 2.3 1.0 NaHCO3 1.0 Vitamin premix2 1.0 Mineral premix3 Chemical composition Basal diet ME, MJ/kg4 DM, g/kg CP, g/kg Ca, g/kg Available phosphorus, g/kg Lys, g/kg4 Met + Cys, g/kg4 Cholesterol, g/kg Total flavonoids, mg/g Total phenolic compounds, mg/g Chlorophyll a, mg/g Chlorophyll b, mg/g Carotenoids, mg/g
11.51 912.3 166.0 40.2 3.0 8.9 7.4 0.15 – – – – –
For measurement of excreta microflora (total anaerobic bacteria (TAB), anaerobic lactic acid bacteria, Clostridium sp. and Escherichia coli), fresh excreta samples were collected from 5 hens per replicate at d 28 and 56 of experiment. Excreta samples from each replicate (5 hens/replicate) were mixed before analysis. The samples collected for microbial analysis were immediately placed on ice until analyses were conducted later on the same day. Blood collection
Gynura procumbens – 923.5 111.7 14.7 6.8 – – 0.06 13.46 54.10 12.99 8.30 4.66
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Dietary treatments were basal diet supplemented with 0 (control), 2.5, 5.0 and 7.5 g/kg diet G. procumbens. 2 Provides per kg diet: retinol, 2700 µg; cholecalciferol, 45 µg; dl-α-tocopheryl acetate, 30 mg; menadione, one milligram; thiamin, one milligram; riboflavin, 10 mg; pyridoxine, 4 mg; cyanocobalamin, 0.02 mg; niacin, 30 mg; pantothenic acid, 12 mg; folic acid, 0.5 mg; biotin, 0.2 mg. 3 Provides per kg diet: 45 mg Fe as ferrous sulphate, 0.25 mg Co as cobalt sulphate, 50 mg Cu as copper sulphate, 15 mg Mn as manganous oxide, 25 mg Zn as zinc oxide, 0.35 mg I as potassium iodide, and 0.13 mg Se as sodium selenite. 4 Based on NRC (1994) values.
egg production and egg weight per treatment group were recorded to determine the hen-d egg production and the egg mass production (g/d/ hen). Eggs from each group were collected at beginning and at 2 weeks interval (for 2 consecutive d) during feeding period for analysis of egg quality (breaking strength, albumin height, yolk colour, shell thickness and shell weight) and egg yolk cholesterol and triglycerides concentrations. Egg shell breaking strength (kg/m2) was measured by using texture analyser (FHK Fujihira Industry co. LTD, Japan). The albumen height was measured with an electronic tripod micrometer. Yolk colour was evaluated by using a colorimetric fan (Roche) and scored according to their intensity. Egg shell weight was measured using an electric weighing balance.
At the beginning and on d 21, 42 and 56 of experimental feeding, a 5 ml blood sample was collected by bronchial wing vein puncture from two randomly selected hens in each replicate using sterilised syringes and needles. After 45 min standing at room temperature, serum was isolated by centrifugation at 1500 g for 15 min. Separated serum samples were stored at −80°C until further analysis. Extraction of yolk and diet lipid One gram of egg yolk was placed into a centrifuge tube and mixed with 15 ml of chloroform:methanol (2:1; v/v), sonicated and filtered as described by Elkin and Rogler (1990). Dietary lipids were extracted by same procedure using 5 g feed samples with 40 ml of chloroform:methanol. Enzymatic and microbial analysis Total cholesterol and triglycerides in the basal diet, G. procumbens, serum and egg yolk extract were determined enzymatically using commercial reagent kits (ASAN Total Cholesterol, AM 202-K and ASAN TG-S, AM 157S-K, respectively, ASAN Pharm. Co. Ltd., Gyeonggi-do, Korea). For analysis of microflora, one gram of mixed excreta samples (d 28 and 56) was diluted with 9 ml of Butterfields’ phosphate buffer solution, followed by further serial dilutions in Butterfields’ phosphate buffer dilution solution. Duplicate culture plates were then inoculated in with 0.1 ml sample and incubated according to standard procedures for different bacteria. The TAB were propagated in tryptic soy agar (Difco Laboratories, Detroit, MI, USA) under anaerobic conditions at 37oC for 48 h; Clostridium sp. in tryptose sulphite cycloserine agar (Oxoid, Hampshire, UK) under anaerobic conditions at 37oC for 48 h and E. coli in violet red bile agar (Difco Laboratories, Detroit, MI, USA) under aerobic conditions at 37oC for 24 h. The anaerobic conditions during the assay of TAB and Clostridium spp. were created by using GasPak anaerobic system (BBL, No. 260 678, Difco, Detroit, MI, USA). The bacterial concentrations were transformed (log) before statistical analysis.
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Statistical analysis The data generated in the present study were subjected to statistical analysis using the generalised linear model (GLM) procedure of SAS (SAS Inst. Inc., Cary, NC, USA) as a completely randomised design. The orthogonal polynomials were used to evaluate the linear and quadratic effects of dietary G. procumbens concentrations (0, 2.5, 5.0 and 7.5 g/ kg diet). For analysis of production performance parameters, a replicate of 10 birds was used as experimental unit. For analysis of egg cholesterol and triglycerides, the individual egg was used as experimental unit, whereas, for analysis of excreta microflora and serum cholesterol and triglycerides, the individual hen was used as experimental unit. The P-values of 0.05) on feed intake of laying hens.
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Excreta microflora Increasing concentrations of G. procumbens in the diets of laying hens linearly reduced (P < 0.05; Table 4) the excreta TAB (d 28), Clostridium sp. and E. coli (d 28 and 56). However, dietary treatments had no effect (linear or quadratic, P < 0.05) on TAB (d 56) and anaerobic lactic acid bacteria (d 28 and 56) populations. Laying performance There was no mortality among any treatment group during 56 d experimental period. Compared with control group, laying hens fed
Egg quality Dietary treatments had no effects on albumin height, shell thickness and shell weight at any period of experiment. At d 28 and 56, hens fed diets supplemented with increasing concentration of G. procumbens had improved (linear, P < 0.05; Table 6) egg yolk colour. At d 56, breaking strength of eggs was linearly increased (P < 0.05) with increasing dietary concentration of G. procumbens. However, dietary treatments had no effects on albumin height, shell thickness and shell weight at any period of experiment.
Table 4. Effect of supplementation with Gynura procumbens on excreta microflora (log10 cfu/g) in laying hens1 P-values3
Gynura procumbens, g/kg Item D 28 Total anaerobic bacteria Anaerobic lactic acid bacteria Clostridium spp. Escherichia coli D 56 Total anaerobic bacteria Anaerobic lactic acid bacteria Clostridium spp. Escherichia coli
0 (Control)
2.5
5.0
7.5
SEM2
Linear
Quadratic
8.49 7.02 6.64 6.47
7.63 6.81 5.91 5.58
7.36 7.23 5.42 5.49
7.19 7.14 5.56 4.98
0.15 0.17 0.15 0.19
0.010 0.214 0.002 0.007
0.098 0.620 0.092 0.208
8.33 7.77 5.99 6.00
7.50 7.64 5.27 5.31
8.13 7.66 4.93 5.15
7.87 7.75 4.65 4.61
0.19 0.07 0.18 0.20
0.613 0.530 0.002 0.007
0.364 0.283 0.625 0.508
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Data represent means based on 4 replicates per treatment. Standard error of the mean. 3 Linear and quadratic effect of increasing dietary concentrations of G. procumbens (0, 2.5, 5.0 and 7.5 g/kg diet). 2
Table 5.
Effect of dietary supplementation with Gynura procumbens on egg production, egg weight, egg mass, feed intake and feed efficiency in laying hens1 P-values3
Gynura procumbens, g/kg Items Overall (1–8 week) Egg production, egg/hen/dy Egg weight, g Egg mass4, g/d/hen Feed intake, g/d/hen Feed efficiency5 1
0(Control)
2.5
5.0
7.5
SEM2
Linear
Quadratic
0.875 58.53 51.22 119.64 2.34
0.876 59.30 51.97 118.78 2.29
0.881 59.52 52.44 117.09 2.23
0.885 59.78 52.92 118.14 2.23
0.02 0.24 0.27 0.42 0.02
0.020 0.081 0.022 0.100 0.013
0.658 0.596 0.768 0.257 0.350
All measurements were done on an as-fresh basis; data represent means based on 4 replicates per treatment. Standard error of the mean. 3 Linear and quadratic effect of increasing dietary concentrations of G. procumbens (0, 2.5, 5.0 and 7.5 g/kg diet). 4 Egg mass = (egg production × egg weight)/100. 5 Feed efficiency = feed intake/egg mass (g/g). 2
GYNURA PROCUMBENS IN LAYING HEN DIETS
Table 6. Effect of dietary supplementation with Gynura procumbens on egg quality parameters (d 28 and 56) of laying hens1 P-value3
Gynura procumbens, g/kg
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Item
0(Control)
2.5
Breaking strength, kg/m2 d 28 3.67 3.76 d 56 3.54 3.78 Albumin height, mm d 28 7.32 7.22 d 56 7.35 7.44 Yolk colour d 28 7.2 8.4 d 56 7.6 8.8 Shell thickness, mm d 28 0.41 0.41 d 56 0.41 0.41 Shell weight, g d 28 7.15 7.21 d 56 7.16 7.32
5.0
7.5 SEM2 Linear Quadratic
3.82 3.94 0.09 3.94 4.14 0.09
0.262 0.01
0.886 0.896
7.44 7.38 0.07 7.48 7.56 0.11
0.541 0.526
0.379 0.971
8.8 9.2
0.22 0.21
0.001 0.001
0.213 0.163
0.41 0.42 0.05 0.42 0.42 0.05
0.309 0.390
0.565 0.846
7.22 7.17 0.11 7.24 7.29 0.06
0.766 0.645
0.828 0.715
9.2 9.6
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All measurements were done on an as-fresh basis; data represent means based on 4 replicates per treatment. 2 Standard error of the mean. 3 Linear and quadratic effect of increasing dietary concentrations of G. procumbens (0, 2.5, 5.0 and 7.5 g/ kg diet).
DISCUSSION With growing concern over the relationship between diets and human health, there is an increasing emphasis on modification of fat and cholesterol contents of edible animal and poultry products. As consumption of eggs has been tripled worldwide over the past 4 decades, a new market for eggs with value-added traits such as reduced yolk cholesterol and fatty acids has emerged (Hernandez et al., 2009). To meet this demand, new approaches to alter nutritive properties of eggs are being developed, which includes exploring phytogenic feed additives that convey positive egg traits (Chowdhury et al., 2005; Islam et al., 2011; Xu et al., 2012). In the current study, we supplemented the medicinal herb G. procumbens to laying hen diets, and its effects on cholesterol metabolism, excreta microflora and laying hen performance were investigated. In the present study, supplementation with G. procumbens to laying hen diets reduced serum cholesterol and triglycerides from d 21 of experimental feeding, whereas, egg yolk cholesterol and triglycerides were reduced from d 28 onwards. Our results suggested that there is a relationship between serum and eggs yolk cholesterol and triglycerides. Some of the previous studies also reported that reduction in serum cholesterol results in reduced yolk cholesterol (Khan et al., 2007; Azeke and Ekpo, 2008). In contrast to the present results, some previous reports suggested that there were no relationships between serum and egg yolk cholesterol concentrations
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(Chowdhury et al., 2002, 2005; Xu et al., 2012). These variations in results might be due to variation in feed additives or genetic difference in breeds of laying hens. In the current study, at d 56, the reductions of egg yolk cholesterol by supplementation of 2.5, 5.0 and 7.5 g/kg diet procumbens were 6.10%, 11.98% or 12.07%, which were greater than 4.1% to 5.5% reduction by including 10 or 30 g/kg diet garlic powder (Sharma et al., 1979) or 5.0% reduction by probucol (Waldroup et al., 1986). Reduction of yolk cholesterol by 7.5 g/kg diet G. procumbens supplementation was quite similar to those obtained by Salma et al. (2007), observing a 13.0% reduction in egg yolk cholesterol by 0.4 g/kg diet Rhodobacter capsulatus. A recent study in the author’s lab reported 9.33% to 19.29% reduction in egg yolk cholesterol with supplementation of 0.5 and 1.5 g/kg diet R. capsulatus KCTC-2583 (Lokhande et al., 2013). The hypocholesterolaemic and other beneficiary effects of G. procumbens demonstrated in the present study are not clearly understood. However, our results indicated that G. procumbens is a rich source of antioxidants like total flavonoid, total phenolic compounds and carotenoids, which are speculated to be associated directly or indirectly with the hypocholesterolaemic and other beneficiary performances of laying hens. Akowuah et al. (2002) and Rosidah et al. (2008) also have reported that G. procumbens is a rich source of phenolic compounds and flavonoids. Previously it has been reported that antioxidant compounds present in the phytogenic feed additives can reduce egg cholesterol concentrations (Du and Ahn, 2000; Khan et al., 2007). Another mechanism through which G. procumbens may exert its hypocholesterolaemic action is via carotenoid. The G. procumbens is a rich source of carotenoids (Kaewseejan et al., 2012), and some of the carotenoids are known as hypocholesterolaemic agents. It has reported that dietary carotenoids had hypocholesterolaemic effects in rats (Amen and Lachance, 1974; Yeum and Russell, 2002). Furthermore, it has been speculated that active chemical ingredients present in G. procumbens may change enzymes, which are associated in regulating cholesterol synthesis, oxidation or elimination of cholesterol in laying hens. Intestinal and excreta microflora populations are indicative of gut health in animals and poultry. In the current study, laying hens fed diets supplemented with G. procumbens had reduced pathogenic bacteria, E .coli and Clostridium sp., but had no change in concentration of beneficial bacteria like anaerobic lactic acid bacteria. These beneficial effects of G. procumbens might be due to the presence of the various active chemical constituents including flavonoids, phenolics, saponins, tannins and terpenoids (Akowuah et al., 2002).
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Results obtained herein are consistent with data reported by Islam et al. (2011), who reported reduced pathogenic microflora in laying hens fed phytogenic feed additive, Nigella sativa. In the current study, the improved performance of laying hens with supplementation of G. procumbens might be associated with improved gut microbial balance. In the present study, increasing concentrations of G. procumbens showed linear improvement in egg production performance of laying hens. These results are in good agreement with data reported by Xu et al. (2012), who observed increased egg production, egg weight and reduced feed efficiency in laying hens fed diets supplemented with phytogenic feed additives, milled fuzhuan tea or its water-soluble extract. Similarly, it was reported that laying hens-fed diets supplemented with up to 40 g/kg diet tamarind (a phytogenic feed additive) had increased egg production and egg mass and reduced feed efficiency as compared to hens-fed control diet (Chowdhury et al., 2005). The improved laying performance in hens-fed diets supplemented with G. procumbens could be associated with improved gut health, which is indicated by reduced population of pathogenic microorganisms (E. coli and Clostridium sp.) in G. procumbens supplemented hens as compared to control. Moreover, improved production performance might be due to overall improvement of health of G. procumbens-supplemented laying hens, as it has been reported that G. procumbens has antioxidant (Rosidah et al., 2008), anti-hyperglycaemic (Akowuah et al., 2002), anti-inflammatory (Iskander et al., 2002), anti-ulcerogenic (Mahmood et al., 2010), anti-carcinogenic (Agustina et al., 2006) capabilities. Our results also showed that G. procumbens is a rich source of Ca, available P, total phenolic compounds, flavonoids and carotenoids. In this study, supplementation of G. procumbens to laying hen diets improved the yolk colour and the breaking strength of the eggs. The improved yolk colour in the present study might be due to high chlorophyll a (12.99 mg/g), chlorophyll b (8.30 mg/g) and carotenoids (4.66 mg/g) content of the G. procumbens. Like the present study, Kaewseejan et al. (2012) also observed that G. procumbens is a rich source of carotenoids. Previously it has been observed that dietary supplementation of external carotenoids improved carotenoids contents and pigmentation of eggs yolk in quail (Karadas et al., 2006). Increased breaking strength of the eggs in G. procumbens supplemented groups might be due to greater calcium (14.7 g/kg) and phosphorus (6.8 g/kg) content in the G. procumbens supplemented diet than in the control diet (0% G. procumbens diet).
The results obtained in the present experiment indicate that dietary supplementation of G. procumbens could reduce the egg yolk cholesterol, suppress harmful excreta microflora and improve layers performance. However, further detailed studies are needed to identify the bioactive compounds of G. procumbens being responsible for the hypocholesterolaemic effects.
ACKNOWLEDGEMENT The authors are thankful to the Institute of Animal Resources, Kangwon National University, Chuncheon, Republic of Korea, for providing technical facilities to conduct this experiment.
FUNDING This study was supported by the 2011 Research Grant from Kangwon National University, Chuncheon, Republic of Korea.
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GYNURA PROCUMBENS IN LAYING HEN DIETS GRIFFIN, H.D. (1992) Manipulation of egg yolk cholesterol: A physiologist’s view. World’s Poultry Science Journal, 48: 101– 112. doi:10.1079/WPS19920010 HERNANDEZ, J., HAMELIN, C., HAYAKAWA, T., IBARRA, B. & SOUZA, R. (2009) Adding value to egg products. World Poultry, 25: 24–26. ISKANDER, M.N., SONG, Y., COUPAR, I.M. & JIRATCHARIYAKUL, W. (2002) Anti-inflammatory screening of the medicinal plant Gynura procumbens. Plant Foods for Human Nutrition, 57: 233– 244. doi:10.1023/A:1021851230890 ISLAM, M.T., SELIM, A.S.M., SAYED, M.A., KHATUN, M.A., SIDDIQUI, M.N., ALAM, M.S. & HOSSAIN, M.A. (2011) Nigella sativa L. supplemented diet decreases egg cholesterol content and suppresses harmful intestinal bacteria in laying hens. Journal of Animal and Feed Sciences, 20: 587–598. KAEWSEEJAN, N., PUANGPRONPITAG, D. & NAKORNRIAB, M. (2012) Evaluation of phytochemical composition and antibacterial property of Gynura procumbens. Asian Journal of Plant Science, 11: 7782. KARADAS, F., GRAMMENIDIS, E., SURAI, P.F., ACAMOVIC, T. & SPARKS, N.H. (2006) Effects of carotenoids from lucerne, marigold and tomato on egg yolk pigmentation and carotenoid composition. British Poultry Science, 47: 561–566. doi:10.1080/ 00071660600962976 KHAN, S.H., SARDAR, S.R. & ANJUM, M.A. (2007) Effects of dietary garlic on performance and serum and egg yolk cholesterol concentration in laying hens. Asian Journal of Poultry Science, 1: 22–27. doi:10.3923/ajpsaj.2007.22.27 LOKHANDE, A., INGALE, S.L., LEE, S.H., KIM, J.S., CHAE, B.J. & KWON, I.K. (2013) The effects of dietary Rhodobacter capsulatus KCTC-2583 on cholesterol metabolism, egg production and quality parameters during the late laying periods in hens. Asian-Australasian Journal of Animal Science, 26: 831–837. doi:10.5713/ajas.2012.12559 MAHMOOD, A.A., MARIOD, A.A., AL, B.F. & ABDEL, W.S.I. (2010) Anti-ulcerogenic activity of Gynura procumbens leaf extract against experimentally induced gastric lesions in rats. Journal of Medicinal Plants Research, 4: 685–691. MATHUR, S. & SINGH, R. (2005) Antibiotic resistance in food lactic acid bacteria – a review. International Journal of Food Microbiology, 105: 281–295. doi:10.1016/j.ijfoodmicro. 2005.03.008 MUSTAFA, R.A., HAMID, A.A., MOHAMED, S. & BAKAR, F.A. (2010) Total phenolic compounds, flavonoids and radical scavenging activity of 21 selected tropical plants. Journal of Food Science, 75: C28–C35. doi:10.1111/j.1750-3841.2009.01401.x NRC (1994) Nutrient Requirement of Poultry. 9th edn. (Washington, DC, National Academy Press).
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PERRY, L.M. (1980) Medicinal Plants of East and Southeast Asia: Attributed Properties and Uses, p. 94 (USA, The MIT press). ROSIDAH, YAM, M.F., SADIKUN, A. & ASMAWI, M.Z. (2008) Antioxidant potential of Gynura procumbens. Pharmaceutical Biology, 46: 616–625. doi:10.1080/13880200802179642 SALMA, U., MIAH, A.G., TAREQ, K.M.A., MAKI, T. & TSUJII, H. (2007) Effect of dietary Rhodobacter capsulatus on egg-yolk cholesterol and laying hen performance. Poultry Science, 86: 714–719. doi:10.1093/ps/86.4.714 SHARMA, R.K., SINGH, R.A., PAL, R.N. & AGRAWAL, C.K. (1979) Cholesterol content of chicken eggs as affected by feeding garlic, sarpagandha, and nicotinic acid. Haryana Agriculture Journal of Research, 9: 263–265. USDA (1991) Nutrient Content of Foods. Dairy and Egg Products Handbook 8-1. (Washington, DC, United States Department of Agriculture). WALDROUP, P.W., NDIFE, L.I., HELLWIG, H.M., HEBERT, J.A. & BERRIO, L. (1986) Influence of Probucol ((4,4’Isopropylidine dithio)-bis(2,6-di-t-butyl-phenol)) on Egg Yolk Cholesterol Content and Performance of Laying Hens. Poultry Science, 65: 1949–1954. doi:10.3382/ ps.0651949 WANG, S. & TONG, J. (2002) Review in manipulation of cholesterol metabolism in laying hens. Acta Zoonutrimenta Sinica, 14: 7–11. WEGGEMANS, R.M., ZOCK, P.L. & KATAN, M.B. (2001) Dietary cholesterol from eggs increase the ratio of total cholesterol to high-density lipoprotein cholesterol in humans. A metaanalysis. American Journal of Clinical Nutrition, 73: 885–891. WORLD HEALTH ORGANISATION (2003) Diet, nutrition and the prevention of chronic diseases. Report of the joint WHO/FAO expert consultation. Geneva. XU, X., HU, Y., XIAO, W., HUANG, J., HE, X., WU, J., RYAN, E.P. & WEIR, T.L. (2012) Effects of fermented Camilla sinensis, Fuzhuan tea, on egg cholesterol and production performance in laying hens. Agriculture and Food Science, 1: 6–10. YANG, C.M., CHANG, K.W., YIN, M.H. & HUANG, H.M. (1998) Methods for the determination of the chlorophylls and their derivatives. Taiwania, 43(2): 116–122. YEUM, K.J. & RUSSELL, R.M. (2002) Carotenoid bioavailability and bioconversion. Annual Review of Nutrition, 22: 483–504. doi:10.1146/annurev.nutr.22.010402.102834 ZHISHEN, J., MENGCHENG, T. & JIANMING, W. (1999) The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry, 64(4): 555–559. doi:10.1016/S0308-8146(98) 00102-2
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effects of dietary supplementation with Gynura procumbens (Merr.) on egg yolk cholesterol, excreta microflora and laying hen performance a
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A. Lokhande , S. L. Ingale , S. H. Lee , S. Sen , C. Khong , B. J. Chae & I. K. Kwon
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Department of Animal Products and Food Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea b
Department of Animal Resources Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea Accepted author version posted online: 10 Jul 2014.Published online: 15 Aug 2014.
Click for updates To cite this article: A. Lokhande, S. L. Ingale, S. H. Lee, S. Sen, C. Khong, B. J. Chae & I. K. Kwon (2014) Effects of dietary supplementation with Gynura procumbens (Merr.) on egg yolk cholesterol, excreta microflora and laying hen performance, British Poultry Science, 55:4, 524-531, DOI: 10.1080/00071668.2014.938020 To link to this article: http://dx.doi.org/10.1080/00071668.2014.938020
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British Poultry Science, 2014 Vol. 55, No. 4, 524–531, http://dx.doi.org/10.1080/00071668.2014.938020
Effects of dietary supplementation with Gynura procumbens (Merr.) on egg yolk cholesterol, excreta microflora and laying hen performance A. LOKHANDE, S. L. INGALE1, S. H. LEE1, S. SEN1, C. KHONG1, B. J. CHAE1 AND I. K. KWON
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Department of Animal Products and Food Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea, and 1Department of Animal Resources Science, College of Animal Life Sciences, Kangwon National University, Chuncheon, Republic of Korea
Abstract 1. The present study investigated the effects of dietary supplementation with Gynura procumbens on egg yolk and serum cholesterol and triglycerides, excreta microflora, laying performance and egg quality. 2. A total of 160 Hy-Line Brown layers (45 weeks old) were randomly assigned into 4 treatments on the basis of laying performance. Each treatment had 4 replicates with 10 birds each. 3. Dietary treatments were basal diet supplemented with 0 (control), 2.5, 5.0 and 7.5 g/kg diet G. procumbens during 56-d feeding period. 4. Serum (d 21, 42 and 56) and egg yolk (d 28, 42 and 56) cholesterol and triglycerides concentrations were linearly reduced with increasing dietary concentrations of G. procumbens. 5. Increasing dietary concentrations of G. procumbens linearly reduced the excreta total anaerobic bacteria (d 28), Clostridium sp. and Escherichia coli (d 28 and 56) populations. 6. Overall egg production and egg mass were linearly increased, and overall feed efficiency was linearly improved with increase in dietary G. procumbens. 7. Dietary increasing concentrations of G. procumbens linearly improved egg yolk colour (d 28 and 56) and breaking strength of eggs (d 56). 8. The results obtained in the present experiment indicate that dietary supplementation with G. procumbens could reduce the egg yolk cholesterol, suppresses harmful excreta microflora and improves layers performance.
INTRODUCTION Chicken eggs are one of the most widely consumed animal food products and generally considered as an excellent source of calcium, phosphorus, high-quality proteins, unsaturated fats, folates and other B vitamins for persons of all age group (USDA, 1991). However, chicken egg yolk is rich source of cholesterol (213 mg/ egg; USDA, 1991), and over 95% of yolk cholesterol is associated with triglyceride-rich lipoprotein (Griffin, 1992). To avoid the elevation of blood cholesterol and heart-related diseases of human, it has been recommended to restrict
dietary cholesterol intake to below 200 mg/d for individuals at high risk of cardiovascular disease, to below 300 mg/d for healthy individuals (WHO, 2003) and limit the consumption of eggs (Weggemans et al., 2001). The poultry industry has continued to seek for production of “designer” eggs with value-added traits such as decreased cholesterol, triglycerides and increased omega-3 fatty acids to meet out the demand of health conscious consumers (Hernandez et al., 2009). Previous attempts to lowering egg yolk cholesterol have centred mostly on genetic selection, pharmacological intervention or alteration of the laying hen diets with feed additives that convey
Correspondence to: I. K. Kwon, Department of Animal Products and Food Science, Kangwon National University, Chuncheon, 200-701, Republic of Korea. E-mail: [email protected] Accepted for publication 10 April 2014. Lokhande and Ingale have contributed equally to this work.
© 2014 British Poultry Science Ltd
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GYNURA PROCUMBENS IN LAYING HEN DIETS
positive eggs traits (Wang and Tong, 2002). However, genetic selection had limited cholesterol reducing effect (Ansah et al., 1985), and use of pharmaceuticals causes serious problem like drug residues in eggs (Mathur and Singh, 2005). In this scene, use of phytogenic feed additives to reduce egg cholesterol could be an attractive alternative. Previous studies have been reported that supplementation of garlic paste (Chowdhury et al., 2002), Fuzhuan tea (Xu et al., 2012), tamarind (Chowdhury et al., 2005) had potential to reduce serum and egg yolk cholesterol concentrations. Gynura procumbens (Merr.) is an annual evergreen herbaceous plant, widely found in SouthEast Asia, especially Indonesia, Malaysia and Thailand. It has been extensively used in folk medicines as remedy for eruptive fevers, rash, kidney diseases, hypertension, diabetes and cancer (Perry, 1980). It has been reported that G. procumbens is a rich source of total phenolic compounds and flavonoids and has high free radical scavenging activity (Mustafa et al., 2010). Pharmacological studies have indicated that G. procumbens has antioxidant (Rosidah et al., 2008), anti-hyperglycaemic (Akowuah et al., 2002), antiinflammatory (Iskander et al., 2002), anti-ulcerogenic (Mahmood et al., 2010) and anti-carcinogenic (Agustina et al., 2006) capabilities. Despite the various medicinal and pharmacological uses of G. procumbens over a long time period, there is no report on the effects of G. procumbens on cholesterol metabolism and performance of laying hens. Therefore, the aim of the present study is to investigate the effects of dietary supplementation with G. procumbens on egg yolk and serum cholesterol and triglycerides, excreta microflora laying performance and egg quality
MATERIALS AND METHODS The protocol for this experiment was approved, and birds were cared for according to the guidelines of the Institutional Animal Care and Use Committee of Kangwon National University, Chuncheon, Republic of Korea. Plant material Gynura procumbens seedlings were grown at Kangwon National University Farm, Chuncheon, Republic of Korea. Whole plants of G. procumbens, excluding roots, were harvested and transported to the laboratory. Plants were air-dried under shade for 3–4 d and then dried using a forced air drying oven at 60°C for 72 h, and ground in Wiley mill (Thomas Model 4 Wiley Mill, Thomas Scientific, Swedesboro, NJ, USA) using a 1-mm screen and stored at 4°C until further use.
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Preparation of the extract A total of 50 g of ground G. procumbens was extracted with 500 ml of pure methanol at 40°C for 2 h. The extract was then concentrated in vacuum at 40°C in a rotary evaporator and stored at −20°C until analysed for total phenolic compounds and flavonoids. Analytical compositions of G. procumbens Gynura procumbens were analysed in triplicate for dry matter (DM) (Method 930.15), crude protein (CP) (Method 990.03), crude fat (Method 954.02), Ca and P (Method 985.01) using AOAC (2007). Total phenolic compounds were determined using Folin and Ciocalteu’s phenol reagent and tannic acid standard (Sigma Aldrich, St. Louis, MO, USA) according to the procedure described by AOAC (2005). The total flavonoids content was measured using aluminium chloride colorimetric method with quercetin (Sigma Aldrich, USA) as a standard according to the procedure described by Zhishen et al. (1999). Chlorophyll a, b and carotenoids contents were measured according to the procedure of Yang et al. (1998). The analytical compositions of G. procumbens are shown in Table 1. Birds, diets and management A total of 160 Hy-Line Brown layers (45 weeks old) were randomly assigned to 4 treatments on the basis of laying performance. Each treatment had 4 replicates with 10 hens each (40 hens per treatment). Two hens were confined in a cage, size 35 × 35 × 40 cm, and each group of 10 birds (5 cages) shared a common feed trough between them forming one experimental unit. Dietary treatments were basal diet supplemented with 0 (control), 2.5, 5.0 and 7.5 g/kg diet G. procumbens. All the birds were fed isocaloric (Metabolisable energy, ME, 11.51 MJ/kg) and isonitrogenous (CP, 166.0 g/kg) diets in mash form for 56 d. Diets were formulated to meet or exceeded the nutrient requirements recommended by NRC (1994). Ingredients and chemical composition of basal diet and G. procumbens are shown in Table 1. The birds were provided daily ad libitum feed and clean drinking water during 56-d feeding period. Laying hens were exposed to a 16-h incandescent light period. Sample collection The laying hens were weighed at the beginning and on d 28 and 56 of experimental feeding. Feed consumption was recorded weekly, and feed conversion efficiency (feed intake/egg mass) was calculated during 56-d experimental period. Daily
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Table 1. Ingredients and chemical composition of basal diet and G. procumbens (as-fed basis)1
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Items
Basal diet
Ingredients, g/kg Maize 481.3 Wheat 50.0 Soybean meal, 450 g/kg 203.0 Rape seed meal 20.0 Maize gluten meal 59.0 Distillers dried grains with solubles 30.0 Rice bran 30.0 Animal fat 16.2 Choline chloride, 500 g/kg 0.6 0.9 L-Lysine, 240 g/kg Methionine hydroxyl analogue, 0.8 880 g/kg Limestone 95.3 Dicalcium phosphate 7.6 Sodium chloride 2.3 1.0 NaHCO3 1.0 Vitamin premix2 1.0 Mineral premix3 Chemical composition Basal diet ME, MJ/kg4 DM, g/kg CP, g/kg Ca, g/kg Available phosphorus, g/kg Lys, g/kg4 Met + Cys, g/kg4 Cholesterol, g/kg Total flavonoids, mg/g Total phenolic compounds, mg/g Chlorophyll a, mg/g Chlorophyll b, mg/g Carotenoids, mg/g
11.51 912.3 166.0 40.2 3.0 8.9 7.4 0.15 – – – – –
For measurement of excreta microflora (total anaerobic bacteria (TAB), anaerobic lactic acid bacteria, Clostridium sp. and Escherichia coli), fresh excreta samples were collected from 5 hens per replicate at d 28 and 56 of experiment. Excreta samples from each replicate (5 hens/replicate) were mixed before analysis. The samples collected for microbial analysis were immediately placed on ice until analyses were conducted later on the same day. Blood collection
Gynura procumbens – 923.5 111.7 14.7 6.8 – – 0.06 13.46 54.10 12.99 8.30 4.66
1
Dietary treatments were basal diet supplemented with 0 (control), 2.5, 5.0 and 7.5 g/kg diet G. procumbens. 2 Provides per kg diet: retinol, 2700 µg; cholecalciferol, 45 µg; dl-α-tocopheryl acetate, 30 mg; menadione, one milligram; thiamin, one milligram; riboflavin, 10 mg; pyridoxine, 4 mg; cyanocobalamin, 0.02 mg; niacin, 30 mg; pantothenic acid, 12 mg; folic acid, 0.5 mg; biotin, 0.2 mg. 3 Provides per kg diet: 45 mg Fe as ferrous sulphate, 0.25 mg Co as cobalt sulphate, 50 mg Cu as copper sulphate, 15 mg Mn as manganous oxide, 25 mg Zn as zinc oxide, 0.35 mg I as potassium iodide, and 0.13 mg Se as sodium selenite. 4 Based on NRC (1994) values.
egg production and egg weight per treatment group were recorded to determine the hen-d egg production and the egg mass production (g/d/ hen). Eggs from each group were collected at beginning and at 2 weeks interval (for 2 consecutive d) during feeding period for analysis of egg quality (breaking strength, albumin height, yolk colour, shell thickness and shell weight) and egg yolk cholesterol and triglycerides concentrations. Egg shell breaking strength (kg/m2) was measured by using texture analyser (FHK Fujihira Industry co. LTD, Japan). The albumen height was measured with an electronic tripod micrometer. Yolk colour was evaluated by using a colorimetric fan (Roche) and scored according to their intensity. Egg shell weight was measured using an electric weighing balance.
At the beginning and on d 21, 42 and 56 of experimental feeding, a 5 ml blood sample was collected by bronchial wing vein puncture from two randomly selected hens in each replicate using sterilised syringes and needles. After 45 min standing at room temperature, serum was isolated by centrifugation at 1500 g for 15 min. Separated serum samples were stored at −80°C until further analysis. Extraction of yolk and diet lipid One gram of egg yolk was placed into a centrifuge tube and mixed with 15 ml of chloroform:methanol (2:1; v/v), sonicated and filtered as described by Elkin and Rogler (1990). Dietary lipids were extracted by same procedure using 5 g feed samples with 40 ml of chloroform:methanol. Enzymatic and microbial analysis Total cholesterol and triglycerides in the basal diet, G. procumbens, serum and egg yolk extract were determined enzymatically using commercial reagent kits (ASAN Total Cholesterol, AM 202-K and ASAN TG-S, AM 157S-K, respectively, ASAN Pharm. Co. Ltd., Gyeonggi-do, Korea). For analysis of microflora, one gram of mixed excreta samples (d 28 and 56) was diluted with 9 ml of Butterfields’ phosphate buffer solution, followed by further serial dilutions in Butterfields’ phosphate buffer dilution solution. Duplicate culture plates were then inoculated in with 0.1 ml sample and incubated according to standard procedures for different bacteria. The TAB were propagated in tryptic soy agar (Difco Laboratories, Detroit, MI, USA) under anaerobic conditions at 37oC for 48 h; Clostridium sp. in tryptose sulphite cycloserine agar (Oxoid, Hampshire, UK) under anaerobic conditions at 37oC for 48 h and E. coli in violet red bile agar (Difco Laboratories, Detroit, MI, USA) under aerobic conditions at 37oC for 24 h. The anaerobic conditions during the assay of TAB and Clostridium spp. were created by using GasPak anaerobic system (BBL, No. 260 678, Difco, Detroit, MI, USA). The bacterial concentrations were transformed (log) before statistical analysis.
GYNURA PROCUMBENS IN LAYING HEN DIETS
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Statistical analysis The data generated in the present study were subjected to statistical analysis using the generalised linear model (GLM) procedure of SAS (SAS Inst. Inc., Cary, NC, USA) as a completely randomised design. The orthogonal polynomials were used to evaluate the linear and quadratic effects of dietary G. procumbens concentrations (0, 2.5, 5.0 and 7.5 g/ kg diet). For analysis of production performance parameters, a replicate of 10 birds was used as experimental unit. For analysis of egg cholesterol and triglycerides, the individual egg was used as experimental unit, whereas, for analysis of excreta microflora and serum cholesterol and triglycerides, the individual hen was used as experimental unit. The P-values of 0.05) on feed intake of laying hens.
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Excreta microflora Increasing concentrations of G. procumbens in the diets of laying hens linearly reduced (P < 0.05; Table 4) the excreta TAB (d 28), Clostridium sp. and E. coli (d 28 and 56). However, dietary treatments had no effect (linear or quadratic, P < 0.05) on TAB (d 56) and anaerobic lactic acid bacteria (d 28 and 56) populations. Laying performance There was no mortality among any treatment group during 56 d experimental period. Compared with control group, laying hens fed
Egg quality Dietary treatments had no effects on albumin height, shell thickness and shell weight at any period of experiment. At d 28 and 56, hens fed diets supplemented with increasing concentration of G. procumbens had improved (linear, P < 0.05; Table 6) egg yolk colour. At d 56, breaking strength of eggs was linearly increased (P < 0.05) with increasing dietary concentration of G. procumbens. However, dietary treatments had no effects on albumin height, shell thickness and shell weight at any period of experiment.
Table 4. Effect of supplementation with Gynura procumbens on excreta microflora (log10 cfu/g) in laying hens1 P-values3
Gynura procumbens, g/kg Item D 28 Total anaerobic bacteria Anaerobic lactic acid bacteria Clostridium spp. Escherichia coli D 56 Total anaerobic bacteria Anaerobic lactic acid bacteria Clostridium spp. Escherichia coli
0 (Control)
2.5
5.0
7.5
SEM2
Linear
Quadratic
8.49 7.02 6.64 6.47
7.63 6.81 5.91 5.58
7.36 7.23 5.42 5.49
7.19 7.14 5.56 4.98
0.15 0.17 0.15 0.19
0.010 0.214 0.002 0.007
0.098 0.620 0.092 0.208
8.33 7.77 5.99 6.00
7.50 7.64 5.27 5.31
8.13 7.66 4.93 5.15
7.87 7.75 4.65 4.61
0.19 0.07 0.18 0.20
0.613 0.530 0.002 0.007
0.364 0.283 0.625 0.508
1
Data represent means based on 4 replicates per treatment. Standard error of the mean. 3 Linear and quadratic effect of increasing dietary concentrations of G. procumbens (0, 2.5, 5.0 and 7.5 g/kg diet). 2
Table 5.
Effect of dietary supplementation with Gynura procumbens on egg production, egg weight, egg mass, feed intake and feed efficiency in laying hens1 P-values3
Gynura procumbens, g/kg Items Overall (1–8 week) Egg production, egg/hen/dy Egg weight, g Egg mass4, g/d/hen Feed intake, g/d/hen Feed efficiency5 1
0(Control)
2.5
5.0
7.5
SEM2
Linear
Quadratic
0.875 58.53 51.22 119.64 2.34
0.876 59.30 51.97 118.78 2.29
0.881 59.52 52.44 117.09 2.23
0.885 59.78 52.92 118.14 2.23
0.02 0.24 0.27 0.42 0.02
0.020 0.081 0.022 0.100 0.013
0.658 0.596 0.768 0.257 0.350
All measurements were done on an as-fresh basis; data represent means based on 4 replicates per treatment. Standard error of the mean. 3 Linear and quadratic effect of increasing dietary concentrations of G. procumbens (0, 2.5, 5.0 and 7.5 g/kg diet). 4 Egg mass = (egg production × egg weight)/100. 5 Feed efficiency = feed intake/egg mass (g/g). 2
GYNURA PROCUMBENS IN LAYING HEN DIETS
Table 6. Effect of dietary supplementation with Gynura procumbens on egg quality parameters (d 28 and 56) of laying hens1 P-value3
Gynura procumbens, g/kg
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Item
0(Control)
2.5
Breaking strength, kg/m2 d 28 3.67 3.76 d 56 3.54 3.78 Albumin height, mm d 28 7.32 7.22 d 56 7.35 7.44 Yolk colour d 28 7.2 8.4 d 56 7.6 8.8 Shell thickness, mm d 28 0.41 0.41 d 56 0.41 0.41 Shell weight, g d 28 7.15 7.21 d 56 7.16 7.32
5.0
7.5 SEM2 Linear Quadratic
3.82 3.94 0.09 3.94 4.14 0.09
0.262 0.01
0.886 0.896
7.44 7.38 0.07 7.48 7.56 0.11
0.541 0.526
0.379 0.971
8.8 9.2
0.22 0.21
0.001 0.001
0.213 0.163
0.41 0.42 0.05 0.42 0.42 0.05
0.309 0.390
0.565 0.846
7.22 7.17 0.11 7.24 7.29 0.06
0.766 0.645
0.828 0.715
9.2 9.6
1
All measurements were done on an as-fresh basis; data represent means based on 4 replicates per treatment. 2 Standard error of the mean. 3 Linear and quadratic effect of increasing dietary concentrations of G. procumbens (0, 2.5, 5.0 and 7.5 g/ kg diet).
DISCUSSION With growing concern over the relationship between diets and human health, there is an increasing emphasis on modification of fat and cholesterol contents of edible animal and poultry products. As consumption of eggs has been tripled worldwide over the past 4 decades, a new market for eggs with value-added traits such as reduced yolk cholesterol and fatty acids has emerged (Hernandez et al., 2009). To meet this demand, new approaches to alter nutritive properties of eggs are being developed, which includes exploring phytogenic feed additives that convey positive egg traits (Chowdhury et al., 2005; Islam et al., 2011; Xu et al., 2012). In the current study, we supplemented the medicinal herb G. procumbens to laying hen diets, and its effects on cholesterol metabolism, excreta microflora and laying hen performance were investigated. In the present study, supplementation with G. procumbens to laying hen diets reduced serum cholesterol and triglycerides from d 21 of experimental feeding, whereas, egg yolk cholesterol and triglycerides were reduced from d 28 onwards. Our results suggested that there is a relationship between serum and eggs yolk cholesterol and triglycerides. Some of the previous studies also reported that reduction in serum cholesterol results in reduced yolk cholesterol (Khan et al., 2007; Azeke and Ekpo, 2008). In contrast to the present results, some previous reports suggested that there were no relationships between serum and egg yolk cholesterol concentrations
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(Chowdhury et al., 2002, 2005; Xu et al., 2012). These variations in results might be due to variation in feed additives or genetic difference in breeds of laying hens. In the current study, at d 56, the reductions of egg yolk cholesterol by supplementation of 2.5, 5.0 and 7.5 g/kg diet procumbens were 6.10%, 11.98% or 12.07%, which were greater than 4.1% to 5.5% reduction by including 10 or 30 g/kg diet garlic powder (Sharma et al., 1979) or 5.0% reduction by probucol (Waldroup et al., 1986). Reduction of yolk cholesterol by 7.5 g/kg diet G. procumbens supplementation was quite similar to those obtained by Salma et al. (2007), observing a 13.0% reduction in egg yolk cholesterol by 0.4 g/kg diet Rhodobacter capsulatus. A recent study in the author’s lab reported 9.33% to 19.29% reduction in egg yolk cholesterol with supplementation of 0.5 and 1.5 g/kg diet R. capsulatus KCTC-2583 (Lokhande et al., 2013). The hypocholesterolaemic and other beneficiary effects of G. procumbens demonstrated in the present study are not clearly understood. However, our results indicated that G. procumbens is a rich source of antioxidants like total flavonoid, total phenolic compounds and carotenoids, which are speculated to be associated directly or indirectly with the hypocholesterolaemic and other beneficiary performances of laying hens. Akowuah et al. (2002) and Rosidah et al. (2008) also have reported that G. procumbens is a rich source of phenolic compounds and flavonoids. Previously it has been reported that antioxidant compounds present in the phytogenic feed additives can reduce egg cholesterol concentrations (Du and Ahn, 2000; Khan et al., 2007). Another mechanism through which G. procumbens may exert its hypocholesterolaemic action is via carotenoid. The G. procumbens is a rich source of carotenoids (Kaewseejan et al., 2012), and some of the carotenoids are known as hypocholesterolaemic agents. It has reported that dietary carotenoids had hypocholesterolaemic effects in rats (Amen and Lachance, 1974; Yeum and Russell, 2002). Furthermore, it has been speculated that active chemical ingredients present in G. procumbens may change enzymes, which are associated in regulating cholesterol synthesis, oxidation or elimination of cholesterol in laying hens. Intestinal and excreta microflora populations are indicative of gut health in animals and poultry. In the current study, laying hens fed diets supplemented with G. procumbens had reduced pathogenic bacteria, E .coli and Clostridium sp., but had no change in concentration of beneficial bacteria like anaerobic lactic acid bacteria. These beneficial effects of G. procumbens might be due to the presence of the various active chemical constituents including flavonoids, phenolics, saponins, tannins and terpenoids (Akowuah et al., 2002).
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Results obtained herein are consistent with data reported by Islam et al. (2011), who reported reduced pathogenic microflora in laying hens fed phytogenic feed additive, Nigella sativa. In the current study, the improved performance of laying hens with supplementation of G. procumbens might be associated with improved gut microbial balance. In the present study, increasing concentrations of G. procumbens showed linear improvement in egg production performance of laying hens. These results are in good agreement with data reported by Xu et al. (2012), who observed increased egg production, egg weight and reduced feed efficiency in laying hens fed diets supplemented with phytogenic feed additives, milled fuzhuan tea or its water-soluble extract. Similarly, it was reported that laying hens-fed diets supplemented with up to 40 g/kg diet tamarind (a phytogenic feed additive) had increased egg production and egg mass and reduced feed efficiency as compared to hens-fed control diet (Chowdhury et al., 2005). The improved laying performance in hens-fed diets supplemented with G. procumbens could be associated with improved gut health, which is indicated by reduced population of pathogenic microorganisms (E. coli and Clostridium sp.) in G. procumbens supplemented hens as compared to control. Moreover, improved production performance might be due to overall improvement of health of G. procumbens-supplemented laying hens, as it has been reported that G. procumbens has antioxidant (Rosidah et al., 2008), anti-hyperglycaemic (Akowuah et al., 2002), anti-inflammatory (Iskander et al., 2002), anti-ulcerogenic (Mahmood et al., 2010), anti-carcinogenic (Agustina et al., 2006) capabilities. Our results also showed that G. procumbens is a rich source of Ca, available P, total phenolic compounds, flavonoids and carotenoids. In this study, supplementation of G. procumbens to laying hen diets improved the yolk colour and the breaking strength of the eggs. The improved yolk colour in the present study might be due to high chlorophyll a (12.99 mg/g), chlorophyll b (8.30 mg/g) and carotenoids (4.66 mg/g) content of the G. procumbens. Like the present study, Kaewseejan et al. (2012) also observed that G. procumbens is a rich source of carotenoids. Previously it has been observed that dietary supplementation of external carotenoids improved carotenoids contents and pigmentation of eggs yolk in quail (Karadas et al., 2006). Increased breaking strength of the eggs in G. procumbens supplemented groups might be due to greater calcium (14.7 g/kg) and phosphorus (6.8 g/kg) content in the G. procumbens supplemented diet than in the control diet (0% G. procumbens diet).
The results obtained in the present experiment indicate that dietary supplementation of G. procumbens could reduce the egg yolk cholesterol, suppress harmful excreta microflora and improve layers performance. However, further detailed studies are needed to identify the bioactive compounds of G. procumbens being responsible for the hypocholesterolaemic effects.
ACKNOWLEDGEMENT The authors are thankful to the Institute of Animal Resources, Kangwon National University, Chuncheon, Republic of Korea, for providing technical facilities to conduct this experiment.
FUNDING This study was supported by the 2011 Research Grant from Kangwon National University, Chuncheon, Republic of Korea.
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