Effects of star anise (Illicium verum Hook.f.) essential oil on laying performance and antioxidant status of laying hens

Effects of star anise (Illicium verum Hook.f.) essential oil on laying performance and antioxidant status of laying hens Caiyun Yu,∗ Jiandong Wei,† Chongwu Yang,‡ Zaibin Yang,∗,1 Weiren Yang,∗ and Shuzhen Jiang∗ ∗

College of Animal Sciences and Technology, Shandong Agricultural University, Tai’an 271018, Shandong, P.R. China; † Huanshan Group Co., Ltd, Qing’dao 266000, Shandong, P.R. China; and ‡ Department of Animal Sciences, University of Manitoba, Canada improve (P < 0.10) egg mass, average egg weight, and ADFI in day 1 to day 28. Supplementation of SAO linearly increased (P < 0.05) activities of total superoxide dismutase (T-SOD) (day 28 and day 56) and glutathione peroxidase (GSH-PX) (day 56) in serum, GSH-PX (day 28 and day 56) in liver and total antioxidant capacity (T-AOC) (day 56) in serum and liver, but linearly reduced (P < 0.05) concentrations of malondialdehyde (MDA) (day 28 and day 56) in liver. Supplementation of SAO linearly increased (P < 0.05) T-SOD activity at day 14 and day 28, reduced (P < 0.05) MDA concentration at day 42 and day 56 of the experiment in yolk. Increasing content of SAO linearly (P < 0.05) increased T-SOD activity in yolk of eggs stored at day 0, 14, 28, 42, and 56, decreased MDA content of eggs stored at day 42 and 56, whether laying hens fed diets for 28 or 56 d. Dietary supplementation of SAO enhanced laying performance and overall antioxidant status of laying hens in a dose-dependent manner.

ABSTRACT To investigate the effects of dietary supplementation of star anise oil (SAO) on performance and antioxidant status of laying hens, a total of 864 Hy-Line brown laying hens at 26 wk of age were randomly allocated to 4 treatments with 6 replicates of 36 birds. Dietary treatments were non-star anise oil supplementation and supplemented with SAO at the level of 200, 400, and 600 mg/kg diet. The birds were fed the diets for 56 d. Average egg weight, average daily feed intake (ADFI), egg mass, laying rate, and feed conversion of each replicate were measured. Blood and liver samples from 12 birds were obtained, 72 eggs were picked out, per treatment at day 28 and day 56 of the experiment, and eggs stored for 56 d, to determine antioxidant status in serum, liver, and yolk. All laying hens had similar average egg weight, egg mass, laying rate, and feed conversion in day 29 to day 56 or the entire period of the experiment but significant difference on ADFI in day 1 to day 56. However, increasing diet concentration of SAO tended to

Key words: laying hen, star anise oil, laying performance, antioxidant status 2018 Poultry Science 0:1–10 http://dx.doi.org/10.3382/ps/pey263

INTRODUCTION

try industry, such as chicken meat and eggs. However, synthetic antioxidants have been restricted for use because of their toxicity (Halvorsen et al., 2002; Ding et al., 2017). Use of natural antioxidants replacing synthetic antioxidants in animal feed has gained interesting attention (Dragland et al., 2003). Plants, including spices, medical herbs, and their essential oils, may be considerable alternatives with the feature of safe, less toxic, and residue free for synthetic chemical feed additives (Hashemi et al., 2008). Star anise (Illicium verum) commonly used as a spice is a small anise-scented star-shaped fruit of an aromatic evergreen tree of Illiciaceae family (Wong et al., 2014). Star anise, as a medicinal plant, has been reported to play a key role in stimulating digestion and antibacterial activity (Singh et al., 2002), growth promoting (Kassie, 2008), and antioxygenic effects (Padmashree et al., 2007). Essential oil of star anise is faint yellow with a highly aromatic odor and anise flavor, may be widely used in pharmaceutical or food industry

Body metabolic processes commonly produce oxidation stress, as a formation of free radicals causing lipid peroxidation and body damage if exist in excessive levels. The products of lipid peroxidation can have a bad influence on broiler performance as a result of decreasing the nutrient content in feed (Zhao et al., 2011; Delles et al., 2014) and shelf life problems in foods. The shorten shelf-life of foods is the result of autooxidation of fats and oils, causing oxidative rancidity (Semwal et al., 1997). Therefore, synthetic antioxidants are widely used to alleviate oxidation damage by preventing the formation of free radicals and extend the shelf-life of lipid-containing foods in commercial poul C 2018 Poultry Science Association Inc. Received March 12, 2018. Accepted May 26, 2018. 1 Corresponding author: [email protected]

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YU ET AL.

for replacing synthetic antioxidant to prevent the harmful effects on public health. The positive effects of star anise oil (SAO) have been reported widely like antimicrobial, antifungal, anti-inflammatory, antimycotoxigenic, and growth promoting effects (Jr et al., 2000; Ciftci et al., 2005; Aly et al., 2016). Most of the research work has demonstrated the extraction of SAO (Illicium verum) by different methods, constituent determination of the essential oil, and the effects of that on serum and liver antioxidant status of broilers. It is reported that antioxidant ability of spices may contribute to extending shelf life of products (Prakash et al., 2011). However, there has been little research on the star anise and its volatile oil as feed additives for enhancing laying performance and antioxidant status of serum, liver and extremely egg yolk in laying hens. The objectives of this study were to assay the practical efficacy of supplemented SAO on laying performance and antioxidant status of laying hens.

MATERIALS AND METHODS Preparation of Star Anise Oil The fresh star anise (approximately 400 kg) was obtained from Shenniu Animal Health Products Co. Ltd (Dezhou, Shandong, China) and dried at 65◦ C in a mechanical drier (DHG-9030A, Jingmi Instruments Co. Ltd., Shanghai, China). The dried star anise (Illicium verum) was smashed with Chinese herb micromill (ZSJB-30B, Create Mechanical Manufacturing Co. Ltd., Zhang jiagang, Jiangsu, China) to pass a 40 mesh screen and was stored in airtight plastic bags in the dark at room temperature for preparation of SAO. Essential oil was extracted from star anise using 1,000 kg capacity of gallipot in a 1:5 solid–liquid ratio after soaking 24 h by hydro-distillation (TN-1/250, Yikun Electric Co. Ltd., Wenzhou, Zhejiang, China). The distillate transferred to a separating funnel and the aqueous layer was discarded and the upper volatile oil layer was dried using anhydrous sodium sulfate. The star anise oil contained (per kilogram) 916.6 g of transanethole (NPC, 2010), 39.2 g of anisaldehyde (Ding et al., 2008), and 11.8 g of estragole (Siano et al, 2003) which was subjected to gas-chromatographic analysis. All the star anise oils were kept in glass bottles in the dark and stored at 4◦ C until use.

Experimental Animals, and Management The animal care and use protocol was reviewed and approved by Shandong Agricultural University Animal Nutrition Research Institute (Tai-an, Shandong, P.R. China, ANRI-2010-6). A total of 864 Hy-Line brown laying hens at 26 wk of age obtained from commercial farm were randomly allocated to 4 dietary treatments with 6 replicates of 36 laying hens for each diet (each with 12 cages, 3 birds/cage). Birds were fed mash corn–soybean meal based diets supplemented with 0,

200, 400, and 600 mg of above-prepared SAO/kg of diet for 56 d in 2 phases after 1 wk of adaptation. All experimental diets were isonitrogenous and isocaloric and were formulated to meet nutrient requirements recommended by NRC (1994) and its ingredients and compositions (Table 1). Star anise oil was first mixed with soybean oil that was subsequently mixed with other ingredients of each diet every 15 d and was stored in airtight plastic bags prior to feeding. All birds were randomly kept in an environmentally controlled room with ad libitum feeding and watering and with the temperature between 21◦ C and 26◦ C and 16 h/d of illumination (10 to 20 lx) throughout the entire experimental period. All feeding conditions among treatments were the same throughout the whole experiment. Mortality and health status were visually recorded daily to correct feed consumption throughout the whole experiment. Feed residues were collected and weighed weekly to estimate average daily feed intake (ADFI). Eggs from each replicate were counted and weighed daily for calculating average egg weight, daily egg mass, laying rate, and feed conversion.

Sample Collection On day 28 and 56 of the experiment, 12 birds (2 birds per replicate) were randomly selected from each treatment after fasting for 12 h and blood samples (5.0 mL) were taken from wing vein using sterilized needles into nonheparinized tube. After that, the blood samples were incubated at 37◦ C for 2 h, subsequently centrifuged at 1,500 × g for 10 min and the resultant serum was stored in 1.5-mL Eppendorf tube at −20◦ C for antioxygenation assay (Zhao et al., 2011). The birds after bleeding were slaughtered by cervical dislocation, removing the liver into 1.5-mL Eppendorf tube at −20◦ C, homogenized using pachinko with icecold physiological saline (0.9%; pH = 7.4) in proportion of 1:9 for 3 min by mechanical homogenizer (Q24RC, Xin beixi Biotechnology Co. Ltd., Jinan, Shandong, China) at ice-water bath condition, then stored the supernatants at −20◦ C for antioxidant status estimation after centrifuged at 3,000 × g for 10 min at 4◦ C (Ding et al., 2017). All of the samples were kept on ice during the preparation process. In addition, 12 eggs (2/replicate) were randomly collected from each treatment respectively on day 14, 28, 42, and 56 of the experiment. The yolk of each egg was manually isolated and subsequently, homogenized with ice-cold physiological saline (0.9%; pH = 7.4) in the ratio of 1:9 for 3 min with a vortex mixer (XH-D, Woxin Instrument Co. Ltd., Wuxi, China), the homogenates were centrifuged at 1,500 × g for 10 min at 4◦ C, subsequently stored the resultant supernatants at −20◦ C for total superoxide dismutase (T-SOD) assay, and then homogenized the yolk with absolute ethyl alcohol in proportion of 1:9 for 3 min with the same vortex mixer,

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STAR ANISE OIL ON LAYING PERFORMANCE AND ANTIOXIDATION Table 1. Ingredients and nutrient composition of the basal diets1 ( %). Dietary SAO concentration (mg/kg) Item Ingredient Corn Soybean meal (44% CP) Wheat bran Fish meal Soy oil Dicalcium phosphate Limestone Sodium chloride DL-Methionine Premix2 Star anise oil concentration Chemical composition, analyzed ME, calculated (kcal/kg) CP Calcium Total phosphorus Lys Met Thr Trp Met+Cys

0

200

400

600

60.00 21.50 2.00 2.10 3.00 1.50 8.50 0.30 0.10 1.00 –

60.00 21.50 2.00 2.10 2.98 1.50 8.50 0.30 0.10 1.00 0.02

60.00 21.50 2.00 2.10 2.96 1.50 8.50 0.30 0.10 1.00 0.04

60.00 21.50 2.00 2.10 2.94 1.50 8.50 0.30 0.10 1.00 0.06

2,864 16.46 3.65 0.73 0.92 0.35 0.63 0.19 0.65

2,864 16.46 3.65 0.73 0.92 0.35 0.63 0.19 0.65

2,864 16.46 3.65 0.73 0.92 0.35 0.63 0.19 0.65

2,863 16.46 3.65 0.73 0.92 0.35 0.63 0.19 0.65

1 Control group was fed the basal diet. The other treatment diets were the same basal diet supplemented with 200, 400, and 600 mg of star anise oil/kg of the basal diet. 2 Supplied per kilogram of diet: vitamin A, 12,200 IU; cholecalciferol, 4,200 IU; vitamin E, 30 IU; vitamin K3 , 4.5 mg; thiamin, 2.3 mg; riboflavin, 8.8 mg; pantothenic acid, 7 mg; pyridoxine, 4.0 mg; cobalamin, 0.016 mg; niacin, 30 mg; choline chloride, 650 mg; biotin, 0.20 mg; folic acid, 0.25 mg; Mn, 40 mg; Fe, 58 mg; Zn, 40 mg; Cu, 8 mg; I, 0.60 mg; Se, 0.30 mg.

centrifuged and stored the supernatants as the above method for determination of malondialdehyde (MDA) content (Zhao et al., 2011). On day 28 and 56 of the experiment, 60 eggs (10/replicate) of each treatment were randomly picked out, marked, and stored in plastic case respectively. The yolk of 12 eggs (2/replicate) of each treatment was manually isolated upon preparing the yolk samples subjected to the above methods every 14 d after until day 56 for determination of yolk antioxidant status at different store time.

Assay of Antioxidant Enzymes in Serum, Liver and in Egg Yolk The already prepared serum samples and liver supernatants were analyzed for antioxidant enzyme activities of T-SOD, glutathione peroxidase (GSH-PX) for total antioxidant capacity (T-AOC), and content of MDA using the same procedures as described by Zhang et al. (2009) with assay kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Activity of T-SOD is expressed as units per mL of serum and per mg protein of liver. The GSH-PX can accelerate the reaction of hydrogen peroxide and prototyping glutathione (GSH) to generate water and oxidizided glutathione (GSSG). Consumption of GSH was used to determine the GSH-PX activity at optical density (OD) 412 nm. The content of MDA was detected with thiobarbituric acid method, monitoring the change of absorbance at 532 nm with the spectropho-

tometer (UV-2000, UNICO Instruments Co., Ltd) as described by Placer et al. (1966). One unit of T-AOC activity was defined as the amount of enzyme per 1 mL serum or 1 mg liver protein that could increase OD by 0.01 at 37◦ C per minute and the detection of T-AOC activity referenced to Zhang et al.(2009). The egg yolk was analyzed for T-SOD activity and content of MDA with the same method as the serum assay.

Data Calculations and Statistical Analyses Daily egg mass was the multiplication of laying rate with average egg weight as grams per day per laying hen, laying rate was counted as ratio of total number of eggs to total number of laying hens as percentage, and feed conversion as ratio of ADFI relative to egg mass. All data were analyzed with ANOVA using the GLM procedure of SAS (SAS Institute, 2000) and as repeated measures with a model containing treatment and time. The data were analyzed as a completely randomized design with per replicate as random factor to examine the overall effect of treatments. The significance of differences among treatments was tested using Tukey’s HSD tests following single degree of freedom contrasts. All statements of significance were based on a probability of P < 0.05. Orthogonal polynomial (ORPOLY) contrasts using contrast coefficients were used to determine linear and quadratic responses of laying hens to SAO levels (SAS Institute, 2000). In addition, relative antioxidant status from dietary SAO concentration was determined, using non-supplemented

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YU ET AL. Table 2. Laying rate, average egg weight, egg mass, ADFI, and feed conversion of laying hens fed diets with different concentration of star anise oil supplementation.1 Dietary SAO concentration (mg/kg) 0

200

400

600

SEM2

With vs. without SAO

Linear

Quadratic

91.0 58.8 53.5b 129.6 2.43

91.7 58.8 54.0a,b 130.1 2.45

92.1 58.9 54.6a 131.1 2.44

92.0 59.1 54.4a 131.2 2.41

0.395 0.090 0.246 0.437 0.014

0.182 0.232 0.085 0.240 0.434

0.755 0.062 0.335 0.252 0.181

0.381 0.159 0.309 0.081 0.154

92.7 58.9 54.6 116.2 2.14

92.7 59.0 54.7 116.8 2.14

93.0 59.0 54.8 116.5 2.12

92.9 59.1 55.1 117.2 2.09

0.741 0.104 0.485 0.479 0.025

0.705 0.709 0.826 0.300 0.389

0.431 0.469 0.605 0.655 0.528

0.448 0.749 0.619 0.400 0.162

91.8 58.8 54.0 122.3b 2.28

92.2 58.9 54.3 122.4b 2.29

92.1 59.0 54.3 122.9a,b 2.29

92.8 59.1 54.7 124.1a 2.27

0.507 0.080 0.328 0.209 0.015

0.585 0.298 0.165 0.006 0.174

0.652 0.145 0.954 0.587 0.889

0.420 0.322 0.585 0.486 0.580

Item 1 to 28 d Laying rate (%) Average egg weight (g) Egg mass (g/d) ADFI (g/d) Feed conversion (g:g) 29 to 56 d Laying rate (%) Average egg weight (g) Egg mass (g/d) ADFI (g/d) Feed conversion (g:g) 1 to 56 d Laying rate (%) Average egg weight (g) Egg mass (g/d) ADFI (g/d) Feed conversion (g:g)

Effect (P-value)

Means within a row with different superscripts tended to be differ (0.05 < P < 0.10). Data are means for 6 replicates of 36 laying hens/replicate. 2 SEM, standard error of the means. a,b 1

SAO group as the standard concentration by multiple linear regression and a slope ratio method (Littell et al., 1995; Spears et al., 2004). Total superoxide dismutase activity and MDA concentration of egg yolk on day 0, 14, 28, 42, and 56 of the storage time after laying hens fed diets for 28 or 56 d respectively were used in the multiple linear regression analysis.

RESULTS

(P < 0.01) or quadratically (P < 0.01) increased the activities of T-AOC and GSH-PX at day 56, T-SOD activity at day 28 and day 56 in serum. All treatments had common MDA concentration in serum of laying hens at day 28 and day 56 of the experiment. In addition, sampling time has significant effects on activities of T-SOD (P = 0.005), T-AOC (P = 0.033), and GSH-PX (P < 0.001). Supplementation with 600 mg SAO/kg of diet of laying hens appeared highest serum antioxidant status.

Laying Performance Every laying hen was healthy and no mortality occurred during the whole experimental period (data not shown). Egg mass of laying hens tended to be improved (P = 0.085) from day 1 to day 28 and the ADFI was increased (P < 0.01) from day 1 to day 56 of the experiment by dietary supplementation of SAO (Table 2). Increasing concentration of SAO from 200 to 600 mg/kg of diet, tended to linearly increased (P = 0.062) average egg weight and quadratically increased (P = 0.081) ADFI at day 1 to day 28. There were no significant differences in ADFI, average egg weight, egg mass, laying rate, and feed conversion of laying hens in day 29 to day 56 or the entire period of the experiment.

Serum Antioxidant status Without consideration of content, laying hens consuming diets with SAO had higher (P < 0.001) serum activities of T-SOD and GSH-PX at day 28 and day 56 of the experiment, and T-AOC at day 56 of the experiment compared with that of control (Table 3). As compared with the control treatments, addition of SAO in the laying hens diets from 200 to 600 mg/kg linearly

Liver Antioxidant status Regardless of the supplementation level, laying hens fed diets with SAO increased activities of T-AOC (P < 0.001) at day 56 and GSH-PX (P < 0.01) at day 28 and day 56 of the experiment, reduced (P < 0.001) MDA concentration at day 28 and day 56 of the experiment in liver (Table 4). Increasing SAO content from 200 to 600 mg/kg of diet of laying hens, linearly or quadratically increased activity of T-AOC (P < 0.001) at day 56 and GSH-PX (P < 0.05) at day 28 and day 56, decreased MDA content at day 28 (P < 0.05) and day 56 (P < 0.001), and tended to linearly (P = 0.058) increase the activity of T-SOD at day 28 of the experiment in the liver. Activities of T-SOD, T-AOC, and GSH-PX of laying hens in the liver were significant affected (P < 0.001) by sampling time. In conclusion, the highest antioxidant status was recorded in the supplementation 600 mg/kg diet.

Egg Yolk Antioxidant status The effects of laying hens fed diets with different concentration of SAO to day 0, 14, 28, 42, and

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STAR ANISE OIL ON LAYING PERFORMANCE AND ANTIOXIDATION Table 3. Antioxidant enzymatic activity and concentration of MDA in the serum of laying hens fed diets with different concentration of star anise oil supplementation on day 28 and 56 of the experiment.1 Dietary SAO concentration (mg/kg) Item2 28 d4 T-SOD (U/mL) MDA (nmol/mL) T-AOC (U/mL) GSH-PX (U/mL) 56 d4 T-SOD (U/mL) MDA (nmol/mL) T-AOC (U/mL) GSH-PX (U/mL)

Effects (P-value)

0

200

400

600

SEM3

With vs. without SAO

415.2c 5.43 7.19 5,365b

444.2b 5.39 7.15 6,194a

454.4a,b 5.4 7.23 6,286a

463.9a 5.37 7.4 6,397a

1.796 0.209 0.134 54.755

< 0.001 0.849 0.919 < 0.001

0.007 0.434 0.562 0.223

0.004 0.732 0.843 0.486

449.5c 5.42 7.23b 13,520c

457.9b,c 5.39 7.43b 17,920b

463.7b 5.41 10.29a 19,520a,b

489.9a 5.4 10.36a 20,267a

2.068 0.14 0.101 239.652

< 0.001 0.363 < 0.001 < 0.001

< 0.001 0.291 < 0.001 0.001

< 0.001 0.581 < 0.001 0.005

Linear

Quadratic

Means within a row with different letters differ (P < 0.05). Data are means for 6 replicates of 2 laying hens/replicate. 2 T-SOD, Total superoxide dismutase; MDA, Malondialdehyde; T-AOC, Total antioxidant capacity; GSH-PX, Glutathione peroxidase. 3 SEM, standard error of the means. 4 SAO supplemented dose × time interaction (28 d vs. 56 d): T-SOD (P = 0.005); MDA (P = 0.488); T-AOC (P = 0.033); GSH-PX (P < 0.001). a–c 1

Table 4. Antioxidant enzymatic activity and concentration of MDA in the liver of laying hens fed diets with different concentration of star anise oil supplementation on day 28 and 56 of the experiment.1 Dietary SAO concentration (mg/kg) Item2

Effects (P-value)

0

200

400

600

SEM3

With vs. without SAO

31.9 1.86a 1.37 18.5c

31.9 1.43b 1.39 19.9b,c

32.6 1.35b 1.40 20.5b

32.9 1.31b 1.43 22.8a

0.168 0.017 0.020 0.366

0.116 < 0.001 0.769 0.004

0.058 0.024 0.591 0.019

0.148 0.079 0.862 0.047

40.3 2.04a 2.41d 42.3c

40.6 2.02a 2.58c 52.0b

40.0 1.61b 2.82b 52.5a,b

40.3 1.27c 2.95a 56.5a

0.475 0.023 0.014 0.709

0.983 < 0.001 < 0.001 < 0.001

0.858 < 0.001 < 0.001 0.015

0.926 < 0.001 < 0.001 0.026

Linear

Quadratic

4

28 d T-SOD (U/mg of protein) MDA (nmol/mg of protein) T-AOC (U/mg of protein) GSH-PX (U/mg of protein) 56 d4 T-SOD (U/mg of protein) MDA (nmol/mg of protein) T-AOC (U/mg of protein) GSH-PX (U/mg of protein)

Means within a row with different letters differ (P < 0.05). Data are means for 6 replicates of 4 laying hens per replicate. 2 T-SOD, Total superoxide dismutase; MDA, Malondialdehyde; T-AOC, Total antioxidant capacity; GSH-PX, Glutathione peroxidase. 3 SEM, standard error of the means. 4 SAO supplemented dose × time interaction (28 d vs. 56 d): T-SOD (P < 0.001); MDA (P = 0.106); T-AOC (P < 0.001); GSH-PX (P < 0.001). a-d 1

56 of the experiment on the activity of T-SOD and concentration of MDA in the yolk were shown (Tables 5 and 6). Dietary supplementation of SAO increased (P < 0.01) activity of T-SOD, reduced (P < 0.05) MDA concentration at day 14, day 28, day 42, and day 56 of the experiment in yolk of laying hens compared with the control treatment (Table 5). Increasing content of SAO from 200 to 600 mg/kg of diet, linearly increased (P < 0.05) activity of T-SOD at day 14 and day 28, quadratically increased (P < 0.001) activity of T-SOD at day 14, and linearly (P < 0.05) or quadratically (P < 0.05) decreased MDA content at day 42 and day 56 of the experiment in yolk of laying hens. The T-SOD activity and MDA content in yolk of laying hens were affected (P < 0.01) by sampling time. Antioxidant status of egg yolk in supplemental SAO group relative to the control treatments was estimated from activity of T-SOD and content of MDA on day

0, 14, 28, 42, and 56 of the experiment using multiple linear regression and a slope ration method. Compared with the diet supplemented 0 mg/kg of SAO (1.00), relative T-SOD activity was 2.69, 2.76, and 2.70 (P < 0.001) and relative content of MDA was 9.21, 10.97, and 11.67 (P < 0.001), for the supplementation of 200, 400, and 600 mg/kg of SAO, using slope ratios for T-SOD and MDA, respectively (Table 6). The effects of the eggs of laying hens fed diets with different concentration of SAO for 28 d and 56 d were stored to day 0, 14, 28, 42, and 56, on the activity of T-SOD and concentration of MDA in the yolk were manifested (Tables 7 and 8). As compared with the control treatments, laying hens fed the SAO supplemented diets for 28 or 56 d had higher (P < 0.01) TSOD activity and lower (P < 0.05) MDA concentration in yolk of eggs stored on day 0, 14, 28, 42, and 56, respectively.

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YU ET AL. Table 5. The activity of T-SOD and concentration of MDA in the yolk of laying hens fed diets with different concentration of star anise oil supplementation on day 0, 14, 28, 42, and 56 of the experiment.1 Dietary SAO concentration (mg/kg) Item2

0

T-SOD (U/mL) 0 d4 99.7 14 d 100.8c 28 d 106.1d 42 d 108.7b 56 d 109.4b MDA (nmol/mL) 0 d4 104.7 14 d 104.1a 28 d 103.5a 42 d 103.4a 56 d 102.4a

Effects (P-value)

200

400

600

SEM3

With vs. without SAO

Linear

Quadratic

99.6 104.7b,c 113.5b,c 124.2a 126.7a

99.7 119.9a,b 123.0a,b 128.9a 132.8a

99.7 125.9a 127.0a 132.7a 133.2a

4.225 4.328 4.427 3.624 2.171

0.989 0.003 0.007 0.001 < 0.001

0.835 < 0.001 0.030 0.097 0.061

0.916 < 0.001 0.080 0.255 0.105

104.7 95.1a,b 86.5b 85.2b 84.5b

104.8 87.6a,b 83.1b 81.3b 77.9b

104.7 86.7b 83.8b 78.0b 77.2b

4.396 4.375 3.726 3.670 3.345

0.965 0.026 0.008 < 0.001 < 0.001

0.763 0.242 0.280 0.001 0.023

0.842 0.442 0.362 0.007 0.044

Means within a row with different letters differ (P < 0.05). Data are means for 6 replicates of 4 laying hens per pen. 2 T-SOD, Total superoxide dismutase; MDA, Malondialdehyde. 3 SEM, standard error of the means. 4 SAO supplemented dose × time interaction: T-SOD (P < 0.001); MDA (P < 0.01). a–d 1

Table 6. Estimated relative antioxidant status of dietary star anise oil concentration, based on multiple linear regression of T-SOD and MDA in the yolk of laying hens.

Dependent variable1 T-SOD (U/mL)

MDA (nmol/mL)

Dietary SAO concentration (mg/kg)

Slope ± S.E.

P-value2

0 200 400 600 0 200 400 600

0.195 ± 0.102 0.525 ± 0.090 0.539 ± 0.077 0.527 ± 0.084 − 0.039 ± 0.119 − 0.359 ± 0.082 − 0.428 ± 0.087 − 0.455 ± 0.058

< 0.001

Relative activity or content

< 0.001

1.00 2.69 2.76 2.70 1.00 9.21 10.97 11.67

T-SOD, Total superoxide dismutase; MDA, Malondialdehyde. 1 Day 0, 14, 28, 42, and 56 values of the experiment were used in regression analysis. 2 P-value for slope differences among star anise oil concentration.

Ranging SAO content from 200 to 600 mg/kg of diet, T-SOD activity was linearly (P < 0.05) increased in yolk of eggs stored on day 0, 14, 28, 42, and 56, and tended to quadratically (P < 0.10) increased stored on day 0, 14, 28, and 42, yet MDA concentration was linearly decreased (P < 0.05) stored on day 42 and 56 in yolk of eggs of laying hens fed the SAO supplemented diets for 28 d. The activity of T-SOD in yolk tended to be linearly increased stored on day 0 (P = 0.061), day 14 (P = 0.059), and day 28 (P = 0.080), the content of MDA was linearly (P < 0.05) decreased stored on day 0, 14, 28, 42, and 56 in yolk of laying hens fed the SAO supplemented diets for 56 d, as the concentration of SAO increased from 200 to 600 mg/kg of diet. The T-SOD activity and MDA content in yolk of laying hens were affected (P < 0.001) by the eggs stored time whether laying hens fed diets for 28 d or 56 d, and there was significant difference on T-SOD activity (P = 0.032) and same MDA concentration (P = 0.668) in yolk of stored eggs of laying hens fed diets for 28 d vs. 56 d.

As shown in Table 8, antioxidant status in yolk of laying hens fed diets with different concentration of SAO for 28 d and 56 d relative to the control treatments was estimated from activity of T-SOD and content of MDA in the eggs stored to day 0, 14, 28, 42, and 56, respectively, using multiple linear regression and slope ratios method. Compared to non-supplemented group (1.00), relative T-SOD activity was increased (P < 0.001), yet relative MDA content decreased (P < 0.001) using slope ratios respectively in the yolk of laying hens fed diets for 28 or 56 d as the content of SAO increased from 200 to 600 mg/kg of diet. This indicated that dietary supplementation of SAO may extend the egg storage time by increasing antioxidant status of egg yolk.

DISCUSSION Effect of Star Anise Oil on Laying Performance of Laying Hens The star anise and SAO may be widely used for growth promoting in poultry as a result of stimulating

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STAR ANISE OIL ON LAYING PERFORMANCE AND ANTIOXIDATION Table 7. The analysis of activity of T-SOD and concentration of MDA of the yolk eggs stored at different time after laying hens fed diets with different concentration of star anise oil supplementation for 28 d and 56 d.1 Dietary SAO concentration (mg/kg)

Item2

0

200

400

600

Effects (P-value) With vs. without SAO

SEM3

Linear

Quadratic

0.007 0.005 0.002 0.009 0.009

0.027 0.033 0.010 0.025 0.048

0.077 0.088 0.075 0.065 0.222

0.008 0.013 0.008 0.006 0.005

0.280 0.397 0.164 0.045 0.001

0.362 0.547 0.183 0.155 0.248

< 0.001 < 0.001 0.001 0.007 0.001

0.061 0.059 0.080 0.123 0.269

0.105 0.124 0.210 0.292 0.491

< 0.001 < 0.001 0.008 0.001 < 0.001

0.023 0.039 0.032 0.031 0.001

0.044 0.392 0.585 0.032 0.006

4

Eggs stored for 56 d after laying hens fed star anise oil diets for 28 d T-SOD (U/mL) 0d 106.1b 113.5a,b 123.0a 127.0a 4.427 14 d 94.3b 99.1a,b 108.3a 111.4a 3.603 28 d 85.1b 88.2b 94.6a,b 98.8a 2.566 81.1a,b 88.1a,b 90.3a 2.865 42 d 77.6b 56 d 58.4c 59.1b,c 61.8a,b 63.1a 1.671 MDA (nmol/mL) 0d 103.5a 86.5b 83.1b 83.8b 3.726 14 d 107.3a 95.1a,b 87.6b 88.9b 4.484 28 d 132.5a 119.5a,b 112.9b 107.2b 5.162 42 d 150.9a 128.4a,b 120.2a,b 110.9b 12.149 56 d 195.0a 180.8a,b 157.2b,c 148.4c 9.683 Eggs stored for 56 d after laying hens fed star anise oil diets for 56 d4 T-SOD (U/mL) 0d 109.4b 126.7a 132.9a 133.2a 2.171 b a a 112.2 113.0 118.6a 2.513 14 d 100.7 28 d 98.5b 107.2a,b 110.1a 114.6a 2.738 42 d 88.9b 99.8a,b 102.1a,b 107.4a 3.636 56 d 69.9b 80.5a 84.4a 84.9a 2.766 MDA (nmol/mL) 0d 102.4a 84.5b 77.9b 77.2b 3.345 14 d 105.8a 90.9b 86.2b 87.9b 2.436 104.3b 101.5b 96.1b 6.275 28 d 126.4a 42 d 149.2a 123.9b 118.1b 104.7b 7.313 178.6a,b 155.5b,c 138.7c 8.466 56 d 199.6a

Means within a row with different letters differ (P < 0.05). Data are means for 6 replicates of 4 laying hens per pen. 2 T-SOD, Total superoxide dismutase; MDA, Malondialdehyde. 3 SEM, standard error of the means. 4 Eggs stored at different time after laying hens fed star anise oil diets for 28 d: T-SOD × time interaction (P < 0.001); MDA × time interaction (P < 0.001); Eggs stored at different time after laying hens fed star anise oil diets for 56 d: T-SOD × time interaction (P < 0.001); MDA × time interaction (P < 0.001); Stored eggs after fed diets for 28 d vs. 56 d: T-SOD (P = 0.032); MDA (P = 0.668). a–c 1

digestion and antimicrobial effects (Ciftci et al., 2005; Kassie, 2008). The high active ingredient in star anise such as trans-anethole has positive effects on nutrient digestibility (Jamroz and Kamel, 2002), and increasing activities of pancreatic lipase and amylase (Ramakrishna et al., 2003) of broilers. Broilers (Ross-308) supplemented with 400 mg of SAO/kg of diets had higher average daily gain by 15% in comparison with that in control group (Ciftci et al., 2005). Bayram et al. (2007) showed that laying performance of laying hens such as feed intake, average egg weight, and feed conversion rate was improved by supplemented star anise seed at 40 g/kg. The result of this study demonstrated that egg mass tended to be improved, average egg weight tended to be linearly increased, and ADFI tended to be quadratically increased by the addition of SAO in the laying hens diets from 200 to 600 mg/kg for 28 d, and the overall ADFI was increased by SAO supplementation for 56 d. However, all treatments had same laying rate and feed conversion. It appeared that higher egg mass may be due to the increased average egg weight by addition of SAO into diet. The increased ADFI is

likely attribute to a stimulating effect of appetite as a result of aromatic and anise flavor (Wang et al., 2011), besides the antimicrobial effects of SAO and stimulating effect on digestive system such as increasing the secretions of digestive enzymes, intestinal mucous, and stabilize microbial balance in the gut (Lee et al., 2001; Mansoub, 2011). Various factors like animal age, environmental factors, diet type, and so on could influence the efficacy of volatile oil of star anise (Amad et al., 2011).

Effect of Star Anise Oil on Antioxidant Status in Serum and Liver of Laying Hens Changes of antioxidant enzyme activities and some reduced nonenzymatic antioxidants can lead to oxidative stress. Normal biological processes continuously produce reactive oxygen species, which can be obliterated by the body’s antioxidant enzymes, including SOD, GSH-PX, and some protective compounds (Bou et al., 2009). It is well recognized that antioxidant

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YU ET AL. Table 8. Estimated relative antioxidant status of dietary star anise oil concentration, based on multiple linear regression of T-SOD and MDA in the yolk of laying hens. Dependent variable1

Dietary star anise oil concentration (mg/kg)

Slope ± S.E.

Eggs stored for 56 d after laying hens fed star anise oil diets for 28 d T-SOD (U/mL) 0 − 0.801 ± 0.073 200 − 0.905 ± 0.082 400 − 1.018 ± 0.062 600 − 1.064 ± 0.075 MDA (nmol/mL) 0 1.470 ± 0.217 200 1.584 ± 0.124 400 1.291 ± 0.122 600 1.079 ± 0.135 Eggs stored for 56 d after laying hens fed star anise oil diets for 56 d T-SOD (U/mL) 0 − 0.649 ± 0.068 200 − 0.748 ± 0.067 400 − 0.771 ± 0.064 600 − 0.768 ± 0.069 MDA (nmol/mL) 0 1.699 ± 0.184 200 1.579 ± 0.171 400 1.336 ± 0.096 600 0.998 ± 0.127

P-value2 < 0.001

< 0.001

< 0.001

< 0.001

Relative activity or content 1.00 1.13 1.27 1.33 1.00 1.08 0.88 0.73 1.00 1.15 1.19 1.18 1.00 0.93 0.79 0.59

T-SOD, Total superoxide dismutase; MDA, Malondialdehyde. 1 Day 0, 14, 28, 42, and 56 values of the storing time after laying hens fed star anise oil diets for 28 d or 56 d were used in regression analysis. 2 P-value for slope differences among star anise oil concentration.

status of poultry can be manifested by measuring 4 main antioxidant enzymes including T-SOD, MDA, T-AOC, and GSH-PX. Total superoxide dismutase and GSH-PX are natural scavengers to free radical via converting oxygen radicals to hydrogen peroxide to eliminate the damaging reactive oxygen species from the cellular environment (Fattman et al., 2003). Malondialdehyde has been widely used as biomarker for the evaluation of lipid peroxidation in biological process, although it is believed to be associated with free radical damage with increase in various diseases (Koracevic et al., 2001). Overall antioxidant defense systems including enzymatic and nonenzymatic can be indicated by T-AOC, the higher T-AOC in laying hens supplemented with SAO reflects greater antioxidant defense. The value of antioxidant enzyme and MDA in serum of laying hens measured in this study was close to reported for the same breed, age, and feeding conditions (Zhao et al., 2011). The findings observed in this study, laying hens fed the diets supplemented of SAO from 200 to 600 mg/kg for 28 d and 56 d, increased activities of GSH-PX and T-AOC in serum and liver, increased activities of T-SOD in serum and decreased MDA content in liver was in accordance with Ding et al. (2017), who also observed increased activities of T-SOD, and GSH-PX in serum, reduced MDA content and increased T-SOD and GSH-PX activities in liver of broilers supplemented SAO at the level of 200 mg/kg of diet at 21 and 42 d of age. The T-AOC capacity was interestingly increased in serum and liver of laying hens of the experiment by supplementation of SAO indicated the enhanced antioxidant status. Supplementation of SAO to the diet of laying hens indicated higher capacity in clearing out the reactive oxygen species by increasing the activities of SOD and GSH-PX and reduced MDA content in this study. This indicated that dietary sup-

plementation of SAO may improve antioxidant defense of laying hens by increasing antioxidant status of serum and liver. Alhajj et al. (2017) also observed broilers fed diets containing 6 and 8 g of star anise/kg had higher T-AOC activities in serum at 42 d compared to control group, as well as in liver tissue at 21 and 42 d of age. Although star anise has been used as a spice and herbal medicine for many years, no research reported its effects on laying hens as feed additive. Wong et al. (2014) and Aly et al. (2016) demonstrated that using the stable free radical-2, 2-diphenyl-1-picrylhydrazyl (DPPH) measured the antioxidant activity of SAO by radical scavenging ability with the method of Brandwilliams et al. (1995). The major compounds contained in SAO such as trans-anthole, limonene, and estragole may contribute to the enhanced antioxidant status of laying hens (Padmashree et al., 2007; Zhai et al., 2009).

Effect of Star Anise Oil on Antioxidant Status in Yolk of Laying Hens This study showed that the T-SOD activity was increased but the MDA content was decreased in yolk of laying hens by supplementation of SAO. This indicated that the essential oil enhanced the antioxidant status of yolk. Furthermore, It was observed that laying hens supplemented SAO at the rate of 600 mg/kg appeared highest antioxidant status in yolk. The radical scavenging effects of the SAO on the β -carotene radical were dose-dependent observed by Aly et al. (2016) and a dose-dependent increase of egg yolk antioxidant status in response to dietary antioxidants in poultry (Sahin et al., 2008) may explain the findings. An interesting finding of this study was decreased TSOD activity and increased MDA content when stored

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STAR ANISE OIL ON LAYING PERFORMANCE AND ANTIOXIDATION

eggs from 0 to 56 d in all groups of the experiment, yet the SAO supplemented group had higher T-SOD activity and lower MDA content in comparison with that in control. This indicated that SAO may extend the store time of eggs by enhancing the antioxidant status of yolk. Trans-anethole containing over 90% of compounds is the major component of SAO (Domiciano et al., 2013). It was observed that the antioxidant activity of SAO measured using DPPH method was comparable with the BHT (synthetic antioxidant), especially extracting the essential oils at 50◦ C and 60◦ C (Wong et al., 2014). Information on the impact of the oil on antioxidant status of yolk in laying hens is limited. The enhanced antioxidant status in yolk is likely due to trans-anethole, estragole, limonene, linalool, and cis-anethole contained in SAO (Dzamic et al., 2009). Prakash et al. (2011) reported that antioxidant ability of spices may contribute to extending shelf life of products. Egg of laying hens containing high fat results in shorting shelf life and the mechanism of antioxidants was scavenging free radical to prevent lipid oxidation and rancidity. As a consequence, star anise essential oil used as natural antioxidants added to laying hens feed may be beneficial in extending shelf life of eggs.

CONCLUSIONS AND IMPLICATION Supplementation of SAO at the level of 200 to 600 mg/kg to diet tended to improve ADFI and egg mass of laying hens without affecting laying rate and or feed conversion rate. Inclusion of SAO in the diet at these levels also enhanced oxidative stability in serum, liver, and yolk of laying hens. Dietary supplementation of SAO may extend the egg storage time by increasing antioxidant status of egg yolk. This study showed that star anise essential oil used as an antioxidant added to laying hens feed may be beneficial in improving antioxidant defense of laying hens and extending shelf life of eggs.

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