Optimization of dietary zinc for egg production and antioxidant capacity in Chinese egg-laying ducks fed a diet based on corn-wheat bran and soybean meal

Optimization of dietary zinc for egg production and antioxidant capacity in Chinese egg-laying ducks fed a diet based on corn-wheat bran and soybean meal W. Chen,∗,†,‡,§,# S. Wang,∗,†,‡,§,# H. X. Zhang,∗,†,‡,§,# D. Ruan,∗,†,‡,§,# W. G. Xia,∗,†,‡,§,# Y. Y. Cui,∗,†,‡,§,# C. T. Zheng,∗,†,‡,§,#,1 and Y. C. Lin∗,†,‡,§,#,1 ∗

Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China; † State Key Laboratory of Livestock and Poultry Breeding, Guangzhou 510640, China; ‡ Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture, Guangzhou 510640, China; § Guangdong Public Laboratory of Animal Breeding and Nutrition, Guangzhou 510640, China; and # Guangdong Key Laboratory of Animal Breeding and Nutrition, Guangzhou 510640, China take did not differ among the groups of graded Zn supplementation. The egg quality was not affected by dietary Zn, including the egg shape index, Haugh unit, yolk color score, egg composition, and shell thickness. The activities of plasma activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) increased in a quadratic manner (P < 0.001) with increasing supplemental Zn. Plasma concentration of Zn increased quadratically (P < 0.05) as dietary Zn increased. The hepatic activity of Cu/Zn-SOD and GSHPX increased quadratically (P < 0.05) with increasing dietary Zn. Plasma Zn concentrations were positively correlated with activities of T-SOD (P < 0.05), and positively with plasma Cu. Plasma concentration of reduced glutathione was correlated with plasma Cu. In conclusion, supplementation of Zn at 30 or 45 mg/kg to a corn-wheat bran and soybean basal diet may improve the productive performance and enhance the antioxidant capacity.

ABSTRACT The aim of this study was to evaluate the effect of zinc supplementation on productive performance and antioxidant status in laying ducks. Five-hundred-four laying ducks were divided into 7 treatments, each containing 6 replicates of 12 ducks. The ducks were caged individually and fed a corn-soybean meal and wheat bran basal diet (37 mg Zn/kg) or the basal diet supplemented with 15, 30, 45, 60, 75, or 90 mg Zn/kg (as zinc sulfate). During the early laying period of 10 d (daily egg production <80%), egg production, daily egg mass, and FCR increased quadratically with increasing dietary Zn levels (P < 0.05). The highest egg production and daily egg weight were obtained when 30 or 45 mg Zn/kg diet was supplemented, with lowest FCR. Similarly, the highest egg production and daily egg mass were observed in the group supplemented with 30 or 45 mg Zn/kg during the peak laying period of the subsequent 120 d (daily egg production >80%). Average egg weight and feed in-

Key words: laying ducks, zinc, superoxide dismutase, glutathione peroxidase 2017 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pex032

INTRODUCTION

eral biochemical and different clinical manifestations of Zn deficiency have been reported in birds. For example, Zn deficiency in chickens causes loss of appetite, reduces efficiency of feed utilization (Ensminger et al., 1990), and inhibits bone growth (Wang et al., 2002) and, thus, leads to growth retardation (Morrison and Sarett, 1958; Reed et al., 2015). A Zn-deficient breeder diet can lead to decreased egg production, eggshell quality, and hatchability (Kienholz et al., 1961). Embryos of Zn-deficient eggs have skeletal abnormalities, and the hatched chicks might be unable to stand, eat, or drink (Van Campen and Scaife, 1967; Seeling et al., 1975). Toxicity also was observed, however, in chickens fed excessive zinc, as reflected in reduced egg production (Kienholz et al., 1961; Shippee et al., 1979) or growth (Roberson and Schaible, 1960).

An important function of Zn is its participation in the antioxidant defense system. Zinc deficiency increases oxidative damage of cell membranes caused by free radicals (Oteiza et al., 1996; Prasad et al., 2009). The mechanism by which Zn exerts antioxidant function is probably through its being a co-factor of the antioxidant enzyme superoxide dismutase (SOD), thereby maintaining protein sulfydryl groups (Bray and Bettger, 1990; Oteiza et al., 1996; Powell, 2000). Sev C 2017 Poultry Science Association Inc. Received May 18, 2016. Accepted February 24, 2017. 1 Corresponding author: [email protected]; [email protected]

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An optimal dietary supply of Zn is needed, therefore, for productive performance and maximizing profitability in the poultry industry. The NRC (1994) recommends a level of 25 to 70 mg Zn/kg in various poultry diets but the existing data are limited to chicken layers and broilers, turkey, quail, and meat ducks. No research has been reported for laying ducks in the last decade, despite their increasing numbers and importance in Asia where more than 4 million tonnes of eggs are produced annually. Based on the requirements of crude protein and metabolic energy for laying ducks established in this laboratory, diets containing about 37 mg Zn/kg diet seem to be sufficient for laying ducks, as the NRC (1994) recommends 35 to 39 mg Zn/kg for Leghorn-type laying hens. It is not known if this level of Zn is optimal nor if supplementation of typical diets can improve the productive performance of laying ducks with respect to the physiological differences between landfowl and waterfowl. The objective of the present study, therefore, was to evaluate the effect of dietary zinc supplementation on the productive performance and antioxidant capacity in egg-laying ducks, by employing an unsupplemented basal diet containing 37 mg Zn/kg, similar to that recommended in laying hens, and 6 graded levels of supplemental Zn.

MATERIALS AND METHODS Animal Management and Diet This experiment started in August and ended in January the following year in South China. The temperature varied from 15◦ C to 33◦ C with the seasons during the experimental period. All animal care procedures were approved by the institutional animal care and use committee of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences. This trial used a randomized complete block design with 7 dietary treatments. The treatments consisted of a nonsupplemented basal diet (control) and that diet supplemented with 6 graded concentrations (ranging from 15 to 90 mg/kg) of Zn (added as ZnSO4 .H2 O). The basic feed was a typical corn-wheat bran and soybean meal diet (Table 1) analyzed to contain 37.4 mg/kg Zn (Table 1). Supplementation with 15, 30, 45, 60, 75, or 90 mg/kg Zn resulted in 53.61, 64.55, 84.39, 91.16, 112.59, or 131.35 mg Zn/kg respectively, as measured by flame atomic absorption spectrophotometry (AOAC method 986.15, 2006). Five-hundred-four Longyan female ducks (Anas platyrhynchos, a typical breed of laying ducks in South China) at 23 wk of age, 1.36 ± 0.15 kg, were allocated to one of 7 treatments, each consisting of 6 replicate cages of 12 ducks. Each replicate consisted of 3 floors of 4 adjacent cages and each floor shared a deep feed trough enabling individual feeding of the 4 birds. Individual cages were 30 cm wide × 35 cm deep × 35 cm high. All ducks were fed the basal diet during an initial adaptation period of 3 wk after which all had normal behavior

Table 1. Composition and nutrient levels of the basal diet (air-dry basis, %). Ingredients Corn Soybean meal Wheat bran Limestone CaHPO4 NaCl L-Lys.HCl DL-Met Premix1 Total Calculated composition2 ME, MJ/kg CP3 , % Ca3 , % Total P, % Available P, % Lys, % Met, % Met+Cys, % Arg, % Trp, % Thr, % Zn3 , mg/kg Phytic acid4 , g/kg

% 55.5 22.8 10.4 8.45 1.38 0.30 0.02 0.15 1.00 100 10.5 17.3 3.40 0.62 0.35 0.86 0.40 0.67 1.14 0.26 0.64 37.4 12.1

1 Vitamin-trace mineral premix provided the following minerals in milligrams per kilogram of diet: Fe, 52; Cu, 10.4; Mn, 91; Se, 0.20; I, 0.52; Co, 0.26; and the following vitamins per kg of diet: thiamine, 3.0 mg; riboflavin, 9.6 mg; niacinamide, 114 mg; D-pantothenic acid, 28.5 mg; choline chloride, 500 mg; cobalamin, 30 μ g; menadione, 0.96 mg; DL-α -tocopheryl acetate, 6 IU; Vitamin A, 12,000 IU; cholecalciferol D3 , 1,800 IU. 2 Metabolizable energy, available phosphorus, lysine, methionine, methionine, and cysteine were calculated from the chemical analysis of dietary ingredients. 3 Crude protein (CP) and Ca were determined by analysis. Zn content in the basal diet was analyzed by flame atomic absorption spectrophotometry. 4 Phytic acid content in the basal diet was calculated from the ingredients.

and egg production of 30 to 60% (average 55%). Ducks were ranked on initial performance then randomly distributed to the different dietary treatments to obtain similar groups. The experiment consisted of a 10-day early laying period (production 50 to 80%) then a 120day peak laying period when production exceeded 80%. Ducks were provided a quantity of feed meeting their requirement throughout the adaptation and experimental periods; the amount was gradually increased during the early and peak laying period. The daily allowance of feed (average 152 g in 2 feedings at 08:00 and 16:00) was the maximum for individuals without their leaving refusals. Residual feed was measured at 07:00 and used to calculate the previous day’s feed intake. The ducks had ad libitum access to water, and a 17-hour daily photoperiod was provided throughout. The egg numbers and egg weight of each replicate were recorded daily and those laying or not each d were recorded individually. Feed conversion ratio (FCR) was calculated as grams of feed per gram of egg mass. The average daily egg production of all ducks ranged from 68 to 79% during the early laying period and from 83 to 91% for the peak laying period.

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Sample Collection After 120 d of peak laying, 2 ducks within each replicate, with similar BW of 1.26 ± 0.16 kg, were randomly selected for blood and tissue sampling at 9:00 a.m. Five mL of blood were collected from the brachial vein using heparinized tubes (BD Vacutainer Systems, Franklin Lakes, NJ). Blood was centrifuged (3,000 × g for 15 min at 4◦ C) to obtain plasma, which was stored (-80◦ C) in aliquots for later determination of activities of enzymes and concentrations of Cu2+ and Zn2+ ions. After blood sampling, the ducks were killed by cervical dislocation. Liver samples were excised, washed with 0.9% isotonic saline, snap-frozen in liquid nitrogen, and stored at -80◦ C. The whole ovary was dissected and weighed, then the number and weight of large follicles (diameter >1 cm) were recorded for each duck. The right and left tibias of each duck were excised from the fresh carcass, weighed, and circumferences were recorded. The bone breaking strength was determined (Chen et al., 2015) using a Material Testing Machine (Instron Corp., Grove City, PA). Three eggs within each of the 6 replicates were sampled every month to assess egg quality indices (shape index, shell breaking strength, shell thickness, Haugh unit, yolk color, and weights of yolk, albumen, and shell.

Measurement of Malondialdehyde in Plasma and Liver Malondialdehyde (MDA) was measured as thiobarbituric acid-reacting substances (TBARS), as described by Ramaekers et al. (1997). Briefly, 100 μL of plasma were shaken with 100 μL 20% trichloroacetic acid (TCA) in a centrifuge tube and one mL of a solution containing 29 mmol/L thiobarbituric acid in 8.75 mol/L acetic acid was added to the mixture. Samples were heated at 95◦ C for 40 min in a water bath. After cooling, the mixture was centrifuged (2,000 × g for 10 min at 20◦ C) and absorbance of the supernatant was recorded at a wavelength of 535 nm in a spectrophotometer (Spectramax M5, Molecular Devices, Sunnyvale, CA). The total MDA content was determined based on the MDA standards. Liver samples were homogenized in 9 volumes of 0.9% NaCl. After centrifuging at 4,000 × g for 10 min at 4◦ C, the supernatant was taken for subsequent MDA analysis as described above. The results were expressed as nm TBARS/mL for plasma or as nm/mg protein for liver samples.

Enzyme Activity and Glutathione Determination Glutathione peroxidase (GSH-PX) activity for plasma and liver was assayed by the method of Paglia and Valentine (1967). The activities of total superoxide dismutase (T-SOD) and Cu/Zn-SOD in plasma and liver were measured using commercial kits (Jiancheng

Engineering Institute, Nanjing, China). All SOD activity was expressed as unit/mL in plasma. The plasma reduced (GSH) and oxidized glutathione (GSSG) also were assayed using a commercial kit (Jiancheng Engineering Institute, Nanjing, Chin); plasma concentrations of GSSG and GSH were expressed as μmol/L and mg/L, respectively.

Assay of Plasma Concentrations of Zn and Cu Ions The plasma for the analysis of Zn and Cu was deproteinized with trichloroacetic acid and analyzed by inductively coupled plasma optical emission spectroscopy using a PerkinElmer Optima 2100 DV instrument (Perkin Elmer, Wellesley, MA) (Standard Methods, 2005; Silva et al., 2009).

Egg Quality Determination Egg shape was measured as length/width in the egg. Eggs were stored at 4◦ C overnight and then broken onto a level surface. Haugh units and yolk color were measured with an egg analyzer (model EA-01, ORKA Food Technology Ltd., Ramat Hasharon, Israel), after which yolk was separated and weighed. Shells were washed under running water, dried, and weighed. Albumen weight was calculated by subtraction. Shell thickness of the eggs with removed membrane was measured as the mean of 3 locations (air cell, equator, and sharp end) with a digital micrometer (model IT-014UT, Mitutoyo, Kawasaki, Japan). Shell breaking strength of un-cracked eggs was measured using an Egg Force Reader (ORKA Food Technology Ltd., Ramat Hasharon, Israel).

Statistical Analysis The experimental data were analyzed for treatment differences by ANOVA for a randomized block design, using SAS 8.1 software. All the production performance data (egg production, feed intake, egg weight, daily egg mass, and FCR), plasma and liver biochemical parameter data, and bone and egg quality data were analyzed for the effect of dietary treatments. As the treatments were equally spaced, and orthogonal contrasts were used to test for linear and quadratic effects when significant effects of dietary Zn were demonstrated (Eisemann et al., 2013). Pearson’s correlations were employed to examine the associations between antioxidant enzymes and plasma Zn and Cu. All statements of significance are based on a probability of less than 0.05.

RESULTS During the early laying period of 10 d (daily egg production <80%), egg production, daily egg mass, and FCR increased quadratically with increasing

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Table 2. Effects of dietary Zn supplementation on the productive performance of laying ducks.1 Variable

Dietary Zn supplementation, mg/kg 0

30

45

60

75

90

Early laying period (egg production < 80%), 10 d Egg production, % 68.8 74.5 Egg weight, g 58.9 58.1 Daily egg mass, g 40.0 43.5 Average daily feed intake, g 136 135 Feed conversion ratio 3.41 3.16

78.5 58.0 45.7 134 2.94

79.7 58.8 47.4 136 2.89

67.9 57.4 39.5 135 3.39

71.8 57.5 42.5 134 3.27

69.0 57.2 39.5 137 3.49

Peak laying period (egg production > 80%), 120 d Egg production, % 83.0 82.0 Egg weight, g 66.5 67.1 Daily egg mass, g 55.0 55.0 Average daily feed intake, g 152 151 Feed conversion ratio 2.79 2.78

91.2 66.3 59.9 151 2.57

87.3 67.0 58.1 153 2.65

85.1 67.6 57.6 153 2.75

82.5 66.5 54.7 150 2.79

82.5 66.1 54.4 151 2.82

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15

SEM

P-value Zn

Linear

Quadratic

3.036 0.654 1.921 1.433 0.147

0.039 0.43 0.026 0.56 0.030

0.35 – 0.24 – 0.25

0.019 – 0.012 – 0.006

1.904 0.645 1.287 1.236 0.058

0.028 0.64 0.041 0.46 0.065

0.52 – 0.49 – –

0.006 – 0.003 – –

Data are means of 6 replicates of 12 ducks each.

Table 3. Effects of dietary Zn supplementation on the plasma indices of antioxidant and lipid peroxidation in peak laying ducks.1 Variables

Dietary Zn supplementation, mg/kg

T-SOD, U/mL Cu/Zn-SOD, U/mL Mn-SOD, U/mL GSH-PX, U/mL GSSG, μ mol/L GSH, mg/L TBARS, nmol/mL 1

SEM

0

15

30

45

60

75

90

67.5 17.9 49.6 292 10.8 11.1 12.2

63.6 21.1 43.6 314 11.2 11.2 9.85

73.5 20.1 50.2 356 10.7 8.17 9.67

81.5 19.3 62.2 278 10.2 8.47 10.2

79.3 17.0 62.3 304 10.3 9.32 9.37

79.5 18.0 61.4 289 11.7 10.9 9.98

75.4 19.1 56.3 271 12.4 9.90 10.4

2.047 1.891 2.874 13.44 0.500 1.254 1.472

P-value Zn

Linear

Quadratic

< 0.001 0.43 < 0.001 0.002 0.035 0.43 0.85

< 0.001 – < 0.001 0.030 0.040 – –

0.002 – 0.053 0.031 0.012 – –

Data are means of 6 replicates of 2 ducks each.

Table 4. Effects of dietary Zn supplementation on the plasma zinc and copper ion concentration in peak laying ducks.1 Variables

Cu , μ mol/L Zn2+ , μ mol/L 2+

1

Dietary Zn supplementation, mg/kg

SEM

0

15

30

45

60

75

90

8.16 55.1

7.69 74.8

7.84 69.8

9.28 77.5

7.68 67.9

8.04 67.9

8.79 68.3

0.951 4.317

P-value Zn

Linear

Quadratic

0.87 0.029

– 0.30

– 0.011

Data are means of 6 replicates of 2 ducks each.

dietary Zn levels (P < 0.05, Table 2). The highest egg production and daily egg weight, with lowest FCR, were obtained when 30 or 45 mg Zn/kg diet supplementation was used (total dietary Zn was 64.6 or 84.4 mg/kg). Similarly, during the subsequent 120-day peak laying period (daily egg production >80%), highest egg production, and daily egg mass also were observed with the same levels of Zn; average egg weight and feed intake were not affected by graded Zn supplementation. Plasma T-SOD activity responded in a linear or quadratic manner (P < 0.01, Table 3), increasing to the maximal value, about 20% higher than that with the basal diet, at 45 mg/kg supplemental Zn before declining; plasma Cu/Zn-SOD activity was not affected by dietary Zn. Plasma GSH-PX activity increased with supplemental Zn, with the highest value observed at 30 mg/kg added Zn (64.6 mg/kg total). Concentration of oxidized glutathione (GSSG) in plasma showed a dominant linear response to dietary levels of Zn but there were no effects of Zn addition on plasma concen-

trations of reduced glutathione (GSH) or TBARS. The plasma concentration of zinc, but not copper, increased quadratically with increased supplemental Zn, with the highest value at 45 mg/kg added Zn, or 84.4 mg/kg total (Table 4). Simple correlations between variables measured in plasma were examined (Table 5). Plasma Zn concentrations were positively correlated with activities of T-SOD and plasma Cu and GSH, but negatively with plasma GSSG (P < 0.05). Plasma concentration of GSH was correlated with plasma Cu, and T-SOD was correlated with GSH-PX. In the liver, the activity of Cu/Zn-SOD and GSH-PX increased quadratically with increasing Zn (P < 0.05), whereas activity of T-SOD and TBARS content in liver was not affected by supplemental Zn (Table 6). Dietary Zn did not affect the egg shape index, Haugh unit, yolk color, the relative weights of yolk, albumen, shell, or the shell thickness at any location (Table 7), indicating negligible effects of dietary Zn on egg quality. Similarly, dietary Zn did not affect the tibial mass,

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ZINC AND LAYING DUCKS Table 5. Correlation coefficients among the plasma variables in peak laying ducks Variables

Zn

Zn Cu T-SOD Cu/Zn-SOD GPX GSH GSSG ∗

P < 0.05;

Cu

1.00

∗∗

T-SOD

∗∗

Cu/Zn-SOD

∗

0.30 1.00

GSH-PX

− 0.21 − 0.20 − 0.03 1.00

0.24 0.14 1.00

GSH

GSSG

∗

− 0.01 − 0.07 − 0.04 0.27∗ 1.00

0.23 0.40∗∗ − 0.02 − 0.15 − 0.09 1.00

− 0.24∗ − 0.01 − 0.08 0.16 0.03 − 0.03 1.00

P < 0.01, n = 84.

Table 6. Effects of dietary Zn supplementation on the hepatic indices of antioxidant status in peak laying ducks.1 Variable

Dietary Zn supplementation, mg/kg

T-SOD, U/mg pro Cu/Zn-SOD, U/mg pro GSH-PX, U/mg pro TBARS, nm/mg pro 1

SEM

0

15

30

45

60

75

90

231 130 45.4 0.79

225 122 43.5 0.54

240 150 49.9 0.57

226 145 50.6 0.61

225 137 50.6 0.72

230 136 47.5 0.55

219 134 45.3 0.46

8.566 6.025 1.853 0.090

P-value Zn

Linear

Quadratic

0.58 0.049 0.039 0.19

– 0.40 0.40 –

– 0.029 0.004 –

Data are means of 6 replicates of 2 ducks each.

Table 7. Effects of dietary Zn supplementation on indices of egg quality in peak laying ducks.1 Variable

Dietary Zn supplementation, mg/kg

Shape index Haugh unit Yolk color score Egg components, % Yolk Albumen Shell Shell thickness, mm Blunt Middle Sharp Average value Shell breaking strength, N 1

SEM

0

15

30

45

60

75

90

1.34 78.5 5.83

1.35 76.9 4.75

1.34 71.7 4.75

1.35 76.9 4.92

1.34 78.8 4.75

1.36 73.6 4.75

1.32 76.8 4.92

31.4 58.9 9.74

31.3 59.2 9.53

31.1 59.1 9.81

31.7 61.3 9.80

31.1 59.1 9.74

30.6 59.8 9.58

0.35 0.37 0.36 0.36 4.55

0.35 0.36 0.35 0.35 4.31

0.36 0.37 0.36 0.36 4.13

0.36 0.37 0.35 0.36 4.01

0.35 0.37 0.35 0.36 4.44

0.36 0.36 0.35 0.36 4.18

P-value Zn

Linear

Quadratic

0.012 3.386 0.322

0.58 0.74 0.20

– – –

– – –

31.9 58.4 9.67

1.746 3.401 0.269

0.74 0.60 0.63

– – –

– – –

0.35 0.36 0.35 0.36 4.14

0.008 0.005 0.007 0.006 0.253

0.93 0.84 0.78 0.93 0.71

– – – – –

– – – – –

Data are means of 6 replicates of 2 ducks each.

Table 8. Effects of dietary Zn supplementation on tibial mass and breaking strength.1 Item

Dietary Zn supplementation, mg/kg

Tibial mass relative to BW, % Circumference (cm) Bone breaking strength, kg/mm2 1

SEM

0

15

30

45

60

75

90

0.41 1.96 157

0.41 1.96 177

0.42 1.76 170

0.42 1.95 164

0.39 1.94 171

0.43 1.97 175

0.40 1.95 165

0.032 0.074 10.28

P-value Zn

Linear

Quadratic

0.19 0.26 0.81

– – –

– – –

Data are means of 6 replicates of 2 ducks each.

circumference, or breaking strength (Table 8). Although there were no differences in the relative ovarian weight, large follicle number, or large follicle relative weight, these variables tended to have maximal values with 30 and 45 mg/kg supplementation (64.6 and 84.4 mg total Zn/kg, Table 9).

DISCUSSION In practical production, ducks usually have a brief period of early laying, 10 to 21 d, during which average

egg production increases from 50 to 80%. As hypothesized, the productive performance of laying ducks was increased by supplementation of Zn during both the periods of early and peak laying. Similarly, egg production performance of hens was improved by supplementation of a basal diet containing required Zn with an additional 30 mg Zn/kg and 8 mg pyridoxine/kg (Kucuk et al., 2008). The present study also found that the highest productive performance, obtained with 30 or 45 mg/kg Zn supplementation (64.6 and 84.4 mg/kg total dietary Zn) corresponded to the highest plasma

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Table 9. Effects of dietary Zn supplementation on the mass of ovary tissues and follicular characteristics in laying ducks.1 Item

Ovary weight relative to BW, % Large follicle number Large follicle weight relative to BW, % 1

Dietary Zn supplementation, mg/kg

SEM

0

15

30

45

60

75

90

3.82 5.25 3.23

4.45 5.00 3.51

4.77 5.67 4.15

4.70 6.00 4.13

4.61 5.29 3.90

4.02 4.86 3.67

4.53 5.13 3.92

0.376 0.370 0.388

P-value Zn

Linear

Quadratic

0.50 0.25 0.66

– – –

– – –

Data are means of 6 replicates of 2 ducks each.

activities of T-SOD and GSH-PX, suggesting that improved antioxidant capacity may result in higher productive performance. The present findings indicate that optimal Zn provision might help improve the productive performance of birds, especially under summer conditions when oxidative stress status was induced (Sahin et al., 2009). In support of this, supplementation with 40 mg Zn/kg in the organic form increased plasma superoxide dismutase activity and improved growth performance of chickens during a tropical summer (Rao et al., 2016). Recent literature indicated that Zn supplementation of broiler diets was beneficial for performance and/or antioxidant responses (Kope´c et al., 2013). Dietary composition has an effect on Zn absorption, which may lead to differences in the actual requirement. For example, the estimated Zn requirement for broilers using a diet based on corn-soybean meal (Huang et al., 2007; Liao et al., 2012) was higher than those recommended by the NRC (1994), mostly based on purified or semi-purified diets. Estimates determined in diets formulated from casein-dextrose, egg white, or crystalline amino acids may not be applicable to cornsoybean diets because of the absence of phytic acid and fiber. Phytic acid is known to inhibit Zn absorption (Yu et al., 2010), and it is naturally present in cerealbased staples (Gibson et al., 2010), with maize, wheat, sorghum, and millet having high phytic acid contents (Reddy, 2001); higher Zn supplementation would be required, therefore, in diets based on corn-soybean meal. Compared to the diet used here that contained relatively high phytic acid from the wheat bran (average 41.29 g/kg phytic acid, Garc´ıa-Estepa et al., 1999), a lower dietary Zn content would be recommended if combined with an effective phytase, because phytase increases Zn utilization in broiler chickens (Sebastian et al., 1996; Yi et al, 1996) and laying hens (Um and Paik, 1999). There is indirect evidence showing that a Zn-deficient corn-soybean diet supplemented with organic forms of Zn, Mn, and Cu at doses 50 to 75% lower than NRC recommendation is sufficient to maintain laying performance (Gheisari et al., 2011), probably because of higher bioavailability of organic minerals than their inorganic forms (Wedekind et al., 1992; Li et al., 2005). The productive performance of laying ducks here was compromised when Zn supplementation was above 75 mg/kg (total 112 mg /kg diet) contrasting with the lack of effect of dietary Zn in excess of 137 mg/kg on the performance of laying hens (Trindade Neto

et al., 2011). The difference may suggest that laying ducks are more susceptible to dietary Zn supplementation than are chickens. Plasma or serum zinc is the most frequently used index for evaluating the likelihood of zinc deficiency (Prasad et al., 1965; Hackley et al., 1968; Halsted and Smith, 1970; Pilch and Senti, 1985) although they may not necessarily reflect the cellular zinc status (Prasad et al., 1978; Milne et al., 1983; Lukaski et al., 1984). In the present study, plasma Zn level was lowest with the basal diet and increased quadratically with supplemental Zn, peaking in the 45 mg/kg treatment (total 84.4 mg/kg) before concentrations decreased. The lowest plasma Zn concentration and lowest egg production during early and peak laying periods were observed in ducks fed the basal diet (37 mg Zn/kg), which suggests this content of Zn did not meet the actual requirement of the laying ducks. Although dietary Zn deficiency decreased feed intake (MacDonald, 2000), reduced eggshell quality (Kienholz et al., 1961), and caused leg abnormalities (Dewar and Downie, 1984) in other poultry, these abnormal phenomena were not observed in the present work. The lack of effect of dietary Zn on shell thickness, weight, or breaking strength was consistent with a study using hens (Tabatabaie et al., 2007), whereas combinations of organic forms of Zn, Mn, and Cu increased eggshell thickness (Gheisari et al., 2011) and improved eggshell breaking strength (Mabe et al., 2003) in laying hens. Supplementation with mixed minerals in organic form may be more effective than inorganic Zn alone in improving eggshell quality. Zinc may play a key role in suppression of free radicals because it is a cofactor of the main antioxidant enzyme Cu/Zn-SOD and it also inhibits NADPHdependent lipid peroxidation (Prasad and Kucuk, 2002). The plasma antioxidant enzymes GSH-PX and T-SOD and hepatic Cu/Zn-SOD in laying ducks were significantly increased with 30 and 45 mg/kg supplementation (total 64 or 84 mg Zn/kg), but higher dietary Zn may compromise the enzyme activities, similar to that observed in egg production. Also, plasma Zn was shown to be positively correlated with the activities of total SOD, indicating the enhanced antioxidant function of Zn. Dietary Zn-deficiency decreased GSH-PX and SOD and increased oxidative stress in erythrocytes of rats (Taysi et al., 2008), but optimal supply of Zn to rat water increased the GSH-PX activity in plasma as well as in liver (Gala˙zyn-Sidorczuk et al., 2012). In broiler chickens, dietary supplements of Zn from both

ZINC AND LAYING DUCKS

organic and inorganic sources have been demonstrated to increase the activity of Cu/Zn-SOD in various tissues, including liver (Bun et al., 2011), breast muscle, and thigh muscle (Liu et al., 2015). These results together with ours indicate that the antioxidant capacity of birds can be enhanced by optimal supplementation of Zn. It is suggested that dietary supplementation of Zn and Cu should be proportionate (Sandstead, 1995) because oral Zn intakes that are disproportionately high relative to Cu are a conditioning factor in inducing Cu deficiency (Maret and Sandstead, 2006). In this study, the range of dietary Zn fed to the laying ducks (total 37.4 to 131.4 mg/kg) had a negligible effect on plasma Cu. In conclusion, the present study demonstrated that plasma Zn concentrations in laying ducks were correlated with activities of T-SOD, and positively with plasma Cu and GSH. Supplementation of a corn-wheat bran and soybean meal basal diet with 30 or 45 mg Zn/kg (total contents of 64.6 to 84.4 mg/kg) is recommended for maximal egg yield and antioxidant capacity in early and peaking laying ducks. Dietary supplementation of Zn did not affect the egg quality and bone quality.

ACKNOWLEDGMENTS We sincerely thank Dr. W. Bruce Currie (Emeritus Professor, Cornell University) for his help in presentation of this manuscript. This work was supported by the Fund for China Agricultural Research System (CARS-43-13), the Science and Technology Planning Project of Guangdong Province (2016A020210043, 2011A020102009), National Natural Science Foundation of China (Grant No. 31301995), and Operating Funds for Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition (2014B030301054).

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