Regulation of egg quality and lipids metabolism by Zinc Oxide Nanoparticles Yong Zhao,∗,† Lan Li,† Peng-Fei Zhang,∗,† Xin-Qi Liu,∗ Wei-Dong Zhang,∗,† Zhao-Peng Ding,∗ Shi-Wen Wang,∗ Wei Shen,† Ling-Jiang Min,† and Zhi-Hui Hao∗,1 ∗

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, P. R. China; and † Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, Qingdao 266109, P. R. China

Key words: ZnO nanoparticle, hen, yolk lipid, metabolism 2016 Poultry Science 0:1–14 http://dx.doi.org/10.3382/ps/pev436

INTRODUCTION

Yang et al., 2015), rat (Hong et al., 2014; Choi et al., 2015), and on human cells (Kim et al., 2013; Tuomela et al., 2013). It has been estimated that the annual production volume of zinc oxide (ZnO) NP ranks the third highest after SiO2 and TiO2 NPs globally (Bondarenko et al., 2013). ZnO NP have numerous applications: paints, coatings, tire rubber, liquid crystal displays, sunscreen, cosmetics, biocides, etc. Therefore the release of ZnO NP from consumer and household products into the environment may pose a threat to organisms. Bondarenko et al., (2013) and Ma et al. (2013) reviewed the toxicity/ecotoxicity of ZnO NP. Although numerous investigations have reported that ZnO NP cause adverse effects on animals such as mice (Cho et al., 2013; Talebi et al., 2013), rats (Jo et al., 2013), fish (Brun et al., 2014), and Daphnia magna (Lopes et al., 2014; Santo et al., 2014), very few studies aimed to investigate the effects of ZnO NP on farm animals. Furthermore, even though some studies have investigated the effect of ZnO NP on the mouse or rat reproductive system (Talebi

Nanotechnology is still in the early stages of development and has attracted much attention with its numerous advantages as well as the many concerns about its disadvantages. Nanotechnology has been applied in industrial products, drug delivery systems, diagnosis and treatment of disease, and farm animal breeding. Nanoparticles (NP), one avenue of nanotechnology, are defined as having one or more dimension less 100 nm with novel properties of small size, large surface area to volume ratio, and a typical smoothly scaling property. The major concern with NP is the adverse effect on consumers and industrial workers (Stone and Donaldson, 2006) because a number of studies have found adverse effects of NP on mouse (Bargheer et al., 2015;  C 2016 Poultry Science Association Inc. Received September 18, 2015. Accepted November 4, 2015. 1 Corresponding Author: [email protected]

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alytical kits. Yolk triglyceride, total cholesterol, pancreatic lipase, and phospholipids were determined by appropriate kits. β -carotene was determined by spectrophotometry. Lipid metabolism was also investigated with liver, plasma, and ovary samples. ZnO NP did not change the body weight of hens during the treatment period. ZnO NP slowed down egg laying frequency at the beginning of egg laying period but not at later time. ZnO NP did not affect egg protein or water contents, slightly decreased egg physical parameters (12 to 30%) and trace elements (20 to 35%) after 24 weeks treatment. However, yolk lipids content were significantly decreased by ZnO NP (20 to 35%). The mechanism of Zinc oxide nanoparticles decreasing yolk lipids was that they decreased the synthesis of lipids and increased lipid digestion. These data suggested ZnO NP affected egg quality and specifically regulated lipids metabolism in hens through altering the function of hen’s ovary and liver.

ABSTRACT This investigation was designed to explore the effects of Zinc Oxide Nanoparticles (ZnO NP) on egg quality and the mechanism of decreasing of yolk lipids. Different concentration of ZnO NP and ZnSO4 were used to treat hens for 24 weeks. The body weight and egg laying frequency were recorded and analyzed. Albumen height, Haugh unit, and yolk color score were analyzed by an Egg Multi Tester. Breaking strength was determined by an Egg Force Reader. Egg shell thickness was measured using an Egg Shell Thickness Gouge. Shell color was detected by a spectrophotometer. Egg shape index was measured by Egg Form Coefficient Measuring Instrument. Albumen and yolk protein was determined by the Kjeldahl method. Amino acids were determined by an amino acids analyzer. Trace elements Zn, Fe, Cu, and P (mg/kg wet mass) were determined in digested solutions using Inductively Coupled Plasma-Optical Emission Spectrometry. TC and TG were measured using commercial an-

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MATERIALS AND METHODS ZnO Nanoparticle Characterization ZnO NP were made by Beijing DK nano technology Co. LTD (Beijing, P. R. China). The NP morphology, size, and agglomeration were characterized by transmission electron microscopy (TEM; JEM-2100F, JEOL Inc., Japan) and dynamic light scattering in a particle size analyzer (Nano-Zetasizer-HT, Malvern Instruments, Malvern, UK).

Study Design (Diets and Treatments) All animal experimental procedures were followed according to the regulations of the animal ethics committee of the Qingdao Agricultural University. The experiment, lasting 24 weeks, was carried out in a commercial poultry house at Maochangyuan Co. (Qiangdao, China). All hens (Jinghong-1 strain) were housed in an enclosed, ventilated, and conventional caged house with the lighting program which was 16:8 light/dark cycle and 55% relative humidity on average, with free access to diets and water. The composition of the basal diet (corn-soybean base) was previously reported (Sun et al., 2012; Laudadio et al., 2014; Xiao et al., 2015). The feeding time was divided into pre-mature (6 to 18 wk of age) and mature (19 to 30 wk of age) stages. At these two stages the diets were a little different because the nutrition requirement is different (Supplemental Table 1 (Table S1)). The hens started to lay eggs at 18 wk of age.

To compare the different effects of intact NP and Zn2+ , ZnSO4 was used in this investigation to provide solely Zn2+ . In order to determine the dose response effects, the same gradients of concentrations of ZnO NP and ZnSO4 were applied in this study. There were 11 treatments based on the concentration of Zn: (1) control treatment (no Zn added); (2) ZnSO4 10 mg/kg; (3) ZnSO4 -25 mg/kg; (4) ZnSO4 -50 mg/kg; (5) ZnSO4 -100 mg/kg; (6) ZnSO4 -200 mg/kg; (7) ZnONP-10 mg/kg; (8) ZnO-NP-25 mg/kg; (9) ZnO-NP50 mg/kg; (10) ZnO-NP-100 mg/kg; (11) ZnO-NP200 mg/kg. Zn content of the control diet was quantified to be 11.98 ± 0.23 mg/kg. Pullets (n = 528) were randomly assigned to 11 treatments, with 3 replicates per treatment and 16 hens per replicate.

Detection of Nanoparticles in Tissues by Transmission Electron Microscopy and Energy Disperse Spectroscopy The procedure for detection of NP in the tissue samples were described in the reports (Yamashita et al., 2011; Faust et al., 2014). Briefly, ZnO NP treated tissues were collected and fixed for 2 h in 2% glutaraldehyde made in sodium phosphate buffer (pH 7.2). The specimens were washed extensively to remove the excess fixative and subsequently post-fixed in 1%OsO4 for 1 h in the dark. After extensive washes in phosphate buffer, the cells were dehydrated in an increasing graded series of ethanol and infiltrated with increased concentration Spur’s embedding medium in propylene epoxide. Then the specimens were polymerized in embedding medium 12 h at 37◦ C, 12 h at 45◦ C and 48 h at 60◦ C. Fifty nanometer were cut on a Leica Ultracut E equipped with a diamond knife (Diatome, Hatfield, PA), and collected on form var-coated, carbon-stabilized molybdenum (Mo) grids. The sectioncontaining grids were stained with uranyl acetate, allowed to air dry overnight, and imaged on a JEM-2010F TEM (JEOL Ltd., Japan). ZnO nanoparticles in the cells were confirmed by X-MaxN 80 TLE energy disperse spectroscopy (EDS) (Oxford Instruments, UK).

Performance Data Record Body weight was recorded at the beginning and end of the experimental period, and feed intake was determined once a week. The number and weight of eggs laid were registered daily, and hen-day egg production was calculated as the total number of eggs collected divided by the number of live hens. Egg mass was calculated as hen-day egg production multiplied by the average egg weight.

Egg Physical Parameters Analysis Twenty four eggs (8 eggs randomly selected from each replicate) for each treatment were used to determine egg

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et al., 2013; Jo et al., 2013), little is known about the effects on egg quality or oocyte lipid metabolism. In addition it is still not clear whether the toxic effects of ZnO NP are caused by intact NP or Zn2+ dissolved from NP in animals (Brun et al., 2014; Chen et al., 2014). Domestic chicken (Gallus gallus) is an important model organism because it bridges the evolutionary gap between mammals and other vertebrates (International Chicken Genome Sequencing Consortium, 2004). Hen eggs are the very important nutrition source for humans (Attia et al., 2014; Miranda et al., 2015) because eggs are a good source of proteins, lipids, amino acids, and minerals. Hen egg yolk is a rich source of lipids (≈30%) which are required for oogenesis and the early stages of embryogenesis (Moran, 2007; Sieber and Spradling, 2015). The hypothesis for this study was that intact ZnO NP may affect hen egg quality and lipids metabolism because ZnO NP cause known problems with mice, rats, and other organisms. The aim of the present experiment was to explore the effects of intact NP on performance, egg production, egg quality, and lipids metabolism of laying hens fed a gradient of concentrations of ZnO NP. ZnSO4 was used as a control which provides only Zn2+ in the diet.

EGG QUALITY REGULATED BY ZINC OXIDE NANOPARTICLES

Egg Chemical Components Analysis The content of water, protein, amino acids, and trace elements (Zn, Fe, Cu, and P) in egg albumen and yolk, as well as lipid profiles [total cholesterol (TC), triglyceride (TG) and β -carotene] in yolk were analyzed by using twenty four eggs (8 eggs from each replicate). Albumen and yolk protein was determined by Kjeldahl method (Vinkl´ arkov´ a et al., 2015). Amino acids were determined by amino acids analyzer L-8900 (Hitachi, Ltd., Jokyo, Japan). Trace elements Zn, Fe, Cu and P (mg/kg wet mass) were determined in digested solutions using Inductively Coupled Plasma-Optical Emission Spectrometry (Optima 2100, Perkin-Elmer, Shelton, CT) (Osmond-McLeod et al., 2014). TC and TG were measured using commercial analytical kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) (Sun et al., 2012). Yolk TG, TC, pancreatic lipase (PL), and phospholipids (PC) were extracted by 95% Ethanol first then determined by the kits (Palacios and Wang, 2005). β -carotene was determined by spectrophotometry (Ndawula et al., 2004).

Plasma Insulin and Apo-B Determination Plasma insulin and apolipoprotein B (Apo-B) were quantified using ELISA kits from Nanjing Jiancheng Bioengineering Institute by following the manufacturer’s instructions. Six samples from each treatment were determined (Sun et al., 2012).

Liver Enzyme Activities of LPL, HL, and PL Determination Liver lipoprotein lipase (LPL), hepatic lipase (HL), and PL activities were analyzed by the kits from Nanjing Jiancheng Bioengineering Institute by following the manufacturer instructions.

Gene expression Determined by q-RT-PCR Total RNA from liver, ovary, and granulosa cells was isolated by TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and purified with PureLink RNA Mini Kit (Cat: 12183018A; Life Technologies, Carlsbad, CA, USA) following the manufacturers protocol. RNA concentration was determined by Nanodrop 3300 (ThermoScientific, Wilmington, DE). Two micrograms of total RNA was used to make the first strand complementary DNA (cDNA; in 20 μL) using RT2 First Strand Kit (Cat. No: AT311-03, Transgen Biotech, P. R. China) following the manufacturer’s instructions. The generated firststrand cDNAs (20 μL) were diluted to 150 μL with double-deionized water (ddH2O). Then, 1 μL was used for one PCR reaction (in a 96-well plate). Each PCR reaction (12 μL) contained 6mL of quantitative Polymerase Chain Reaction (qPCR) Master Mix (Roche, German), 1 μL of diluted first-stand cDNA, 0.6 μL primers (10 μM), and 4.4 μL of ddH2O. The primers for qPCR analysis were synthesized by Invitrogen and present in Table 2. The qPCR was conducted by the Roche LightCycler 480 (Roche, Germany) with the following program-step 1: 95◦ C, 10 min; step 2: 40 cycles of 95◦ C, 15 s; 60◦ C, 1 min; step 3: dissociation curve, step 4: cool down. Three samples from each treatment of liver, ovarian tissue, and ovarian granulosa cells were analyzed.

Statistical Analyses The quantitative Reverse Transcription Polymerase Chain Reaction (q-RT-PCR) was statistically analyzed using proprietary software from SABiosciences online support. Other data were statistically analyzed by SPSS statistics software (IBM Co., NY) using ANOVA. Comparisons between groups were tested by one-Way ANOVA analysis and least-significant difference (LSD) test. All the groups were compared with each other for every parameter (mean ± SD). Differences were considered significant at P < 0.05.

RESULT Characterization of ZnO NP A photo of the ZnO NP used in this investigation is given in Figure 1A, and the ultra-structures of the NP (analyzed by TEM) are shown in Figure 1B. The morphology of the particles was nearly spherical with a milk white color. The size and the surface area were approximately 30 nm and 50 m2 /g, respectively. And the density of the NP was 5.606 g/cm3 .

Body Weight and Egg Laying Frequency The body weights of hens at the beginning and the end of the experiment is shown in Figure 2. There was no statistical difference between each treatment for hen

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quality traits (Alsaffar et al., 2013) included egg weight, yolk weight, albumen weight, shell weight, egg shape index, breaking strength, shell thickness, shell color, albumen height, Haugh unit (HU), yolk color. The hens were 30 wk of age. The eggs were collected in the morning of the day. Albumen height, HU, and yolk color score were analyzed by an Egg Multi Tester (EMT-7300, Touhoko Rhythm Co., LTD, Japan). Breaking strength was determined by an Egg Force Reader (ORKA Food Technology Ltd., Israel). Egg shell thickness was measured using an Egg Shell Thickness Gouge (ORKA Food Technology Ltd., Israel). Shell color was detected by a spectrophotometer (CM-2600d Konica Minolta Sensing, InC., Japan). Egg shape index was measured by Egg Form Coefficient Measuring Instrument (FHK, Fujihira Industry CO., LTD., Japan).

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Figure 3. Effects of ZnO NP and ZnSO4 treatments on egg production. Y axis is the egg production (eggs/hen/day) and X axis shows the treatment groups at different periods of time. a–c Means at that time period for egg production not sharing a common superscript are different (P < 0.05).

Figure 2. Effects of ZnO NP and ZnSO4 treatments on hens’ body weight. Y axis is the body weight (kg), and X axis shows the treatment groups.

body weight. The hens started to lay eggs at the beginning of 18 weeks of age. Figure 3 shows the egg laying frequency (eggs/hens/day) for all treatments at 3 different time points (average): 18 to 22 weeks of age, 23 to 26 weeks of age, and 27 to 30 weeks of age. Compared with control or ZnSO4 treatments, ZnO-NP-10, 25, 50, and 100 mg/kg decreased the egg laying frequency at 18 to 22 weeks of age (Figure 3A; P < 0.05). However, at 23 weeks of age and later, the egg laying frequency was not different between treatments.

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Figure 1. ZnO NP in ovary and liver. A. TEM photo of ZnO NP and their characteristics. B. TEM photo of ZnO NP in ovary indicated by the white arrow. C. EDS picture of ZnO NP in ovary, where three Zn peaks have shown.

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EGG QUALITY REGULATED BY ZINC OXIDE NANOPARTICLES

tration in the ovary and yolk compared to that in the control group. The concentration of Zn in the yolk was higher than that in the ovary (by almost double). The concentration of Zn was elevated as the concentrations of ZnSO4 and ZnO NPs increased in the liver. However, the concentration of Zn was higher in ZnO NP treatments than in the same concentration of ZnSO4 treatments. The concentration of Zn was the highest in the ZnO-NP-200 mg/kg treatment in the liver (Figure 4; P < 0.05). The concentrations of Zn in the ZnSO4 200 mg/kg and ZnO-NP-100 mg/kg groups were similar in the liver.

Figure 4. Effects of ZnO NP and ZnSO4 treatments on Zn contents in liver, ovary and yolk. Z axis is the content (mg/kg wet mass), and Y axis is the treatment (concentration of Zn; mg/kg), X axis shows the treatment groups. a–c Means in liver for Zn content not sharing a common superscript are different (P < 0.05).

Intact NP in Ovary and Liver The NP were found in the ovary (Figure 1C, indicated by white arrow) and liver. The NP in the organs were confirmed by EDS with Zn (Figure 1D). Three standard Zn peaks were present.

Concentration of Zn in Liver, Ovary and Yolk As shown in Figure 4, all the treatments of ZnSO4 and ZnO NP did not significantly change Zn concen-

In total 11 egg physical parameters were analyzed in this study. Almost all ZnSO4 and ZnO NP treatments decreased albumen height and HU (Table 1; P < 0.05, different letters mean significant difference between treatments) compared to that in control treatment. Compared to ZnSO4 -200 mg/kg, ZnO-NP-200 mg/kg decreased egg weight, yolk weight, egg shape index, and increased shell color (Figure 5 A, B, C, and D; P < 0.05) and decreased shell weight (Table 1; P < 0.05). All the other parameters: egg albumen weight, breaking strength, shell thickness, and yolk color were not changed much by ZnSO4 or ZnO NP treatments (Table 1). At concentration 100 mg/kg and 200 mg/kg, ZnSO4 and ZnO NP increased yolk water content compared to the control treatment (Table 1; P < 0.05). There was no large difference between different treatments for water content in the egg albumen or protein content in the

Table 1. Effects of ZnO NP on egg quality parameters, water and protein content. Zn concentration (mg/kg) Items Albumen weight(g)

Treatment group

ZnSO4 ZnO-NP Shell weight(g) ZnSO4 ZnO-NP Breaking strength ZnSO4 ZnO-NP Shell thickness(mm) ZnSO4 ZnO-NP Albumen height(mm) ZnSO4 ZnO-NP H.U. ZnSO4 ZnO-NP Yolk color ZnSO4 ZnO-NP Yolk H2O (%) ZnSO4 ZnO-NP Albumen H2O (%) ZnSO4 ZnO-NP Yolk protein (%) ZnSO4 ZnO-NP Albumen protein (%) ZnSO4 ZnO-NP a–f

0 35.09 ± 1.02 6.68 ± 0.11c,d 3.42 ± 0.12c 0.403 ± 0.005a,c 9.92 ± 0.35a 99.99 ± 1.36b 5.02 ± 0.24a,b 43.38 ± 0.92d,f 86.94 ± 0.29a,b 16.21 ± 0.35a,b 9.49 ± 0.70

10 34.53 34.34 6.61 6.69 3.51 3.57 0.407 0.398 9.23 9.46 97.25 98.39 5.09 4.87 45.49 42.31 87.53 86.75 16.23 14.90 9.45 10.63

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.69 0.68 0.14d 0.09c,d 0.18b,c 0.08b,c 0.010a,c 0.004b,c 0.23b,c 0.23a,b 1.05a,c 1.01a,b 0.19a,b 0.25a,b 0.43b,d 1.76f 0.09a 0.16a,b 0.25a 0.38b 0.44 0.34

25 35.67 34.82 7.09 6.93 3.81 3.64 0.407 0.420 9.19 9.16 96.68 96.83 4.90 4.74 43.46 48.01 87.48 87.52 15.86 15.29 10.32 10.19

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.99 0.68 0.16a,b 0.12a–d 0.15a,b 0.11b,c 0.006a,c 0.011a 0.18b,c 0.21b,c 0.88a,c 0.88a,c 0.20a,b 0.20b 0.70e,f 0.12a 0.40a 0.22a 0.49a,b 0.84a,b 0.34 0.12

Means for one parameter not sharing a common superscript are different (P < 0.05).

50 35.02 35.56 6.82 7.04 3.65 3.64 0.398 0.412 9.22 9.29 97.08 96.99 5.01 4.71 45.44 45.33 87.31 86.50 14.96 15.72 10.45 9.39

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.74 0.59 0.13b–d 0.11a–d 0.11b,c 0.10b,c 0.006b,c 0.006a,b 0.20b,c 0.24b,c 0.85a,c 1.13a,c 0.19a,b 0.16b 0.40b,c 0.41c–e 0.14a,b 0.87b 0.22b 0.52a,b 1.86 0.26

100 34.73 36.23 7.07 6.84 4.04 3.75 0.403 0.402 8.74 9.26 94.72 96.90 5.41 5.05 47.28 47.00 87.56 87.19 15.53 15.58 10.11 9.68

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.56 0.59 0.17a–c 0.14a–d 0.11a 0.10a–c 0.004a,c 0.007a,c 0.20c 0.18b,c 0.95c 0.77a,c 0.16a 0.19a,b 0.43a,b 0.26a–c 0.10a 0.13a,b 0.38a,b 0.52a,b 0.26 0.54

200 36.22 34.81 7.21 6.74 3.81 3.54 0.408 0.392 9.53 9.38 97.86 98.03 4.93 5.06 47.67 47.19 87.16 87.38 15.85 15.16 10.41 10.78

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.73 0.85 0.19a 0.16b–d 0.15a,b 0.18b,c 0.007a,c 0.005c 0.27a,b 0.16a,b 1.04a,b 0.63a,b 0.20a,b 0.23a,b 0.17a 0.85a–c 0.10a,b 0.17a,b 0.23a,b 0.36a,b 0.35 0.26

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Egg physical Parameters and Egg Chemical Components Altered by ZnO NPs

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egg albumen and yolk except for the 10 mg/kg ZnO NP treatment which decreased yolk protein in comparison with those on the same concentration of Zn-SO4 (Table 1). ZnSO4 -25 mg/kg and ZnO-NP-10 mg/kg slightly increased all the amino acids analyzed in egg albumen, while only ZnO-NP-10 mg/kg slightly elevated all the amino acid concentrations in the egg yolk (Figure 5E and F, Tables 2 and 3; P < 0.05). Almost all of ZnSO4 and ZnO NP treatments decreased trace elements Fe and Zn in egg albumen compared to that in the control treatment (Table 4; P < 0.05). ZnO-NP50, 100, and 200 mg/kg decreased Cu in egg albumen in comparison with the ZnSO4 10 mg/kg and ZnO NP

25 mg/kg treatments (Table 4; P < 0.05). Total phosphate (P) was not changed much by all the treatments (Table 4). The concentrations of the trace elements were much higher in egg yolk than that in egg albumen.

Total cholesterol, Triglyceride, Phospholipids (Phosphatidylcholine) in Egg Yolk Compared to the control treatment, different concentrations of ZnSO4 treatments did not significantly

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Figure 5. Effects of ZnO NP and ZnSO4 treatments on: A. Egg weight, Y axis is the weight (g); B. Yolk weight, Y axis is the weight (g); C. Egg shape index, egg shape index = egg length/egg width; Y axis is the egg shape index; D. Egg color, Y axis is the reading from spectrophotometer for egg color; E. Relative total amino acid contents in egg albumen, Y axis is the relative amount (% of wet mass). F. Relative total amino acid content in egg yolk, Y axis is the relative amount (% of wet mass); and X axis is the treatment (concentration of Zn; mg/kg) for all the parameters. a–b Means for each parameter not sharing a common superscript are different (P < 0.05).

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EGG QUALITY REGULATED BY ZINC OXIDE NANOPARTICLES Table 2. Effects of ZnO NP on egg albumen amino acid content (% of wet mass). Treatments (Concentration of Zn; mg/kg) AA

Treatment group

ALA ARG ASP CYS GLU GLY

ILE LEU LYS MET PRO RHE SER THR TYR VAL a–c

10

0.688 ± 0.038

a,c

0.667 ± 0.040a,c 1.176 ± 0.065b,c 0.327 ± 0.019b,c 1.540 ± 0.083b,c 0.420 ± 0.023b,c 0.268 ± 0.016b,c 0.605 ± 0.033b,c 1.060 ± 0.064b,c 0.807 ± 0.045a,b 0.537 ± 0.044a,b 0.388 ± 0.023b 0.690 ± 0.037b,c 0.765 ± 0.042b,c 0.523 ± 0.030b,c 0.447 ± 0.024a,b 0.710 ± 0.040b,c

0.692 0.823 0.690 0.785 1.176 1.203 0.337 0.365 1.570 1.863 0.400 0.470 0.273 0.313 0.618 0.700 1.100 1.303 0.815 0.928 0.510 0.512 0.398 0.455 0.727 0.855 0.780 0.920 0.537 0.620 0.460 0.535 0.710 0.817

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

25 a,c

0.066 0.146a,b 0.071a,c 0.149a,c 0.065b,c 0.058a,b 0.033b,c 0.061b,c 0.147b,c 0.343a,b 0.039a,b 0.081b 0.027b,c 0.059a 0.059b,c 0.131a,b 0.109b,c 0.240a 0.075a,b 0.168a 0.062a,b 0.101a,b 0.040b 0.085b 0.060b,c 0.143a 0.074b,c 0.162a,b 0.053b,c 0.111a,b 0.041a,b 0.086a 0.064b,c 0.145a,b

0.875 0.662 0.855 0.675 1.213 1.372 0.395 0.295 1.952 1.525 0.507 0.378 0.332 0.263 0.743 0.583 1.297 1.095 0.938 0.793 0.647 0.393 0.492 0.388 0.788 0.662 0.980 0.802 0.653 0.532 0.542 0.445 0.888 0.662

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

50 a

0.112 0.030b,c 0.111a 0.047a,c 0.119b 0.251a,c 0.044a 0.019c 0.251a 0.069b,c 0.063a 0.017b,c 0.043a 0.012b,c 0.098a 0.026b,c 0.164a 0.051b,c 0.118a 0.038a,b 0.088a 0.034b 0.064a 0.022b 0.094a,b 0.040b,c 0.123a 0.049b,c 0.080a 0.028b,c 0.062a 0.019b 0.110a 0.030b,c

0.653 0.697 0.612 0.658 1.470 1.143 0.298 0.335 1.477 1.588 0.368 0.402 0.238 0.263 0.538 0.568 0.970 1.117 0.692 0.790 0.463 0.487 0.357 0.385 0.595 0.683 0.745 0.792 0.493 0.533 0.402 0.442 0.632 0.667

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

100 b,c

0.028 0.039a,c 0.027b,c 0.038b,c 0.184a,b 0.050b,c 0.012c 0.015b,c 0.061b,c 0.084b,c 0.017b,c 0.022b,c 0.010c 0.014b,c 0.024b,c 0.033b,c 0.043c 0.066b,c 0.028b 0.029a,b 0.023b 0.035b 0.017b 0.024b 0.024c 0.035b,c 0.031b,c 0.041b,c 0.020b,c 0.028b,c 0.017b 0.023b 0.027c 0.035b,c

0.598 0.633 0.567 0.598 1.100 1.193 0.288 0.308 1.360 1.282 0.345 0.365 0.220 0.237 0.490 0.515 0.888 0.988 0.642 0.693 0.418 0.415 0.332 0.345 0.562 0.598 0.682 0.722 0.452 0.483 0.375 0.400 0.583 0.607

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

200 c

0.013 0.054c 0.013b 0.053b,c 0.047c 0.059a,c 0.006c 0.024c 0.032c 0.178c 0.008c 0.032b,c 0.005a 0.022c 0.012c 0.048c 0.023c 0.095c 0.014b 0.065b 0.009b 0.046b 0.010b 0.032b 0.014c 0.064c 0.016c 0.064b,c 0.012c 0.042b,c 0.008b 0.035b 0.015c 0.051c

0.718 0.743 0.668 0.693 1.015 1.072 0.337 0.355 1.602 1.658 0.412 0.428 0.270 0.272 0.600 0.613 1.103 1.140 0.802 0.762 0.462 0.487 0.388 0.412 0.708 0.630 0.793 0.820 0.533 0.553 0.447 0.447 0.707 0.727

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.036a,c 0.031a,c 0.034a,c 0.031a,c 0.024b,c 0.092b,c 0.016b,c 0.016b,c 0.073b,c 0.063b,c 0.020b,c 0.019b,c 0.012b,c 0.011b,c 0.028b,c 0.025b,c 0.057b,c 0.051b,c 0.037a,b 0.032a,b 0.019b 0.018b 0.021b 0.018b 0.033b,c 0.031b,c 0.038b,c 0.032b,c 0.027b,c 0.024b,c 0.022a,b 0.018b 0.034b,c 0.030b,c

Means for one amino acid not sharing a common superscript are different (P < 0.05).

change the contents of TC (Figure 6A) or TG (Figure 6B). Concentrations of 10 to 100 mg/kg of ZnO NP did not affect the content of TC compared to that in the control treatment either. However, TC was less in ZnO-NP-25, 100, and 200 mg/kg treatments than that in the ZnSO4 -200 mg/kg treatment (Figure 6A; P < 0.05). The amount of TG was decreased in the ZnO NP treatments. The ZnO-NP-200 mg/kg treatment significantly decreased TG compared to the control, 10, 25, 50, and 200 mg/kg of ZnSO4 treatments (P < 0.05). However, there was no significant difference for TG among ZnO NP treatments (Figure 6B). Concentrations of 100 and 200 mg/kg of ZnO NP treatment significantly decreased phospholipids compared with that in the ZnSO4 -100 or 200 mg/kg treatments (Figure 6C; P < 0.05). PC was also significantly reduced by ZnO-NP-100 and 200 mg/kg treatment compared to that in the ZnSO4 -100 mg/kg treatment (Figure 6D; P < 0.05). β -carotene is also an important component of egg yolk. ZnSO4 or ZnO NP treatment did not change the β -carotene content in the yolk (Figure 6E).

Plasma TG, TC, and PC Reduced by ZnO NP Compared with the control, ZnSO4 and ZnO NP treatments did not change blood TC, however, the content of TC was reduced approximately 25% by the ZnO-NP-100 and 200 mg/kg treatments compared to that in the ZnSO4 treatments (Figure 7A; P < 0.05). ZnSO4 and ZnO NP treatments altered blood TG, PC similarly to that for TC (Figure 7B, C, and D). These data suggest that the decrease of blood lipids might result in a reduction of yolk lipids.

The Expression of Genes Related to Lipid Synthesis Down-Regulated in Liver by ZnO NP Of the twelve important genes FANS, DECR1, ECI1, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, GPAM, and AGPAT3 related to TG synthesis, 10 genes were down regulated by ZnO-NP-100 and ZnO-NP-200 mg/kg (Table 5). All

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HRS

ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP

0

8

ZHAO ET AL.

Table 3. Effects of ZNO NP on egg yolk amino acid content (% of wet mass). Treatments (Concentration of Zn; mg/kg) AA

Treatment group

ALA ARG ASP CYS GLU GLY

ILE LEU LYS MET PRO RHE SER THR TYR VAL a–c

10

0.823 ± 0.017

a,b

1.207 ± 0.024a,b 1.512 ± 0.028a,b 0.300 ± 0.005a,b 1.813 ± 0.044a,b 0.485 ± 0.008a,b 0.423 ± 0.009a,b 0.905 ± 0.015a,b 1.608 ± 0.030a,b 1.278 ± 0.022a 0.410 ± 0.010a,b 0.647 ± 0.015a 0.743 ± 0.015a,b 1.327 ± 0.030a,b 0.848 ± 0.016a,b 0.695 ± 0.012a,b 0.885 ± 0.015a,b

0.828 0.960 1.197 1.348 1.493 1.733 0.310 0.297 1.775 2.077 0.500 0.557 0.410 0.447 0.918 1.012 1.613 1.910 1.270 1.360 0.413 0.420 0.637 0.710 0.757 0.773 1.295 1.515 1.002 0.988 0.700 0.775 0.902 1.000

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

25 a,b

0.017 0.134a 0.018a,b 0.206a 0.031a,b 0.236a 0.009a,c 0.043a,b 0.047a,b 0.293a 0.012a,b 0.078a 0.008a,b 0.067a 0.012a,b 0.146a 0.024a,b 0.269a 0.019a 0.191a 0.018a,b 0.066a,b 0.014a,b 0.103a 0.017a,b 0.100a 0.023a,b 0.213a 0.170a 0.136a,b 0.010a,b 0.110a 0.017a,b 0.138a

0.735 0.840 1.063 1.097 1.345 1.500 0.248 0.317 1.655 1.903 0.437 0.485 0.387 0.382 0.783 1.012 1.385 1.602 1.117 1.175 0.340 0.413 0.587 0.608 0.668 0.767 1.177 1.293 0.748 0.812 0.632 0.670 0.775 0.850

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

50 b

0.131 0.051a,b 0.171b,c 0.059a,b 0.246b 0.076a,b 0.039b,c 0.035a,c 0.304a,b 0.121a,b 0.078b 0.030a,b 0.037a 0.018a,b 0.123b,c 0.146a 0.247b 0.072a,b 0.187a,b 0.053a,b 0.063b 0.058a,b 0.090a,b 0.033a,b 0.090a,b 0.061a 0.211b 0.068a,b 0.135b 0.037a,b 0.078b 0.028a,b 0.137b 0.045a,b

0.687 0.783 0.945 1.103 1.232 1.425 0.232 0.287 1.592 1.810 0.397 0.460 0.320 0.375 0.660 0.772 1.262 1.553 0.943 1.143 0.320 0.385 0.495 0.603 0.535 0.683 1.110 1.268 0.723 0.805 0.550 0.658 0.682 0.792

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

100 b

0.020 0.032a,b 0.031b,c 0.049a,b 0.034b 0.057a,b 0.009b 0.014a,b 0.049b 0.073a,b 0.010b 0.018a,b 0.010c 0.018a,b 0.018c 0.032b,c 0.032b 0.062a,b 0.027b 0.048a,b 0.017b 0.018a,b 0.019b 0.026a,b 0.012b 0.026a,b 0.033b 0.060a,b 0.032b 0.031a,b 0.014b 0.025a,b 0.016b 0.032b

0.778 0.758 1.040 1.063 1.413 1.368 0.292 0.283 1.790 1.712 0.450 0.445 0.353 0.362 0.745 0.740 1.402 1.473 1.032 1.078 0.422 0.368 0.577 0.568 0.607 0.608 1.205 1.258 0.767 0.785 0.607 0.618 0.768 0.765

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

200 a,b

0.104 0.029b 0.097b,c 0.041b,c 0.170a,b 0.054b 0.047a,b 0.011a,b 0.224a,b 0.062a,b 0.062a,b 0.019a,b 0.038b,c 0.016b,c 0.082b,c 0.028b,c 0.156b 0.066b 0.100b 0.052b 0.085a,b 0.017a,b 0.058a,b 0.022a,b 0.085a,b 0.032a,b 0.112a,b 0.50a,b 0.077a,b 0.034a,b 0.057b 0.023b 0.094b 0.030b

0.797 0.770 0.820 1.035 1.352 1.375 0.342 0.270 1.783 1.712 0.458 0.460 0.315 0.352 0.695 0.752 1.298 1.457 0.948 1.013 0.468 0.410 0.472 0.598 0.767 0.572 1.013 1.070 0.672 0.790 0.532 0.577 0.802 0.775

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.043a,b 0.012a,b 0.102c 0.023b,c 0.083b 0.034b 0.024a 0.009a,b 0.100a,b 0.046a,b 0.024a,b 0.011a,b 0.030c 0.005b,c 0.058b,c 0.020b,c 0.113b 0.041b 0.090b 0.015b 0.037a 0.013a,b 0.052b 0.021a,b 0.037a 0.025b 0.116b 0.149b 0.068b 0.026a,b 0.051b 0.011b 0.047b 0.015b

Means for one amino acid not sharing a common superscript are different (P < 0.05).

Table 4. Effects of ZNO NP on trace element contents of egg albumen and yolk. Zn concentration (mg/kg) Treatment group

Items Cu in yolk (ng/g) Cu in albumen (ng/g) Fe in yolk (mg/kg) Fe in albumen (mg/kg) Zn in albumen (mg/kg) P in yolk (mg/kg) P in albumen (mg/kg) a–c

ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP

0 1951.8 ± 21.9

10 a,b

171.0 ± 25.6a,b 78.3 ± 2.7a,b 4.59 ± 1.41a 2.03 ± 0.19a 6683.8 ± 101.5b 123.3 ± 6.4a,b

1888.9 2020.8 191.9 158.9 69.3 76.4 2.56 4.10 1.84 1.29 6714.4 6983.1 115.5 132.8

± ± ± ± ± ± ± ± ± ± ± ± ± ±

25 b

30.6 67.6a,b 18.6b 12.6a,b 1.9b 4.5a,b 0.34b 0.89a,b 0.16a,b 0.13b 48.8b 113.9a 6.9b 4.4a

2090.1 2015.2 170.1 191.0 77.8 80.2 2.68 3.18 1.46 1.39 6934.5 6924.6 124.2 135.2

± ± ± ± ± ± ± ± ± ± ± ± ± ±

50 a

60.9 25.2a,b 12.3a,b 21.5a 5.0a,b 5.9a 0.29b 0.42a,b 0.18b 0.15b 43.3a 97.5a 2.5a,b 3.1a

1942.1 2004.8 167.4 131.4 73.6 79.2 2.63 2.70 1.62 1.29 6779.9 7120.7 133.7 128.5

± ± ± ± ± ± ± ± ± ± ± ± ± ±

100 a,b

57.3 50.6a,b 8.1a,b 15.0b 5.0a,b 1.9a,b 0.31b 0.26b 0.22a,b 0.11b 113.1a,b 69.5a 3.4a 4.0a

2035.4 2009.6 175.5 130.7 76.0 75.4 3.63 3.09 1.58 1.45 6554.1 6975.4 134.8 129.2

± ± ± ± ± ± ± ± ± ± ± ± ± ±

200 a,b

88.4 54.7a,b 8.9a,b 13.7b 3.7a,b 2.9a,b 0.73a,b 0.08a,b 0.13b 0.15b 281.9b 177.4a 2.8a 6.1a

2104.6 1993.3 178.7 139.0 79.2 72.1 3.14 2.73 1.51 1.43 6899.8 7019.5 137.6 129.4

± ± ± ± ± ± ± ± ± ± ± ± ± ±

70.7a 72.4a,b 22.8a,b 13.9b 1.5a,b 2.4a,b 0.35a,b 0.32b 0.09b 0.10b 84.0a,b 86.0a 1.2a 5.2a

Means for one parameter not sharing a common superscript are different (P < 0.05).

the important four genes LSS, CYP51A1, NSDH1, and DHCR7 involved in TC synthesis were decreased by ZnO-NP-100 and 200 mg/kg treatments. Two (PCYT1A, PCYT2) of the nine genes CKA, PCYT1A, PCYT1B, PCYT2, BHMT, CHPT1, CEPT1, CHDH, and ETNK related to PC synthesis were reduced by ZnO-NP-100 and 200 mg/kg treatments. Nuclear re-

ceptor subfamily 1, group H, member 3 (NR1H3) is an important regulator for the expression of genes related to lipids synthesis. It was also down regulated by ZnO-NP-100 and 200 mg/kg treatments (Table 5). The data suggested that lipids synthesis in the liver was decreased, which contributed to the reduction of lipids in plasma and egg yolk.

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HRS

ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP ZnSO4 ZnO-NP

0

EGG QUALITY REGULATED BY ZINC OXIDE NANOPARTICLES

9

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Figure 6. Effects of ZnO NP and ZnSO4 treatments on egg yolk: A. TC; B. TG; C. Phospholipids; and D. PC. Y axis is the content (mmol/g set mass), and X axis is the treatment (concentration of Zn; mg/kg). a–c Means for each parameter not sharing a common superscript are different (P < 0.05).

Expression of Genes Involved in Phospholipids Synthesis and Genes Related to Phospholipids Digestion and Lipids Transportation in Ovary and Ganulosa Cells The genes expression of PCYT1A and PCYT2 was decreased in ovarian tissues and granulosa cells (GC) by ZnO-NP-100, 200 mg/kg treatments compared to that in ZnSO4 -200 mg/kg treatment. Another gene,

CEPT1, for phospholipids synthesis was reduced by ZnO-NP-100, 200 mg/kg treatments in the ovary also (Table 5). The gene involved in phospholipids digestion, phospholipase D1 (PLD1), was up regulated by ZnO-NP-100 mg/kg in GC. The gene expression of VLDL/VTG receptor VLDLR, major receptor for lipids transportation to oocytes, was dramatically increased by ZnO-NP-100 and 200 mg/kg treatments in GC which indicated that ovary used the feedback mechanism to increase lipids transportation in order to compensate for the decreased lipids in the blood (Table 5).

10

ZHAO ET AL.

Expression of Genes Involved in Hormones Production in Ovary Reduced

ever there was no difference between ZnSO4 and ZnO NP treatments or the control group (Figure 7F).

The four genes related to steroid hormone production (CYP11A1, StAR, aromatase, and HSD3β 1) were down regulated by ZnO-NP-100 and 200 mg/kg treatments in the ovary. This indicated that ZnO NP might decrease hormones (progesterone, estrogen) production in ovary which contributed to the reduction in yolk lipids.

Activity of Lipoprotein Lipase, Hepatic Lipase, and Pancreatic Lipase in Liver

Plasma Insulin Activity Insulin may regulate the contents of TC, TG, and PC in the blood and therefore insulin was determined in the plasma. ZnSO4 did not affect plasma insulin levels. ZnO-NP-100 and 200 mg/kg treatments increased plasma insulin concentration above that in the ZnSO4 25 and 200 mg/kg treatments (Figure 7E). This indicated that insulin might not the major factor for the reduction of yolk lipids by ZnO NP treatments. Apo-B was also analyzed in the plasma because it is the major component of VLDL to deliver lipids to the yolk. Higher concentrations of ZnO NP and ZnSO4 slightly increased Apo-B in the plasma compared to that in the 25 mg/kg ZnO NP treatment group. How-

ZnSO4 treatments did not affect LPL activity in the liver significantly, however, as the concentration of ZnO NP increased, the activity of LPL in the liver elevated. ZnO-NP-50 and 100 mg/kg treatments significantly increased LPL activity in the liver compared to that in the control or ZnSO4 -100 mg/kg treatments (Figure 8A). HL activity was significantly elevated by ZnSO4 -25, 100, 200 mg/kg and ZnO-NP-10, 50 mg/kg treatments compared to that in the control treatment (Figure 8B; P < 0.05). Another lipids digestion enzyme, PL, was also analyzed in the liver samples. As the concentrations increased, ZnO NP showed a different trend as PL activity was higher in the ZnO-NP-10 and 25 mg/kg groups than in the ZnSO4-10, 25, 50 mg/kg and ZnO-NP-50 mg/kg treatments (Figure 8C; P < 0.05).

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Figure 7. Effects of ZnO NP and ZnSO4 treatments on lipids, insulin and Apo-B in plasma: A. TC; B. TG; C. Phospholipids; and D. PC, Y axis is the content (mmol/g). E. Plasma Insulin activity, Y axis is the activity (mIU/L; F. Apo-B content, Y axis is the concentration (μ g/mL); and X axis is the treatment (concentration of Zn; mg/kg) for all parameters. a–b Means for each parameter not sharing a common superscript are different (P < 0.05).

EGG QUALITY REGULATED BY ZINC OXIDE NANOPARTICLES

11

– 0.005 2.33

0.008 0.031 9.36 1.59 0.001 0.001 10.22 1.98

0.046 0.031 −1.58 −1.72 – –

– −2.17 −2.68 –

0.010 0.001

0.100 0.001 0.001 0.083 −4.16 −5.29 −2.42 −3.45

PC synthesis PCYT1A PCYT2 Transportation VLDLR PCTP PC degradation PLD1 0.004 0.021 0.010 −2.19 −1.73 −2.41 0.032 0.022 0.024 −1.52 −1.34 −1.72

0.001 0.001 0.001 0.002 0.001 0.018

0.090 0.014 0.126

−4.95 −1.79 −2.19 – −2.36

−2.96 −3.22 −3.54 −2.84 −3.09

0.037 0.001 0.050 0.035 −1.71 −1.73

0.022 0.001 0.003

0.017

−3.06 −1.5

0.002 0.001 0.015 0.002 0.001 0.001 0.001 0.012 0.002

PC synthesis PCYT1A PCYT2 CEPT1 Hormone production CYP11A1 StAR Aromatase HSD3b1 – −2.65 −2.23 −1.86 −3.08 −5.07 −3.48 −3.28 −3.36 −2.36 0.170 0.044

−3.38 −1.78 – – −2.12 −9.32 −2.02 – – −1.54

TG synthesis FASN DECr1 Ecl1 ElovL1 ElovL2 ElovL5 ElovL6 ElovL7 Gpam Agpat3 PC synthesis PCYT1A PCYT2 TC synthesis LSS CYP51A1 NSDH1 DHCR7 NRIH3

Fold change p-value Fold change p-value Gene

Fold change P-value Fold change P-value

Gene

Fold change P-value Fold change P-value

NP-200 NP-100

Ovary

NP-200 Liver

NP-100

Table 5. q-RT-PCR data for liver, ovary, and GC.

Numerous studies have reported that ZnO NP cause adverse effects on organisms such as toxicity on Daphnia magna (Poynton et al., 2011; Lopes et al., 2014), zebrafish embryos (Brun et al., 2014), rat reproductive development (Jo et al., 2013), mouse spermatogenesis (Talebi et al., 2013), and human hepatocyte and immune cells (Kim et al., 2013; Tuomela et al., 2013). However, little is known about the effect of ZnO NP on egg quality. Herein, the effects of ZnO nanoparticles on the egg quality were explored and molecular insights regarding ZnO nanoparticles decreasing egg yolk lipids were investigated. Based on the reports, the concentration of Zn added to the chicken diet was from 50 to 1000 mg/kg (NRC, 1994; Attia et al., 2013), and usually 200 mg/kg was added to the diet for hens (Moreng et al., 1992; Southern and Baker, 1983; Kim and Patterson, 2005). Therefore, a low range of Zn (10 to 200 mg/kg diet) was used in this investigation. It was found that ZnO NP did not change the body weight of the hens during the treatment period. However, ZnO NP slowed down egg laying frequency at the beginning of egg laying period but did not alter it at later time. ZnO NP did not affect egg protein or water content, but slightly decreased egg physical parameters and trace elements after 24 weeks treatment. However, yolk lipids content were significantly decreased by ZnO NP. The metabolisms of lipids include three aspects: (1) mainly synthesis in the liver, (2) transportation and degradation in the blood (Holdsworth et al., 1974), and (3) absorption, usage, and storage in the tissues (Fahy et al., 2005; Athenstaedt and Daum, 2006; Huang and Freter, 2015). Most of the yolk lipids are from the liver, however, a very small amount are made by the ovarian follicles (Christie and Moore, 1972). It was found that the blood concentrations of TC, TG, and PC matched well with that in the egg yolk in this study. This suggests that ZnO NP might regulate both lipids synthesis in the liver and degradation in the blood. The genes involved in TG, TC, and PC synthesis were analyzed by q-RT-PCR. The synthesis of lipids is a very long process and different types of lipids are synthesized by different enzymes (Vieira et al., 1995; Zhou et al., 2008) based on lipid type and different other dietary and husbandry factors (Attia et al., 2014). Compared with ZnSO4 treatments, the major genes for TG, TC, and PC synthesis were reduced by ZnO-NP-100 and 200 mg/kg in the liver, ovary, and GC. These data matched very well with the concentrations of TG, TC, and PC in egg yolk and blood. The results suggested that ZnO NP regulate the bio-synthesis of TG, TC, and PC. Estrogen, produced in the ovary and transported to target organs by blood, is a very important hormone for the development of female reproductive systems. Estrogen was found to regulate the synthesis of lipids and lipoproteins in the liver (Vigo and Vance, 1981). CYP11A1 and StAR play key roles in estrogen and progesterone synthesis. Aromatase is the major

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Gene

GC

NP-100

NP-200

DISCUSSION

12

ZHAO ET AL.

enzyme for estrogen production in the ovary. ZnO-NP100 and 200 mg/kg treatments decreased estrogen producing enzymes, which might result in a reduction of estrogen synthesis. This matched very well with the reduced lipids content in the plasma and egg yolk. Further, insulin has also been found to regulate lipids synthesis in the liver. However, it might not play an important roles for lipids synthesis under ZnO NP treatment (Foretz et al., 1999). The enzymes involved in lipids degradation are the lipases. There are three major lipases: PL, LPL, and HL (Scow et al., 1998; Lowe, 1997; Wong and Schotz, 2002; Lowe, 2002). As the names indicate, pancreatic lipase and hepatic lipase are from the pancreas and liver. Lipoprotein lipase is mainly formed in muscles and fat tissues (Camps et al., 1991). However, it has been reported that LPL also can be produced by the liver, and the liver can absorb LPL and PL from the blood (Merkel et al., 1998). Therefore the amount of these enzymes in the liver is representative of their status in the blood. When lipids are released to the blood from the liver, they form a complex with lipoproteins. Lipoproteins serve as vehicles to deliver lipids to other places (Reardon et al., 1982). TG, TC, and PC are delivered to the plasma and oocytes by complexes of very low density lipoprotein (VLDL)/vitellogenin (VTG) and TG/PC, LDL and TC (Nimpf et al., 1998). Lipases cleave the bonds between lipids and lipoprotein, and digest the ester form of lipids to free acids which can be absorbed to the cells. However, ovarian follicles of laying hen are different. Lipids and lipoprotein complexes are directly taken up by the growing follicles (Perry and Gilbert, 1979). Therefore, increased amount of lipases may reduce the amount of lipids and VLDL/VTG or LDL complexes in the blood. The activities of HL, PL, and LPL in the liver were analyzed in this study. The patterns of HL and PL in the liver did not well match the lipids profile in the egg yolk or blood. LPL was in-

creased by ZnO-NP-50, 100 and 200 mg/kg treatments, therefore LPL might contribute to the decrease of TG, TC, and PC in the blood and egg yolk. Next, the transportation of lipids to yolk was investigated. The lipoproteins and lipid complexes are taken up to the oocytes by receptor mediated endocytosis. The major receptor for the lipids transportation to the yolk is VLDLR. It was very interesting to find that VLDLR was up regulated by ZnO-NP-100 and 200 mg/kg in GC. This suggests that the capability of transportation of lipids to the yolk was increased. A possible feedback mechanism is that the oocytes tried to absorb more lipids from the blood in order to compensate for the decrease of lipids in the blood by ZnONP-100 or 200 mg/kg treatments (Vieira et al., 1995). In conclusion, our study has for the first time investigated the effects of ZnO NP on egg quality and has offered molecular insights into how ZnO NP decrease yolk lipids. ZnO NP reduced yolk lipids through decreasing biosynthesis, increasing lipid degradation. Therefore, ZnO NP might cause adverse effects on hen’s reproductive systems and livers. Further mechanistic studies on how NP from ZnO NP exposure get into the liver and ovary, and how NP interact with targets in the organisms to regulate gene expression and enzymes activity need to be explored.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31402256), Qingdao Technology Innovation Program for Application Foundation (14-2-4-22-jch) and QingDao Agricultural University Outstanding Research Foundation. The TEM and EDS work in this manuscript made use of the resources of the Beijing National Center for Electron Microscopy at Tsinghua University. We would

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Figure 8. Effects of ZnO NP and ZnSO4 treatments on lipases in liver: A. LPL; B. HL; and C. PL. Y axis is the activity (IU/mg protein), and X axis is the treatment (concentration of Zn; mg/kg). a–e Means for each parameter not sharing a common superscript are different (P < 0.05).

EGG QUALITY REGULATED BY ZINC OXIDE NANOPARTICLES

like to thank Ms. Hui-Hua Zhou in Tsinghua University for helping on TEM and EDS analysis. We would like to thank Miss Yu-Na Zhang and Mr. Kui Xu from Core Laboratories of Qingdao Agricultural University for metal elements analysis.

CONFLICT OF INTEREST The authors declare that there are no conflicts of interest.

SUPPLEMENTARY DATA

REFERENCES Attia, Y. A., M. A. Al-Harthi, and M. M. Shiboob. 2014. Evaluation of quality and nutrient contents of table eggs from different sources in the retail market. Ital. J. Anim. Sci. 13:369. Attia, Y. A, E. M. Qota, H. S Zeweil, F. Bovera, A. E. Abd AlHamid, and M. D. Sahledom 2013. Effect of dietary amounts of inorganic and organic zinc on productive and physiologic traits of White Pekin ducklings. Animal. 7:895–900. Alsaffar, A. A., Y.A Attia, M. B. Mahmoud H. S. Zewell, and F. Bovera. 2013. Productive and reproductive performance and egg quality of laying hens fed diets containing different levels of date pits with enzyme supplementations. Trop. Anim. Health Prod. 45:327–334. Athenstaedt, K., and G. Daum. 2006. The life cycle of neutral lipids: synthesis, storage and degradation. Cell Mol. Life Sci. 63:1355– 1369. Bargheer, D., A. Giemsa, B. Freund, M. Heine, C. Waurisch, G. M. Stachowski, S. G. Hickey, A. Eychm¨ uller, J. Heeren, and P. Nielsen. 2015. The distribution and degradation of radiolabeled superparamagnetic iron oxide nanoparticles and quantum dots in mice. Beilstein. J. Nanotechnol. 6:111–123. Bondarenko, O., K. Juganson, A. Ivask, K. Kasemets, M. Mortimer, and A. Kahru. 2013. Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: a critical review. Arch Toxicol. 87:1181– 1200. Brun, N.R., M. Lenz, B. Wehrli, and K. Fent. 2014. Comparative effects of zinc oxide nanoparticles and dissolved zinc on zebrafish embryos and eleuthero-embryos: importance of zinc ions. Sci. Total Environ. 476–477:657–666. Camps, L., M. Reina, M. Llobera, G. Bengtsson-Olivecrona, T. Olivecrona, and S. Vilar´ o. 1991. Lipoprotein lipase in lungs, spleen, and liver: synthesis and distribution. J. Lipid Res. 32:1877–1888. Chen, T., C. Lin, and P. Meng. 2014. Zinc oxide nanoparticles alter hatching and larval locomotor activity in zebrafish (Danio rerio). J. Hazard Mater. 277:134–140. Cho, W. S., B. C. Kang, J. K. Lee, J. Jeong, J. H. Che, and S. H. Seok. 2013. Comparative absorption, distribution and excretion of titanium dioxide and zinc oxide nanoparticles after repeated oral administration. Part Fibre Toxicol. 10:9. Choi, J., H. Kim, P. Kim, E. Jo, H. M. Kim, M. Y. Lee, S. M. Jin, and K. Park. 2015. Toxicity of zinc oxide nanoparticles in rats treated by two different routes: single intravenous injection and single oral administration. J. Toxicol. Environ. Health. A78:226– 243. Christie, W. W., and J. H. Moore. 1972. The lipid components of the plasma, liver and ovarian follicles in the domestic chicken (Gallus gallus). Comp. Biochem. Physiol. B41:287–295.

Fahy, E., S. Subramaniam, H. A. Brown, C. K. Glass, A. H. Merrill, R. C. Murphy, C. R. Raetz, D. W. Russell, Y. Seyama, W. Shaw, T. Shimizu, F. Spener, G. van Meer, M. S. VanNieuwenhze, S. H. White, J. L. Witztum, and E. A. Dennis. 2005. A comprehensive classification system for lipids. J. Lipid Res. 46:839–861. Faust, J., W. Zhang, Y. Chen, and D. Capco. 2014. Alpha-Fe(2)O(3) elicits diameter-dependent effects during exposure to an in vitro model of the human placenta. Cell Biol. Toxicol. 30: 31–53. Foretz, M., C. Guichard, P. Ferr´e, and F. Foufelle. 1999. Sterol regulating element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes. Proc. Natl. Acad. Sci. USA. 96: 12737–12742. Holdsworth, G., R. H. Michell, and J. B. Finean. 1974. Transfer of very low density lipoprotein from hen plasma into egg yolk. FEBS Lett. 39:275–277. Hong, J. S., M. K. Park, M. S. Kim, J. H. Lim, G. J. Park, E. H. Meng, J. H. Shin, M. K. Kim, J. Jeong, J. A. Park, J. C. Kim, and H. C. Shin. 2014. Prenatal development toxicity study of zinc oxide nanoparticles in rats. Int. J. Nanomedicine, 9 (Suppl 2): 159–171. Huang, C., and C. Freter. 2015. Lipid Metabolism, Apoptosis and Cancer Therapy. Int. J. Mol. Sci. 16:924–949. Consortium, International Chicken Genome Sequencing. 2004. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432: 695–716. Jo, E., J. G. Seo, T. Kwon, M. Lee, B. C. Lee, I. Eom, P. Kim, and K. Choi. 2013. Exposure to zinc oxide nanoparticles affects reproductive development and biodistribution in offspring rats. J. Toxicol. Sci. 38:525–530. Kim, A. R., F. R. Ahmed, G. Y. Jung, S. W. Cho, D. I. Kim, and S. H. Um. 2013. Hepatocyte cytotoxicity evaluation with zinc oxide nanoparticles. J. Biomed. Nanotechnol. 9:926–929. Kim, W. K., and P. H. Patterson. 2005. Effects of dietary zinc supplementation on hen performance, ammonia volatilization, and nitrogen retention in manure. J. Environ. Sci. Health B. 40:675– 686. Laudadio, V., E. Ceci, N.M.B. Lastella, and V. Tufarelli. 2014. Effect of feeding low-fiber fraction of air-classified sunflower (Helianthus annus L.) meal on laying hen productive performance and egg yolk cholesterol. Poult Sci. 93:2864–2869. Lopes, S., F. Ribeiro, J. Wojnarowicz, W. L  ojkowski, K. Jurkschat, A. Crossley, A. M. Soares, and S. Loureiro. 2014. Zinc oxide nanoparticles toxicity to Daphnia magna: size-dependent effects and dissolution. Environ. Toxicol. Chem. 33:190–198. Lowe, M.E. 1997. Structure and function of pancreatic lipase and colipase. Annu. Rev. Nutr. 17:141–158. Lowe, M.E. 2002. The triglyceride lipases of the pancreas. J. Lipid Res. 43:2007–2016. Ma, H., P. L. Williams, and S. A. Diamond. 2013. Ecotoxicity of manufactured ZnO nanoparticles–a review. Environ. Pollut. 172:76– 85. Merkel, M., P. H. Weinstock, T. Chajek-Shaul, H. Radner, B. Yin, J. L. Breslow, and I. J. Goldberg. 1998. Lipoprotein lipase expression exclusively in liver, A mouse model for metabolism in the neonatal period and during cachexia. J. Clin. Invest. 102:893– 901. Miranda, J.M., X. Anton, C. Redondo-Valbuena, P. Roca-Saavedra, J. A. Rodriguez, A. Lamas, C. M. Franco, and A. Cepeda. 2015. Egg and Egg-Derived Foods: Effects on Human Health and Use as Functional Foods. Nutrients 7:706–729. Moran, E.T. 2007. Nutrition of the developing embryo and hatchling. Poult. Sci. 85:1043–1049. Moreng, R. E., D. Balnave, and D. Zhang. 1992. Dietary zinc methionine effect on eggshell quality of hens drinking saline water. Poult Sci. 71:1163–1167. Ndawula, J., J. D. Kabasa, and Y. B. Byaruhanga. 2004. Alterations in fruit and vegetable beta-carotene and vitamin C content caused by open-sun drying, visqueen-covered and polyethylene-covered solar-dryers. Afr. Health Sci. 4:125–130. Nimpf, J., R. George, and W. J. Schneider. 1998. Apolipoprotein specificity of the chicken oocyte receptor for low and very low

Downloaded from http://ps.oxfordjournals.org/ at University of California, Santa Barbara on February 18, 2016

Table S1. Ingredient composition of the basal diet for the layers Table S2. Primer sequences for q-RT-PCR Supplementary data is available at PSA Journal online.

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ZHAO ET AL. breeders and post hatch growth of their offsprings. Biol. Trace Elem. Res. 145:318–324. Talebi, A.R., L. Khorsandi, and M. Moridian. 2013. The effect of zinc oxide nanoparticles on mouse spermatogenesis. J. Assist. Reprod. Genet. 30:1203–1209. Tuomela, S., R. Autio, T. Buerki-Thurnherr, O. Arslan, A. Kunzmann, B. Andersson-Willman, P. Wick, S. Mathur, A. Scheynius, H. F. Krug, B. Fadeel, and R. Lahesmaa. 2013. Gene expression profiling of immune-competent human cells exposed to engineered zinc oxide or titanium dioxide nanoparticles. PLoS One. 8: e68415. Vieira, P.-M., A.-V. Vieira, E.-J. Sanders, E. Steyrer, J. Nimpf, and W.-J. Schneider. 1995. Chicken yolk contains bona fide high density lipoprotein particles. J. Lipid Res. 36: 601–610. Vigo, C., and D. E. Vance. 1981. Effect of diethylstilboestrol on phosphatidylcholine biosynthesis and choline metabolism in the liver of roosters. Biochem. J. 200:321–326. ˇ Vinkl´ arkov´ a, B., V. Chrom´ y, L. Sprongl, M. Bittov´ a, M. Rikanov´ a, ˇ I. Ohn´ utkov´ a, and L. Zaludov´ a. 2015. The Kjeldahl Method as a Primary Reference Procedure for Total Protein in Certified Reference Materials Used in Clinical Chemistry. II. Selection of Direct Kjeldahl Analysis and Its Preliminary Performance Parameters. Crit. Rev. Anal. Chem. 45:112–118. Wong, H., and M. C. Schotz. 2002. The lipase gene family. J. Lipid Res. 43:993–999. Xiao, J.F., S.G. Wu, H.J. Zhang, H.Y. Yue, J. Wang, F. Ji, and G.H. Qi. 2015. Bioefficacy comparison of organic manganese with inorganic manganese for eggshell quality in Hy-Line Brown laying hens. Poult Sci. 94:1871–1878. Yamashita, K., Y. Yoshioka, K. Higashisaka, K. Mimura, Y. Morishita, M. Nozaki, T. Yoshida, T. Ogura, H. Nabeshi, K. Nagano, Y. Abe, H. Kamada, Y. Monobe, T. Imazawa, H. Aoshima, K. Shishido, Y. Kawai, T. Mayumi, S. Tsunoda, N. Itoh, T. Yoshikawa, I. Yanagihara, S. Saito, and Y. Tsutsumi. 2011. Silica and titanium dioxide nanoparticles cause pregnancy complications in mice. Nat. Nanotechnol. 6:321–328. Yang, X., H. Shao, W. Liu, W. Gu, X. Shu, Y. Mo, X. Chen, Q. Zhang, and M. Jiang. 2015. Endoplasmic reticulum stress and oxidative stress are involved in ZnO nanoparticle-induced hepatotoxicity. Toxicol. Lett. 234:40–49. Zhou, Y., X. Zhang, L. Chen, J. Wu, H. Dang, M. Wei, Y. Fan, Y. Zhang, Y. Zhu, N. Wang, M. D. Breyer, and Y. Guan. 2008. Expression profiling of hepatic genes associated with lipid metabolism in nephrotic rats. Am, J. Physiol. Renal. Physiol. 295:662–671.

Downloaded from http://ps.oxfordjournals.org/ at University of California, Santa Barbara on February 18, 2016

density lipoproteins: lack of recognition of apolipoprotein VLDLII. J. Lipid Res. 29:657–667. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Osmond-McLeod, M. J., Y. Oytam, J. K. Kirby, L. GomezFernandez, B. Baxter, and M. J. McCall. 2014. Dermal absorption and short-term biological impact in hairless mice from sunscreens containing zinc oxide nano- or larger particles. Nanotoxicology Suppl 1:72–84. Palacios, L.E., and T. Wang. 2005. Egg-yolk lipid fractionation and lecithin characterization. J. the American Oil Chemists’ Society 82:571–578. Perry, M. M., and A. B. Gilbert. 1979. Yolk transport in the ovarian follicle of the hen (Gallus domesticus): lipoprotein-like particles at the periphery of the oocyte in the rapid growth phase. J. Cell Sci. 39:257–272. Poynton, H. C., J. M. Lazorchak, C. A. Impellitteri, M. E. Smith, K. Rogers, M. Patra, K. A. Hammer, H. J. Allen, and C. D. Vulpe. 2011. Differential gene expression in Daphnia magna suggests distinct modes of action and bioavailability for ZnO nanoparticles and Zn ions. Environ. Sci. Technol. 45:762–768. Reardon, M.F., H. Sakai, and G. Steiner. 1982. Roles of lipoprotein lipase and hepatic triglyceride lipase in the catabolism in vivo of triglyceride-rich lipoproteins. Arteriosclerosis 2: 396–402. Santo, N., U. Fascio, F. Torres, N. Guazzoni, P. Tremolada, R. Bettinetti, P. Mantecca, and R. Bacchetta. 2014. Toxic effects and ultrastructural damages to Daphnia magna of two differently sized ZnO nanoparticles: does size matter. Water Res. 53: 339–350. Scow, R. O., C. J. Schultz, J. W. Park, and E. J. BlanchetteMackie. 1998. Combined lipase deficiency (cld/cld) in mice affects differently post-translational processing of lipoprotein lipase, hepatic lipase and pancreatic lipase. Chem. Phys. Lipids. 93: 149–155. Sieber, M. H., and A. C. Spradling. 2015. Steroid Signaling Establishes a Female Metabolic State and Regulates SREBP to Control Oocyte Lipid Accumulation. Curr. Biol. 25:993–1004. Stone, V., and K. Donaldson. 2006. Nanotoxicology: signs of stress. Nat. Nanotechnol. 1:23–24. Southern, L. L., and D. H. Baker. 1983. Zinc toxicity, zinc deficiency and zinc-copper interrelationship in Eimeria acervulina-infected chicks. J. Nutr. 113:688–696. Sun, Q., Y. Guo, S. Ma, J. Yuan, S. An, and J. Li. 2012. Dietary mineral sources altered lipid and antioxidant profiles in broiler