All procedures of our experiments were approved by animal care and welfare committee institute of South China Agricultural University.
Experimental design and diets
Embryonic development and Zn mobilization (Exp. 1) Fertile eggs (n = 72) from 33-wk-old female Muscovy duck breeders were obtained from a commercial breeder farm (WENS Group, Yunfu, Guangdong). Twelve egg embryos, representing the weight distribution of the eggs at set, were selected at incubation day 17 (E17), E20, E23, E26, E29, and E32 (day of hatch) respectively, with 6 replicates of 2 embryo per replicate. The egg, embryo, liver, yolk sac, and yolk-free embryo were weighed and the YSC was separated from the yolk sac, and then weighed and homogenized. The yolk sac and liver were stored at -20°C for Zn analyses. Small pieces of embryonic liver samples from E17, 23, and 29 were rinsed in a 0.9% autoclaved-saline solution and placed in microcentrifuge tubes at -80°C for analysis of mRNA expression of genes related to Zn absorption and transport.
Prediction and effect of maternal marginal Zn deficiency (Exp. 2). A total of 370 26-wk-old Muscovy duck breeders obtained from a commercial duck breeder farm (WENS Group, Yunfu, Guangdong) were housed in caged system for 4 wk period of adaptation. During the adaptation period, all ducks were fed restrictively with a commercial feed at the nutritional level (11.32 MJ metabolizable energy/kg, 180 g crude protein/kg, 7.0 g lysine/kg, 7.2 g methionine + cysteine/kg, 24.0 g calcium/kg, 3.8 g available phosphorus/kg, 40 mg Zn/kg). At 30 wk of age, 324 laying duck breeders were selected, balanced for laying rate and then randomly allotted into 3 dietary Zn levels with 6 replicates of 18 ducks per replicate. The experimental diets included 3 dietary supplemental Zn levels of 0 mg/kg (maternal Zn-deficient group, MZD), 60 mg/kg (maternal Zn-adequate group, MZA), 120 mg/kg (maternal Zn-high group, MZH) as inorganic Zn source. The basal diet was formulated to meet or exceed the nutritional requirements of laying duck breeders except Zn. The composition of the basal diet was shown in Table 1. The analyzed values of Zn contents in diets of MZD, MZA and MZH were 29.2, 63.4, and 163.4 mg/kg respectively. All birds had diets restriction and access to water ad libitum according to the operation manual and guideline of Muscovy duck breeders. The ducks received 16 h of daily lighting from 04:30 am to 20:30 pm. Room temperature and humidity were controlled by the air-conditioner and recorded daily. Manure was removed through an automatic belt system daily. All eggs were collected from each replicate daily and egg production (number of total laid eggs, defective eggs, and average qualified egg weight) were recorded daily. Feed consumption and egg weight were measured weekly. Feed intake was calculated by dividing total feed consumed by numbers of ducks per replicate per day. The eggs were incubated were incubated at a temperature of 37.8°C and a relative humidity of 55 to 60% for 28d. Eggs were candled at E7 and E28 to identify nonviable embryos. All removed eggs on E7 and E28 were counted, opened, and visually evaluated also to determine the true embryonic mortality.
The feed ingredients and diet samples from all the treatments were collected and analyzed for crude protein, calcium, phosphorus, and Zn contents. In each replicate cage, blood samples were collected via a bronchial vein from 2 fasted female duck breeders in each replicate on the last day of each period. Blood samples were separated into plasma and erythrocytes by centrifugation at at 3,000 × g for 15 min at 4°C. Erythrocytes samples were washed three times in cold isotonic saline (0.9 %, v/w), then haemolyzed with a nine-fold volume of phosphate buffer (pH 7.4). The plasma and haemolyzed erythrocytes were stored at -20°C for further analysis.
Twelve eggs from each treatment (2 per replicate) were collected at the last day of 36 wk of age. The separated yolk from 2 eggs per replicate were pooled together and stored at -20°C for Zn analysis. On E29, 24 embryos (4 per replicate) from each treatment were killed by cervical dislocation. Equal weight sub-samples of the livers from the 4 embryos in each replicate were pooled into one sample for analyses. Total one gram liver sample of each replicate was homogenized at 8000 g for 10 sec in 9 mL of 0.9% sodium chloride buffer on ice and centrifuged at 3000 g at 4°C for 15 min, and the resultant supernatant was used for the analyses of antioxidant ability. The liver samples from the embryos were immediately dissected and frozen in liquid nitrogen and then stored at -80°C for further analyses of gene and protein expressions.
Determination of Zn concentration.
Zinc concentrations in samples including diets, plasma, egg yolk, yolk sac, and embryonic liver were measured using an inductively coupled plasma emission spectroscope (model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA) after wet digestions with HNO3 and HClO4 as described by . The total Zn contents of yolk sac and embryonic liver were calculated by multiplying Zn concentration and weight. The relative Zn mobilization rates were calculated as the ratio of Zn content in released Zn from yolk sac per day on average during E17-20, E20-23, E23-26, E26-29, and E29-32, respectively.
Determination of Zn metalloenzyme activities in erythrocytes
ALP activity was measured using a HITACHI 7180 automatic biochemical analyzer (Hitachi Ltd., Tokyo, Japan) with detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). CuZnSOD activity was determined by subtracting manganese superoxide dismutase (MnSOD) activity from total SOD (TSOD) activity according to the nitrite method . 5’-NT activity was assayed by the determination of the Pi liberated from the substrate nuclcotide as described by . Total protein concentration in erythrocytes was determined using a BCA Protein Assay Kit (Cat no. 23225; Pierce). All indices of erythrocytes were expressed as nitrite units per milligram protein.
Determination of indices related to the oxdative damage
The activity of superoxide anion radical production was calculated and expressed as percentage of control (Vitamin C) based on the inhibition rate of superoxide anion radicals from the Xanthine and xanthine oxygenase reaction following the instruction of a commercial assay (A052-1-1, Nanjing Jiancheng Institute of Bioengineering, Jiangsu, China). The malondialdehyde (MDA) and protein carbonyl content (PCC) were determined by thiobarbituric acid colorimetric (A003, Nanjing Jiancheng Institute of Bioengineering) and 2, 4-dinitrophenylhydrazine methods according to kits (A087, Nanjing Jiancheng Institute of Bioengineering), respectively. The 8-hydroxy-2-deoxyguanosine (8-OHdG) was determined using with a commercially available ELISA test kits (H165, Nanjing Jiancheng Institute of Bioengineering). All indices of supernatant were expressed as nitrite units per milligram protein.
Determination of antioxidant enzymes activities
Supernatant of the liver homogenization solution was used to measure the activities of glutathione peroxidase (GSH-Px) and catalase (CAT) using the commercial kits (Nanjing Jiancheng Bioengineering Institute) according to the instructions of the manufacturer. The total SOD and MnSOD activities were measured following the nitrite method described by Zhu et al. (2015), and CuZnSOD activity was calculated by subtracting MnSOD activity from total SOD activity. MT content was determined using a duck MT ELISA Kit (CG3309, Waltham, MA, USA).
RT-qPCR for gene mRNA expression
Total RNA was extracted from the embryonic liver tissues using Trizol reagent (Cat #15596018, Life Technologies) and then reverse-transcription was performed using QuantiTech Reverse Transcription Kit (Cat #205311, Qiagen) following the manufacturer’s protocols with genomic DNA wiping off. The protocol of two-step PCR using ABI Power SYBR Green PCR Master Mix was conducted as described previously . The primer sequences are listed in Supplementary Supplemental Table 1. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used to normalize the expressions of the targeted genes. The 2−△△Ct was used to calculate mRNA level of each target gene.
Western blotting for protein expression
Total protein was extracted with ice-cold RIPA lysis buffer (Cat #P0013B, Beyotime Institute of Biotechnology, Haimen, China). The procedure following the preparation of the protein sample and SDS-PAGE, blotting transfer and detection of the protein specific antibodies were performed as described previously (26). The primary antibodies are listed in Supplemental Table 2.
Data of other indices were analyzed by one-way using the PROC GLM procedure of the SAS (SAS Inst. Inc., Cary, NC). Additionally, the significant effect of dietary Zn on plasma Zn concentration and Zn metalloenzyme activities in erythrocytes was analyzed for each week. The replicate served as the experimental unit. Differences among means were tested by the LSD method, and statistical significance was set at P ≤ 0.05.