Animals, Experimental Design and Treatments
Moringa oleifera leaf powder (MOLP) were obtained from Yunnan Dayaoshan Trading Co.,LTD.(Yunnan, China), and mulberry leaf powder (MLP) were obtained from Danyang Tianyuan Shengshu Ecological Park Co.,LTD. (Zhenjiang, China). Moringa oleifera leaf and mulberry leaf powder were picked, dried, crushed, sifted and stored. The contents of crude protein, crude fat and crude fiber in MOLP were 26.95%, 5.76% and 19.26%, respectively. The contents of crude protein, crude fat and crude fiber in MLP were 13.79%, 1.98% and 24.89%, respectively. The basic diet is a corn-soybean meal diet provided by China Oil and Foodstuffs Corporation. Our experimental animals were provided by Jiangsu Institute of Poultry Science. A total of 210 birds of F1 generation produced from Wenchang Chicken and Rugao Yellow Chicken were randomly assigned to three groups each group consisted of 5 replicates (14 birds each replicate). These groups are: C0. basal corn-soybean meal, T1. feed supplemented with 2.5% MOLP +2.5% MLP, T2. 5% MOLP +2.5% MLP. The experiments lasted 7 weeks including 1week adaptation. The ingredients and chemical composition of the feed are shown in Table 1.
Management of Experimental Birds
All animal care and experimental procedures were approved by the Institutional Animal Care and Use of Committee of Jiangsu University of Science and Technology. Our experiment was carried out in Poultry Institute, Chinese Academy of Agricultural Sciences. All hens were raised in single cage and ad libitum, with the photoperiod regime was 16L:8D throughout the study.
Sample Collection and Analytical Determination
Laying Performance
Daily egg production was monitored during the trail, average egg weight and feed intake were recorded weekly. Laying rate is expressed as average hen-day production, calculated from the total number of eggs divided by the total number of days. Feed intake was recorded weekly and their conversion was determined.
Egg Quality
Freshly laid eggs were collected at the end of 6th week. The internal and external egg quality of 6 randomly selected eggs per group (6 eggs/ replicate) were measured. The eggs were stored at room temperature before measurement. The length and width of the eggs were measured using the electronic digital caliper and the egg shape index (ESI) were calculated (length /width ×100). Eggshell thickness (EST) was measured using the eggshell thickness tester ESTG-1(ORKA Co. Ltd.,) at the blunt, equatorial, and sharp regions to obtain an average value. Eggshell color (ESC) was measured using the spectrophotometer CM-2300D (MINOLTA Co. Ltd.,) and three traits were recorded: lightness of eggshell L*, redness of eggshell a* and yellowness of eggshell b*. Eggshell Strength (ESS) was evaluated using the EggShell Force Gauge EFR-01(ORKA Co. Ltd.,). Egg weight (EW), albumen height (AH), Haugh unit (HU), and yolk color (YC) were measured using the Egg Multi Tester EA-01(ORKA Co. Ltd.,). Then, the yolk weight (YW) and yolk rate (YR) was calculated. Eggshell weight (ESW) is weighed after natural drying.
Sample collection
Thirty hens (2 hens/per replicate, 10 hens per group) were selected after 12 hours fasting at the end of 6th week. Blood were collected from wing vein. Serum were obtained by centrifugation at 4000r/min for 10min of the blood and stored at -20℃. Then the hens were humanely killed by carbon dioxide overdose, and their internal organ including heart, liver, spleen, lung, kidney, abdominal fat were removed and measured, their index was calculated by the following formula: internal organ index% = (internal organ weight/body weight) × 100. Specially, liver tissues were collected and stored at -80℃ until assayed for antioxidant or lipid indicators and their related genes expression analysis.
Antioxidant and lipid indicators
Livers samples were homogenized with saline to make a 10% homogenate with 0.9% sodium chloride buffer with tube embed in ice and centrifuged at 4000 rpm at 4℃ for 10 min. The serum and liver supernatant were used to measure MDA, SOD, T-AOC, GSH, TG, T-CHO, HDLC, LDLC by ELISA method, using commercial kits bought from Nanjing Jiancheng Bioengineering Institute, Nanjing, China.
RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction
Total RNA of liver was extracted using Trizol Reagent (TaKaRa Biotechnology, Dalian, Liaoning, China). Quality and integrity of RNA was assessed by Nanodrop ND- 2000c spectrophotometer (Thermo Scientific, Camden, NJ). The reverse transcription was carried out according to Takara reverse transcription kit protocol (Perfect Real Time, PrimeScriP™ TaKaRa, China). The reaction conditions of reverse transcription were as follows: reaction at 37 ℃ for 15 min, deformation at 85 ℃ for 15 s, and finally cooling to 4 ℃. The real-time quantitative polymerase chain reaction was carried out using the SYBR Premix Ex Taq II kit (TaKaRa,Dalian, China) in an ABI 7300 fluorescence quanti-tative polymerase chain reaction instrument (Applied Biosystems, Foster City, CA). The 20μL reaction system included 10 μL of SYBR Premix Ex Taq buffer, 0.4μL each of forward and reverse primers and ROX,1μL of cDNA template, and 7.8 μL of distilled water. The real-time polymerase chain reaction cycling conditions were as follows: 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 31 s. The relative mRNA expression was determined using β-actin as an internal reference gene. The significance and correlation of quantitative results were analyzed using 2-ΔΔct [17]. Primer sequences are shown in Table 2.