All procedures adapted for the experiment were approved by the Animal Ethics Committee of China Agricultural University, Beijing, China.
Experimental design and animal management
A total of 270 Hy-line gray layer hens with 21-week-old weights and similar egg production rates were selected and housed in a conventional stepped cage in a closed house. The cages were arranged in 3 tiers with 5 cages per tier and 3 birds per cage. One week pre-feeding was carried out, the control group was fed during the pre-feeding. The diet formula of laying hens was formulated with reference to NY /T33-2004 (table 1). After an acclimation period, 270 22-week-old Hy-line Grey hens were randomly divided into three treatment groups according to the principle of uniform egg production rate (47% ± 0.02%) and similar weight (1470 ± 10 g). Control group (feeding basic diet with low soybean meal), 50 ppm SS group [Basic diet supplemented with 50 mg/kg SS], 500 ppm SS group [Basic diet supplemented with 500 mg/kg SS]. There were 6 replicates per treatment and 15 birds per replicate. The test SS was purchased from Xi'an Tongze Biotechnology Co., Ltd. (total SS content is 45.1%). The formal test period was 10 weeks, artificial feeding and the nipple drinker supplies water were used during the test. Eggs were collected and weighed once at 4 pm every day. The temperature of laying hen room was controlled at 25 ± 3°C, besides, 16h light: 8h dark lighting program was used. During formal testing, egg quality was measured every three weeks, egg production and feed efficiency were calculated every week. At the end of the 5th week, eight laying hens with uniform body weight and egg production rate were selected to collect whole blood from the underwing vein, and then extracted lymphocytes and separated serum for detection and analysis. At the same time, six layers with uniform body weight and egg production rate from each group were anesthetized with 50 mg/kg BW of sodium pentobarbital, and then slaughtered to obtain ovaries, fallopian tubes and ileal chyme for detection and analysis. At the end of the trial, eight layers with uniform body weight and egg production rate from each group were selected to be collected blood, and then anesthetized and slaughtered to collect ovaries, fallopian tubes, hypothalamus, liver and spleen for testing and analysis.
Determination of production performance and egg quality
Calculating egg production rate, feed consumption and feed egg ratio in weeks. Egg production rate (%) = total number of eggs laid during the statistical period / (number of housed hens × number of statistical days) × 100%. Average egg production rate during the test period (%) = total number of eggs laid during the test period / (number of hens housed × total days of the test) × 100%. Feed-to-egg ratio (FCR)= total material consumption during the test / total egg weight during the test.
All eggs from each treatment within 24h were randomly extracted to detect eggshell thickness, eggshell strength, Haugh units, albumen height and egg yolk color. Using the egg quality tester DET-6000 (NABEL Co., Ltd, Japan) to measure the eggshell strength (kg/cm2) and egg yolk color after weighing the eggs. Specifically, placing the egg vertically on the eggshell strength tester, with the blunt end up, to measure the pressure on the eggshell surface per unit area. albumen height was determined with the albumen height measuring instrument KIYA-818B (SEISAKUSHO, LTD), and then the Haugh unit was calculated according to the formula. This formula=100 Log(H-1.7W0.37+7.57), where H= the albumen height (mm) and W= the egg weight (g). The thickness of the eggshell was measured with a micrometer. Specifically, taking 3 parts (large, medium and small) from each egg after removing the shell membrane from the eggshell to take the average value after the measurement. By the way, the average value was in millimeters, accurate to 0.01 mm.
Organ index and liver morphology
The ovaries, oviducts, liver and spleen were weighted with an electronic balance (accurate 0.01g). Using a ruler (accurate 0.01 mm) to measure the length of ovary, the total length of the oviduct, the length of the magnum and the shell gland. Ovarian, oviduct and liver weight index (%) = weight of ovary, oviduct and liver (g) / live body weight of birds (g) × 100%. Spleen weight index (‰) = weight of spleen (g) / live body weight of birds (g) × 1000%. At the end of the trial, the samples about 1 cm2 of the liver tip were collected and then suspend it into 4% paraformaldehyde solution. Liver sections were made and then stained with eosin-hematoxylin (HE stain). The OLYMPS BX-41TF microscope was used to observe the infiltration state of inflammatory cells in liver slices. The magnification was 400 times.
Determination of serum hormone levels, immune indexes and biochemical indexes
Blood was collected from the wing vein, and then, serum was separated by centrifugation at 3000 rpm and 4°C for 15 minutes. The contents of Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Estradiol (E2) and Progesterone (P4) in serum were detected by radioimmunoassay according to the Protocols of the kit (from Beijing Northern Biotechnology Institute, Beijing). Detection coefficient of variation was less than 10%. According to the steps of the kit instructions of Nanjing Jian cheng Biotechnology Co., Ltd., an automatic biochemical analyzer (Unicel DXC800, Beckman Coulter, USA) were used to determine the total serum protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and glucose (GLU) contents of laying hens at the end of the trial. The level of globulin was total protein minus albumin.
At the end of the trial, the kits from Nanjing Jian cheng Biotechnology Co., Ltd. were used to detect the contents of lysozyme (LZM) and complement C3 in the serum. The chicken β-defensin 1 enzyme-linked immunoassay kit from Beijing Konka Hong yuan Biotechnology Co., Ltd. was used to detect the content of β-defensin in the serum. The kits from Beijing Solarbio Biotechnology Co., Ltd. were used to detect the levels of immunoglobulin G, immunoglobulin A and immunoglobulin M in the serum. ELISA kits (IDEXX laboratories lnc., Weatbrook, Maine, USA) were used to determine the levels of interleukin-2, interleukin-6, IL-4 and IFN-γ in the serum at 5th and 10th week. The ratio of IFN-γ to IL-4 was calculated.
Peripheral blood lymphocyte ratio and stimulation index
According to the method of Fan Hao (2018) [7], chicken peripheral blood lymphocyte separation solution from Tianjin Hao yang Biological Co., Ltd. was used to separate lymphocytes, and then red blood cell lysate was used to lyse red blood cells. The separated lymphocytes were washed twice in hanks buffer without calcium and magnesium, and then resuspended in RPMI-1640 complete medium. The cell counter was used to make sure the cell concentration as 1×106 cell/m L. Lymphocytes were mixed with CD3, Monocytes/Macrophages or Bu-1 antibodies purchased from Southern Biotech and bathed in water at 37°C for 30 minutes. Hanks solution was used to wash it twice, and then it was fixed with 3% paraformaldehyde. Four-colour flow cytometric analysis was conducted using a Navios EX flow cytometer with 10 colors (Beckman Coulter Corp., Fullerton, CA, USA) at Xi-Yuan Traditional Chinese Medicine Hospital, Chinese Academy of Medicine Science, China. The percentages of CD3+ T cells, Monocytes/Macrophages, and Bu-1 were subsequently calculated. The result was expressed as a percentage. The MTT method was used to determine the stimulation indexs of Concanavalin A (ConA, 45μg/mL) on T cells and lipopolysaccharide (LPS, 25μg/mL) on B cells. The results were expressed in terms of the stimulus index (SI) value [43]. Both ConA and LPS were purchased from Sigma-Aldrich.
Gene expression measurement and analysis
Hypothalamus, ovarian, liver, and spleen samples were collected and placed in RNase-free Centrifuge tube, and then the samples were quickly placed in liquid nitrogen. Taking 100 mg tissue sample into 1mL trizol (Invitrogen Life Technologies, Carlsbad, USA), Total RNA isolation, quantification, cDNA synthesis, and real-time PCR were carried out as previously described Hou (2013) [53]. Total RNA was quantified by using the NanoDrop® ND-2000 UV-VIS spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at an OD of 260 nm, and the purity was assessed by determining the OD260/OD280 ratio. All the samples had an OD260/OD280 ratio above 1.8, corresponding to 90-100% pure nucleic acids. Meanwhile, the integrity of RNA in each sample was assessed using 1% denatured agarose gel electrophoresis. RNA was used for RT-PCR analysis when it had a 28 S/18 S rRNA ratio ≥ 1.8. Total RNA was reverse-transcribed using the PrimeScript® RT reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer's instruction. cDNA was synthesized and stored at -80℃ until use. The RT-PCR analysis of gene expression was performed using primers listed in table 2, and the SYBR® Premix Ex TaqTM (Takara, Dalian, China) on an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA, USA). The total volume of the PCR reaction system was 20 µL. Amplification products were verified by melting curves, agarose gel electrophoresis, and direct sequencing. Results were analyzed by the cycle threshold (CT) method from Fu (2010) [8].
Microbial sequencing and analysis
The ileum chyme of laying hens was collected at end of 5th week. Sequencing and analysis according to the method described by Zhang (2018) [51]. The method was briefly described as that fecal microbial DNA extraction kit (QIAamp Fast DNA Stool Mini Kit, Qiagen Company, Germany) was used to extract microbial DNA from ileal chyme. NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA) was used to determine the concentration of DNA samples, after which 1% agarose gel electrophoresis was used to detect the purity of DNA samples. 16SrDNA gene V3-V4 region universal primers 338 F (5'-ACTCCTACGGGAGGCAGCA-3') and 806 R (5'-GGACTACHVGGGTWTCTAAT-3') were used to amplify bacterial DNA, and then the PCR products were purified, quantified and homogenized to form a sequencing library. HiSeq2500 PE250 was used for on-machine sequencing. Sequencing analysis was completed by Beijing Nuohe Zhiyuan Bio-Information Technology Co., Ltd. Qiime software (Qiime2-2019.7, Nature Biotechnology) was used to generate species abundance tables of different taxonomic levels. The alpha diversity and the beta diversity of the samples were analyzed, and then the UPGMA clustering tree was constructed. Subsequently, LEfSe analysis was performed to find biomarkers (Biomarker) with statistical differences between the groups based on the LDA value. R software (Version 2.15.3) was used to draw Venn diagram, principal coordinate analysis (PCoA) diagram and perform ANOSIM analysis. PICRUSt software was used to perform metagenome function prediction analysis.
Statistical Analysis
Data were presented as means ± SD and analyzed using one-way ANOVA. Differences among treatment means were determined by Duncan’s post hoc test. All statistical analyses were performed by the SPSS 23.0 software (Chicago, IL, USA). Possibility values < 0.05 were taken to indicate statistical significance. Graphpad prism 8.0 software was used to graph the data.