Animals and housing
The animal protocol for our study was approved by the Animal Care and Use Committee of the Feed Research Institute of the Chinese Academy of Agricultural Sciences. A total of 96 healthy young (42 wk of age) and 96 healthy aged (72 wk of age) Hy-Line Brown laying hens were individually divided into 8 replicates of 12 birds each in a randomized block design, and 3 birds were placed in one cage. The layer chicks were commercially obtained from Xiaoming Agriculture and Animal Husbandry Co. Ltd. (Ningxia, China) and were separately reared in two houses with similar configurations. Before this experiment, all the laying hens used in this study were raised in one hen house to acclimate the environment for 4 wks. All birds were fed with the same corn-soybean meal basal diet (Additional file 1) and provided with feed and water ad libitum with exposure to 16 h of light/d and the control temperature. All hens remained in good health during the feeding period. There were no culled birds, and medical intervention was not applied to any bird.
A total of 24 egg samples (8 eggs/replicate/d × 3 d) from each replicate were collected on three successive days and weighed. Another 12 egg samples (4 eggs/replicate/d × 3 d) from each replicate were collected only for the measurement of stiffness, elastic modules and fracture toughness. An automatic-monitoring control system (FRI, CAAS, Beijing, China) was used to record the daily oviposition time and the total time an egg spent in the oviduct could be calculated. Two birds from each replicate were sacrificed at 8.5 h PO corresponding to the initiation stage of eggshell calcification. One sample from the aged hen group was rejected for further analysis, because no egg with incomplete shell was present in hen oviduct. The uterus tissues surrounding the eggs were collected and snapped frozen immediately in liquid nitrogen, then stored at -80°C until further analysis.
Eggshell physical and mechanical properties
Eggshell thickness was measured by the Egg Shell Gauge and breaking strength was determined by Egg Force Reader (Israel Orka Food Technology Ltd., Ramat Hashron, Israel). Shell stiffness was measured by quasi-static compression using a Texture Lab Pro (TMS-Pro, Food Technology Ltd., VA, USA) fitted with a 25-N load cell at a compression speed of 1 mm/min. Three consecutive measurements were carried out at different points around the equator of each egg. The elastic modules and fracture toughness of each egg were calculated according to the formulas as previously described . The elastic modulus (E, N/mm2) reflects the contribution of eggshell material to the overall stiffness characteristics: E = C[(Sd·R)/T2], where Sd = stiffness (N/mm), R = radius of curvature (breadth/2 (mm)), T = thickness of eggshell (mm), and C = non-dimensionalised constant. The C is determined by the shape index (SI), radius of curvature (R), and the thickness (T) of the eggshell and was calculated as follows: C = A(0.408+(3.026T)/R), where A = (-0.666+(1.8666×(SI))-(0.907×(SI)2) + (0.153×(SI)3))/0.444. Fracture toughness, or resistance to fracture (KC, N/mm3/2), reflects the nature and magnitude of inherent defects within a material and was calculated as follows: KC = Knd (F/T3/2), where Knd = 0.777 (2.388 + (2.9934 (6/R))), F = breaking strength (N). After removing egg contents, shell was washed, air-dried at room temperature and weighed. The shell ratio was calculated as shell weight/egg weight ´ 100.
Three pieces of shell sample (~0.5 cm2) from the equatorial section of each egg were assessed for the ultrastructure of the external shell surface and the cross section by scanning electronic microscopy (SEM, FEI Quanta 600, Thermo Fisher Scientific Ltd., Portland, OR, USA). Shell specimens for the ultrastructure of mammillary layer were prepared according to previous methods [62, 63]. Briefly, the shell membrane was carefully removed after soaking in distilled water. Then, each sample was immersed overnight in a solution of 6% sodium hypochlorite, 4.12% sodium chloride, and 0.15% sodium hydroxide, washed with distilled water and air-dried at room temperature. Shell samples were glued on a copper block, coated with gold powder, and analyzed with SEM. Mammillary thickness, the effective thickness (total thickness of palisade, vertical crystal layer and cuticle) and the width of the mammillary knobs were determined and calculated with SEM ruler. Mammillary thickness is the length from the top of the membrane to the bottom of the palisade layer. The width of the mammillary knobs was calculated as follows: width = the length of mammillary knobs/ the number of mammillary knobs. Mammillary knob density was counted and expressed as the number of knobs per unit. The incidence of ultrastructural variants in the mammillary layer was assessed and then was used to calculate the total score according to previously described methods [62, 63].
Calcium and phosphorus contents in diets and eggshell
Diet and eggshell samples were dissolved in a solution of 3 mL HNO3 and 3 mL H2O2 and then digested by the microwave dissolution instrument (MDS-10, Shanghai Xinyi Instrument Technology co., Ltd, Shanghai, China). Calcium and phosphorus contents were measured by flame atomic absorption spectrophotometry (Zeenit700P, Analytik Jena, Germany) and a spectrophotometer (UV-2700, Shimadzu, Japan).
Proinflammatory cytokine contents in uterus tissue
The contents of IL-1β, IL-6 and IFN-α in uterus tissues were measured with ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) as recommended by the manufacturer’s instructions. The results were normalized against total protein content in each sample for comparison.
RNA extraction, library preparation and sequencing
Total RNA in the uterus tissues was extracted using TRIzol reagent according to the manufacturers’ instructions (Tiangen Biotech Co. Ltd., Beijing, China). Before library construction, the RNA integrity was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) with the RNA Nano 6000 Assay Kit. A total of 1 μg RNA per sample was used as input material for the RNA sample preparation. RNA library construction was performed using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB Inc., Ipswich, MA, USA) following the manufacturer’s instruction. Briefly, poly-T oligo-attached magnetic beads were used for the purification of mRNA from total RNA. Fragmentation was performed using divalent cations in NEBNext First Strand Synthesis Reaction Buffer (5×) under elevated temperature. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase and second strand cDNA synthesis was subsequently carried out using DNA Polymerase I and RNase H. The rest of the overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. AMPure XP system (Beckman Coulter, Beverly, USA) was used to purify the library fragments to select cDNA fragments of preferentially 240 bp in length. The size-selected, adaptor-ligated cDNA was mixed with 3 μl USER Enzyme (NEB Inc., Ipswich, MA, USA) and then incubated at 37°C for 15 min and 95°C for 5 min before PCR. The PCR was carried out with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. The products were purified with AMPure XP system (Beckman Coulter, Beverly, USA) and library quality was assessed with Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The clustering of the index-coded samples was performed with a cBot Cluster Generation System and TruSeq PE Cluster Kit v4-cBot-HS (Illumia) and the library preparations were sequenced on an Illumina platform and paired-end reads were generated.
Sequence quality control and functional annotation of DEGs
Raw reads with adapter, ploy-N and of low quality were removed to obtain clean reads and Q20, Q30, GC-content and sequence duplication level of the clean data were assessed. These clean reads were then mapped to the chicken reference genome (Gallus gallus 5.0) using Hisat2 tools soft. Gene function was annotated based on the following databases: Nt (NCBI non-redundant nucleotide sequences), COG and GO. Gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped (FPKM). Differentially expression analysis was performed using the DESeq2 and the resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). The genes at least 1.5-FC and FDR < 0.05 were defined as DEGs between groups. GO analysis of DEGs was performed using the GOseq R packages based Wallenius non-central hyper-geometric distribution  and the KEGG pathway enrichment analysis of DEGs was performed using KOBAS software .
qRT-PCR validation of RNA sequencing results
Eight genes were selected for qRT-PCR validation. The RNA samples were reverse transcribed with the FastQuant RT kit (KR106, Tiangen Biotech Co. Ltd., Beijing, China) to prepare cDNA. The mRNA expression of target genes was examined by qRT-PCR using CFX96 touch RT-PCR detection system (Bio-rad laboratories. Inc., CA) with SuperReal PreMix Plus (SYBR Green, FP205, Tiangen Co., Beijing, China). Primers used in this study are shown in Additional file 2. The reaction conditions were as follows: 95ºC for 15 min; 40 cycles of 95 ºC for 10 s, 60ºC for 30 s. Each sample was measured in duplicate. The size of all amplified products was confirmed by electrophoresis on a 1.5% (w/v) agarose gel with gelred (SolarGelRed Nucleic Acid Gel Stain, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and visualized in Gel Doc XR+ System (Bio-rad laboratories. Inc., CA, USA). The relative mRNA expression levels were normalized to avian β-actin by the 2-ΔΔCt method .
Unpaired t-tests (two tailed) were used to analyze the significant differences between groups using SPSS (version 23.0 for Windows; SPSS Inc., Chicago, IL, USA). Data were presented as mean with standard deviation (SD) and statistical significance was defined as a P value < 0.05.