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 birds used in this study were raised in one hen house to acclimate the environment for 4 wks. All the laying hens were fed with the same corn-soybean meal basal diet (Additional file 4) 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. The egg production in young and aged hen groups were 95.05±1.90% and 86.94±0.49%, respectively. There were no culled birds, and medical intervention was not applied to any bird.
Sample collection
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. A total of 16 birds (8 replicates with 2 birds each) from each group were sacrificed at 8.5 h PO corresponding to the initiation stage of eggshell calcification. The rest of the birds were still raised until elimination. One sample from the aged hen group was rejected for further analysis, because no egg with incomplete shell was present in hen oviduct. The mucosa of uterine 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 and the elastic modules and fracture toughness of each egg were calculated according to the formulas as previously described [67]. After removing egg contents, eggshell was washed, air-dried at room temperature and weighed. The shell ratio was calculated as shell weight/egg weight ´ 100.
Eggshell ultrastructure
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 [68, 69]. 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 knob density was counted and expressed as the number of knobs per unit. Each ultrastructural variant in the mammillary layer was assigned a score depending on its incidence in the eggshell. The total score was calculated by the sum of all variant scores [68, 69].
Calcium and phosphorus contents in diets and eggshell
Diet and eggshell samples were dissolved in a solution containing HNO3 and H2O2 (v/v = 1:1) 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).
Extraction and determination of matrix protein in eggshell
Organic matrix proteins in eggshell were extracted and determined as previously described [70]. Eggshell powder (500 mg, pooled from 24 eggs from each replicate) was weighed and demineralized with 20% acetic acid. These samples were mixed with distilled water (v/v = 1:1) and freeze-dried. Then, they were dissolved in an extraction milieu (4°C; about 12 h) and these extracts were dialyzed (cutoff 3500 Da). Each sample was centrifugated (20 min; 2500 g), and the supernatant was used to determine the protein content (PC0020; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).
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. RNA samples (1 μg) were used for RNA library construction with NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB Inc., Ipswich, MA, USA). The construction involved mRNA purification, fragmentation under elevated temperature, and the synthesis of first and second strand cDNA. Exonuclease/polymerase were used for the conversion of the remainder overhangs into the blunt ends. The cDNA library construction included the adenylation of 3’ ends of DNA fragments, NEBNext Adaptor ligation and PCR. The purification of PCR products was conducted on AMPure XP system (Beckman Coulter, Beverly, USA) and the assessment of library quality was conducted on Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). After clustering, the library sequencing was carried out on an Illumina platform. The sequencing data have been deposited at Sequence Read Archive of National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/bioproject; accession number: PRJNA627801).
Sequence quality control and functional annotation of DEGs
Clean reads were obtained by removing raw reads of low quality, or with adapter and ploy-N. Q20, Q30, GC-content and sequence duplication level of the clean data were assessed. These clean reads were aligned to the reference genome (Gallus gallus 5.0; http://asia.ensembl.org/index.html) using Hisat2 tools soft. Function annotation was performed on the basis of the databases below: Nt (NCBI non-redundant nucleotide sequences; https://www.ncbi.nlm.nih.gov/), COG (Clusters of Orthologous Groups; http://www.ncbi.nlm.nih.gov/COG) and GO (Gene Ontology; http://geneontology.org). Fragments per kilobase of transcript per million fragments mapped (FPKM) were used to estimate the levels of gene expression. Differentially expression was analyzed with the DESeq2 and the resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). GO analysis of DEGs (FC > 1.5, FDR < 0.05) was conducted using the GOseq R packages based Wallenius non-central hyper-geometric distribution [71] and the KEGG pathway (http://www.genome.jp/kegg/) enrichment analysis of DEGs was performed using KOBAS software [72].
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, USA) with a 20 μL PCR reaction mixture (primer concentration: 0.3 μM) according to instructions of the SuperReal PreMix Plus kit (SYBR Green, FP205, Tiangen Co., Beijing, China). Primers used in this study are shown in Additional file 5. 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 [73].
Statistical analysis
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.