Fermentation of Astragalus
L. plantarum (CGMCC 1.557) was purchased from the China General Microbiological Culture Collection Center (CGMCC) (Beijing, China). Te dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus was obtained from Gansu Huisen Pharmaceutical Development Co., Ltd. (Minxian, Gansu, China) and verified by Dr. JingYu Zhang (Henan University of Traditional Chinese Medicine, Zhengzhou, Henan, China). The purchased Astragalus was crushed into powder and filtered with a 100-mesh filter for further studies. The fermentation of Astragalus was performed following the method reported in our previous publications with slight modification (Qiao et al 2018b). Briefly, dried Astragalus powder (7,500 g) was inoculated with L. plantarum (1×106 colony forming unit (CFU) per gram) with a water content of 45%, and Astralagus-L. plantarum mixtures were aliquot into 35 × 45-mm plastic film bags. The bags were sealed for fermentation at 37ºC for 30 days, and then dried out at room temperature for future use.
Experimental Design, Diets and Management
Two hundred and forty healthy Hy-Line Gray hens (351 days, Zhengzhou, China) were acclimated with the basal diets for 7 days. Then, hens were randomly divided into 4 groups (fermented Astragalus group, Astragalus group, L. plantarum group and control group), each containing five replicates, with 12 hens per replicate. The control group was fed with the basal diet; the L. plantarum group was fed with the basal diet supplemented with 2% Lactobacillus solution (5×108 CFU/mL) through uniform spraying; the Astragalus group was fed with the basal diet supplemented with 3‰ Astragalus, and fermented Astragalus group was fed with the basal diet supplemented with 3‰ fermented Astragalus (pre-experimental results showed that supplementing at a rate of 3‰ of diet achieves optimal results). The trial lasted for 35 days (7-day adaptation period and 28-day experimental stage). The hens were housed in a clean environment with good ventilation and artificial lighting allowed 16 h of lighting per day, and with water and food ad libitum. The basal diet of all groups was the same and prepared according to the NRC (1994) laying hen nutrition requirement standard. The composition and nutrient levels of basal diet were showed in Table 1. All animal experiments were conducted according to the Guidelines for the Care and Use of Experimental Animals established and approved by the Laboratory Animal Management Committee of Henan University of Animal Husbandry and Economy (HNMY 1606).
Hen Productivity and Egg Quality
During the experimental period, egg production, broken egg production, egg weight and feed intake were recorded daily. The egg production rate and the feed conversion ratio (FCR) (feed intake/egg weight gain) during day 1 to day 14 and day 15 to day 28 were calculated to assess the laying performance. On day 14 and day 28, five eggs from each replicate were randomly sampled and measured egg quality parameters of egg shape index (ESI), eggshell strength (ESS), eggshell thickness (EST), albumen height (AH), haugh unit (HU), yolk color (YC) and yolk weight (YW).
Serum Antioxidant Indices
On day 14 and day 28, one hen from each replicate was randomly selected. Following blood collection from heart, the serum was isolated and stored at -20 ºC until use. The CAT assay kit, GSH-Px assay kit, SOD assay kit, T-AOC assay kit and MDA assay kit were purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China).
After blood samples collection, liver, spleen, ileum and cecum samples were harvested for interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) mRNA expression evaluation. Total RNA was extracted from these tissues using RNAiso Plus (Takara, Beijing, China) and reverse transcribed into cDNA with PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Beijing, China) according to the manufacturer’s protocol. The primers used in the study were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) and primer sequences are summarized in Table 2. The Real-time qPCR reactions were performed using a SYBR Premix EX Taq Kit (Takara, Beijing, China) in a 7500 Fast Real-Time PCR System (Thermo Fisher). b-actin was used as a housekeeping gene. The relative mRNA expression levels of the target genes compared to the housekeeping gene were calculated using the 2−ΔΔCt method.
Sample Collection and DNA Extraction
On day 14 and day 28, a total of 48 hens were randomly selected (twelve hens per group) and euthanized to collect ileal and cecal contents. The samples were named as 14-d ileum control group (14IA), 14-d ileum Astragalus group (14IB), 14-d ileum L. Plantarum group (14IC), 14-d ileum fermented Astragalus group (14ID), 14-d cecum control group (14CA), 14-d cecum Astragalus group (14CB), 14-d cecum L. Plantarum group (14CC), 14-d cecum fermented Astragalus group (14CD), 28-d ileum control group (28IA), 28-d ileum Astragalus group (28IB), 28-d ileum L. Plantarum group (28IC), 28-d ileum fermented Astragalus group (28ID), 28-d cecum control group (28CA), 28-d cecum Astragalus group (28CB), 28-d cecum L. Plantarum group (28CC), and 28-d cecum fermented Astragalus group (28CD). All collected samples were immediately stored at -20ºC until extraction. DNA extraction was performed with a commercial DNA extraction kit (Tiangen Biotech Corporation, Beijing, China) and quantified by a Qubit 2.0 fluorometer (Invitrogen Corporation, Carlsbad, CA, USA). The extracted DNA was qualitatively assessed by 0.8% agarose gel electrophoresis and spectrophotometry (optical density at 260/280 nm) and stored at -20°C until further analysis.
16S rRNA Gene Sequencing and Analysis
For amplicon library generation, the V4 region of the 16S rRNA gene of all DNA samples was amplified with gene-specific primers (F: 5'- AYTGGGYDTAAAGNG-3'; R: 5'-TACNVGGGTATCTAATCC-3'). PCR amplifications were performed using Q5 high-fidelity PCR DNA polymerase (NEB) and completed under the following conditions: a pre-denaturation at 98°C for 30 s; 27 cycles of 98°C for 15 s, 50°C for 30 s, and 72°C for 30 s; a final extension at 72°C for 5 min. Amplicons were purified using the Axygen AP-GX-250G AxyPrepTM DNA Gel Extraction Kit (Corning Life Sciences, Corning, NY, USA). DNA libraries were validated and quantified using the TruSeq Nano DNA LT Library Preparation Kit (Illumina, San Diego, CA, USA) and Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA). After quantification, the barcoded V4 amplicons were pooled to a final concentration of 2 nmol/L and sequenced using an Illumina MiSeq platform to generate 300 bp paired-end reads. Raw reads were quality-filtered to remove any reads less than 150 bp using Quantitative Insights into Microbial Ecology (QIIME) version 1.8  and clustered into Operational Taxonomic Units (OTUs) based on a 97% similarity threshold. The representative sequence was chosen based on the abundance and was aligned under a given taxonomic classification using the Greengenes database, and low abundance OTUs of archaea and eukaryotes were removed . Alpha-diversity was calculated with Chao1 and ACE estimators, Shannon and Simpson indices. Partial least squares discriminant analysis (PLS-DA) was performed using QIIME software package v1.8 to discriminate between different groups (day 14 and day 28) and to establish b-diversity. The sequences generated in this study have been deposited in the National Center for Biotechnology Information sequence read archive (https://www.ncbi.nlm.nih.gov/biosample) under the accession number SRA: PRJNA533918.
Only for genes mRNA expression assay, data were analyzed and graphed using GraphPad Prism 6.00 (GraphPad Software), and significance levels are indicated as: * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. All other statistical analyses were performed by one-way analysis of variance using SPSS 24.0 software, and all data were expressed as means ± SD, with P<0.05 considered statistically significant.