Fermented Astragalus in diet improved laying performance, egg quality, antioxidant and immunological status and intestinal microbiota in laying hens

In the era of increased antibiotic resistance and ever-stricter control on antibiotic use, it is urgent to develop green, safe, and non-residue alternatives to antibiotics applied to the poultry industry. To this end, we supplied the potential Lactobacillus plantarum (L. plantarum) fermented Astragalus in the diet of laying hens, with a final addition of 3‰. Its effects have been assessed on laying performance, egg quality, antioxidant and immunological status, and intestinal microbiota, and are compared to the control group, to the Astragalus group containing 3‰ unfermented Astragalus, and to the L. plantarum group containing 2% L. plantarum [5 × 108 colony-forming unit (CFU) per milliliter (mL)]. During the second half of the experimental period (15 to 28 days), the egg production rate was considerably higher in the fermented Astragalus group than that in the other groups, with the fermented Astragalus group having the lowest feed conversion ratio. No significant difference (P > 0.05) was noted among treatments on egg quality. Fermented Astragalus-treated hens exhibited significantly increased catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in serum, and reduced malondialdehyde (MDA) in serum. Furthermore, fermented Astragalus supplementation resulted in a significant increase in ileal microbiota abundance relative to control. In conclusion, feeding laying hens with L. plantarum fermented Astragalus has beneficial effects on production, antioxidant potential, immunity, and ileal microbiota. L. plantarum fermented Astragalus is expected to be a novel feed additive used in poultry production.


Introduction
Eggs are one of the most crucial sources of animal protein and nutritional content in human diets. Due to the widespread use of antibiotics in poultry, drug residues in eggs have been gaining worldwide concern over the past few years (Vandemaele et al. 2002). In addition, antibiotic abuse has led to intestinal dysbacteriosis, diarrhea, immunocompromised state (Willing et al. 2011). Thus, it is imperative to develop green, safe, and non-residue alternatives to antibiotics applied to the poultry industry. Traditional Chinese herbal medicines are the gem of China with characteristics of safety, efficiency, and low residue and are commonly used in preventive or therapeutic strategies for animal diseases (Xu et al. 2017). Traditional Chinese herbal medicines have utilised as feed additives for growth promotion and improvement of immunity and various effects, including anti-bacterial, anti-viral and antioxidative activities (Patra et al. 2015;Wang et al. 2015). Since ancient times, traditional Chinese herbal medicines can be processed by microbial fermentation for improving its quality (Zhu et al. 2014). For example, fermentation of Chinese herbal medicine mediated by microbes can degrade macromolecule-materials into small ones and reduce their side effects (Ai et al. 2019). Because microorganisms and their metabolic products can regulate the bioactive products of traditional Chinese herbal medicines, there is a close relationship between microorganisms and traditional Chinese herbal medicines.
Astragalus is a universal traditional Chinese herbal medicine and its main active pharmaceutical ingredients include polysaccharides, saponins, flavonoids, anthraquinones, alkaloids, amino acids, β-sitosterol and metallic elements (Li et al. 2014). Astragalus has been reported to possess anti-inflammatory (Kim et al. 2013), anti-viral (Kallon et al. 2013) and antioxidant (Shahzad et al. 2016) activities and to enhance immunity (Qin et al. 2012), and it has been widely used in livestock. Nevertheless, challenges to the extraction yield of Astragalus functional ingredients are raised due to the recalcitrance of plant cell walls, and novel strategies for the improvement of Astragalus utilization efficiency have to be focused. The trend of microbial fermentation offers the possibility of addressing the above problem. In recent years, research revealed that utilizing the fungus Aspergillus to ferment the Astragalus can significantly increase its phenolic contents and antioxidant activity, and the solid-state bioprocessing strategy could be an innovative approach to enhance the antioxidant activity of Astragalus (Sheih et al. 2011). Our previous studies have verified that the solid fermentation of Astragalus by L. plantarum promotes the extraction yield of Astragalus active components and the production yield of organic acids (Qiao et al. 2018c). Further investigation showed that fermented Astragalus improves broiler growth performance, enhances serum antioxidant status, and reduces fecal pathogenic microbiota of broiler chickens (Qiao et al. 2018b).
Over the last few years, there has meant considerable research on the application of Astragalus polysaccharide as a feed additive in livestock including laying hens. However, there has not been a systematic appraisal of the application of Astragalus fermented by L. plantarum as a feed additive for laying hens. In this study, we investigated the possible effects of Astragalus fermented by L. plantarum on egg production, egg quality, antioxidant status, immune factors expression and gut microbiome of laying hens, combining the classical culture and detection methods with high throughput sequencing.

Fermentation of Astragalus
Lactobacillus 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 (7500 g) was inoculated with L. plantarum (1 × 10 6 CFU per gram) with a water content of 45% and Astragalus-L. plantarum mixtures were divided equally into 35 × 45-mm plastic film bags. The bags were sealed for fermentation at 37 °C 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 four 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 × 10 8 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 the basal diet were shown 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 the heart, the serum was isolated and stored at − 20 °C until use. The CAT assay kit (Catalog number R22072), GSH-Px assay kit (Catalog number R21876), SOD assay kit (Catalog number R22262), T-AOC assay kit (Catalog number R24147) and MDA assay kit (Catalog number R21869) were purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China).

Real-time qPCR
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 (Catalog number 9108, Takara, Otsu, Shiga, Japan) and reverse transcribed into cDNA with PrimeScript ™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Catalog number RR047, Takara, Otsu, Shiga, Japan) 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 TB Green Premix EX Taq (Catalog number RR420, Takara, Otsu, Shiga, Japan) in a 7500 Fast Real-Time PCR System (Thermo Fisher). β-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.
Raw reads were quality-filtered to remove any reads less than 150 bp using Quantitative Insights into Microbial Ecology (QIIME) version 1.8 (Caporaso et al. 2010) 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 (Bokulich et al. 2013). 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 β-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/biosa mple) under the Accession number SRA: PRJNA533918.

Statistical analysis
Only for genes mRNA expression assay, statistical analyses were performed with Student's t-test 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.

Hen productivity and egg quality
The effects of different dietary supplements on the laying hen production performance and egg quality are listed in Tables 3 and 4. During day 1 to day 14, there were no differences in the laying rate and FCR among four groups (P > 0.05), with hens fed with fermented Astragalus had the highest laying rate. During day 15 to day 28, hens fed with fermented Astragalus had the highest laying rate, 7.14% higher than that of the control group (P < 0.05). Although the differences were not statistically significant (P > 0.05), the laying rate of the Astragalus group and L. plantarum group were also increased by 3.25% and 2.99%, respectively in comparison with the control group. Furthermore, the FCR of the fermented Astragalus group was reduced by 6.6% compared with that of the control group (P < 0.05), while the FCR of the Astragalus group and L. plantarum group displayed no significant differences as compared with the controls (P > 0.05). In addition, no significant differences in the phenotype of the egg quality including ESI, ESS, EST, AH, YC, HU, and YW were observed among dietary treatments, suggesting that dietary supplements have no significant effects on egg quality in this study. Therefore, we identified that dietary supplementation of fermented Astragalus can markedly improve egg production and decrease FCR, and the effect is substantially superior to that of Astragalus and L. plantarum.

Serum antioxidant indices
The effects of different dietary supplements on the laying hen antioxidant status are listed in Table 5. The data indicated that all dietary supplementation did not have an effect on the biomarkers of antioxidative stress at day 14 (P > 0.05). However, serum CAT, GSH-Px, SOD, and T-AOC concentrations were increased by 61.5%, 62.4%, 68.0%, and 52.6% (P < 0.05) at the end of experimentation in the fermented Astragalus group as compared with the controls. No statistically significant differences were observed for CAT, GSH-Px, and SOD among the control, Astragalus, and L. plantarum groups (P > 0.05). Among the effects of different dietary supplements on MDA activity in serum of laying hens, hens fed with fermented Astragalus, Astragalus diet were significantly decreased by 54.7% and 43.0% than that of the control treatment (P < 0.05); treatment with L. plantarum diet did not dramatically differ from the control treatment (P > 0.05). The results presented above show that dietary supplementation of fermented Astragalus can markedly improve laying hen antioxidant status, and the effect is significantly superior to that of Astragalus and L. plantarum.

IFN-γ and TNF-α mRNA expression
The expression levels of IFN-γ and TNF-α mRNA in the liver, spleen, ileum, and cecum were assessed at 14 and 28 days. As shown in Figs. 1 and 2, the addition of fermented Astragalus to the diets respectively increased the mRNA content on day 14 of IFN-γ and TNF-α in the ileum by 1.7-fold (P < 0.01) and 3.1-fold (P < 0.001), and the mRNA content on day 14 of IFN-γ in the cecum by 2.1-fold (P < 0.01). Interestingly, we found that the highest amount of IFN-γ and TNF-α mRNA in the liver, spleen, ileum, and cecum were present in the fermented Astragalus group at 28 days.

Sequencing output
A total of 48 intestinal content samples were analyzed by 16S rRNA gene sequencing and produced a total of 2,006,223 high-quality sequences with an average of 41,796 reads. After OUT clustering at 97% sequence identity, a total of 216,116 OTUs were classified into 49,235 phyla, 48,677 classes, 48,634 orders, 40,101 families, 24,072 genera and 4995 species (Fig. 3).

Diversity of intestinal microflora
The α-diversity of ileal and cecal microbiota of four groups on different days are shown in Table 6. For bacteria on day 14, fermented Astragalus treatment reduced the Chao1 and ACE index in the cecum in comparison to the control treatment suggesting that fermented Astragalus decreased the richness of the bacterial communities. On day 28, the fermented Astragalus treatment increased the estimators of diversity (Shannon and Simpson) of the bacterial community in the ileum. PLS-DA was performed to evaluate the similarity (β-diversity) of microbial community structure among groups (Fig. 4). PLS-DA plot defined groups where the samples from different groups occupied distinct positions.

Composition of intestinal microflora
A total of 20 phyla were identified within the intestinal microbiota among 48 samples as shown in Fig. 5. There were three major groups of the intestinal microbiota, including Firmicutes, Bacteroidetes, and Proteobacteria. The relative abundance (%) of cecal bacterial phyla of hens fed with different dietary supplements was presented in approximately the same amount on days 14 and 28. On day 28, fermented Astragalus led to a reduced abundance of ileal Firmicutes, with an increased abundance of ileal Bacteroidetes and Proteobacteria. Genus level analysis showed that the Lactobacillus and Bacteroides accounted for the largest proportion of the intestinal microbiota

Discussion
The present study was undertaken to investigate the effects of L. plantarum fermented Astragalus supplementation on the performance, egg quality, antioxidant status of serum, and gut microbiota in laying hens. When taking out the feeding trial, we observed that diets supplemented with fermented Astragalus increased egg production rate (P < 0.05) and decreased feed conversion rate (P < 0.05), which may likely be attributed to the improvement of laying hen health status. In vivo, free radicals are harmful by-products generated during normal cellular metabolism and are prone to attack unsaturated fatty acid on the biological membrane, triggering lipid oxidation and lipid peroxide accumulation that result in impairment of organism health (Fang et al. 2002). The antioxidant enzymes CAT, GSH-Px, and SOD are associated with free radical scavenging to protect cells from oxidative damage ). In the present study, supplementation with fermented Astragalus resulted in the highest levels of CAT, GSH-Px, SOD, and T-AOC and the lowest level of MDA in the serum (P < 0.05) on day 28. Our findings are consistent with our previous studies on broilers (Qiao et al. 2018a), indicating that Astragalus fermented by L. plantarum can enhance the antioxidant ability of both broilers and laying hens. In our previous  research, we compared the differences between fermented Astragalus and Astragalus. During the fermentation, the pH was markedly reduced, as a consequence of the increase of organic acids content, which could inhibit the growth of miscellaneous bacteria (Qiao et al. 2018c). Fermentation could also elevate the content of Astragalus polysaccharide, total saponins, and total flavonoids in Astragalus, and fermented Astragalus possess more abundant microflora (Qiao et al. 2018c). The increase in the content of Astragalus active components by fermentation may be responsible for the enhancement of the performance, egg quality, and antioxidant status of serum