The Beijing University of Agriculture’s Animal Care and Use Committee approved all experimental protocols (Approval ID: BUA-zc-20200073).

Epimedium, Astragalus, Angelica, Leonurus, and Houttuynia were all purchased from Yiren Pharmaceutical Co., Ltd. (Baoding, Hebei, China). The test bacteria and enzymolysis enzymes were purchased from Beijing challenge Biotechnology Co., Ltd. (Beijing, China). The main components of Angelica, Astragalus, Epimedium, Houttuynia, and Leonurus are Angelica polysaccharide, Astragalus polysaccharide, flavone, chlorogenic acid, and alkaloid, respectively (Table 1). In this experiment, Angelica and Astragalus were treated by fermentation, Leonurus was treated by enzymatic hydrolysis, and Houttuynia and Epimedium were not treated. According to the pre-experiment, the main components of Angelica and Astragalus reach their highest level through fermentation, the main components of Leonurus through enzymatic hydrolysis, and the main components of Houttuynia and Epimedium through no treatment at all. According to the Chinese veterinary medicine code, the mixing ratio of Epimedium, Astragalus, Angelica, Leonurus, and Houttuynia after pretreatment is 1:2:2:1:2. After processing, the mixed herbs were called PCHM. Fermentation conditions: Bacillus licheniformis, Bacillus coagulans, Aspergillus niger, Bacillus subtilis (1:1:1:1), conditions: time: 5 days, temperature: 37°C, water content: 45%. Enzymatic hydrolysis conditions: complex enzyme (pectinase, cellulase, laccase), conditions: time: 4 h, temperature: 55°C, water content: 45%.

A total of three hundred and sixty 68-week-old Jinghong No. 1 laying hens (initial body weights 1.65 ± 0.10 kg) with a similar weight and genetic background were used. A completely randomized design was used to divide the birds into the four treatment groups. Each treatment had six replicates with 15 birds each. All birds were fed a basal diet for 1 week before being assigned a diet containing maize-soybean meal containing 0, 0.2, 0.4, or 0.8% PCHM for 6 weeks. Table 2 displays the composition and nutritional levels of the diet based on maize-soybean meal. The experimental diet feeding period lasts for 6 weeks and the birds in the experiment were exposed to 16-h light cycles and had free access to water and the experimental diets at all times.

Every day, data on egg production and weight were logged. The number of eggs produced was expressed as an average hen-day production, which was derived by dividing the total number of hen-days by the number of eggs. Average egg weight was calculated as total egg weight divided by the number of eggs. Feed consumption was recorded on a replicate basis at weekly intervals. The feed conversion ratio was measured as the amount of feed consumed relative to the amount of eggs produced in kilograms. Egg quality was measured on three eggs collected randomly from each replicate on the 14th, 28th, and 42nd days. One healthy bird was chosen at random from each replicate at the end of the feeding period (one bird per replicate, and 24 birds in total). Exsanguination of the left jugular vein with scalpels was used to collect blood samples, which were then centrifuged at 4°C at 4,000 g for 10 min to separate plasma. Following collection, plasma samples were flash-frozen at −80°C and stored in the freezer until analysis. Birds were euthanized by exsanguination and necropsied, and the liver, magnum, and uterus were separated immediately and quickly frozen at −80°C for further analysis. Approximately 2 cm medial portion sections of the uterus were removed and cleaned thoroughly with 0.9% saline, then placed in 4% formaldehyde solution for tissue fixation and histological measurement.

The rate of egg production was determined as follows: egg production rate = eggs laid per day/(birds counted per day). Feed efficiency was calculated weekly. Haugh units, yolk color, and albumen height were measured using an egg analyzer (Orka Food Technology Ltd., Ramat Hasharon, Israel). Yolk color was defined according to the Roche yolk color fan, where 1 represents bright yellow and 15 represents dark yellow. An egg force reader (Herzliya, Tel Aviv, Israel) was used to measure eggshell strength. The thickness of the eggshell (ST) was calculated as follows: [ST, mm = SW/(ES d)], where SW is the weight of the eggshell, ES is the surface area of the egg, and d is the density of the material (2.3 g/cm³ for calcium carbonate).

Uterine tissues were fixed in 4% paraformaldehyde and embedded in paraffin, and the 5 µm-thick sections were stained with hematoxylin-eosin (H&E). Samples were trimmed, dehydrated, embedded, sliced, dyed, and sealed in strict accordance with the standard operating procedures for pathological experiment detection of the service-bio unit (Liu et al., 2022). Histological samples were evaluated for degree of injury, using variables such as the integrity of tissue structure and the numbers of infiltrated inflammatory cells (service-bio, Wuhan, China).

The plasma, liver, magnum, and uterus tissue concentrations of glutathione peroxidases (GSH-Px), catalase (CAT), superoxide dismutase (SOD), and the ability to scavenge superoxide anion radicals and the hydroxyl radical malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay (ELISA) using a commercial ELISA kit for chicken (Nanjing Jiancheng Bioengineering Institute, Nanjing, China; catalog no. A003-1-2, A007-1-1, A001-1-2, A005-1-2, A052-1-1, A018-1-1) according to the manufacturer’s instructions. T-SOD activity was determined by using the xanthine/xanthine oxidase method, which is based on the inhibition of nitroblue tetrazolium formazan. The activity of GSH-Px was measured using H₂O₂ as a substrate in the presence of reduced glutathione. The GSH-Px activity was reported in micromoles of oxidized NADPH per minute.

Total RNA was extracted from 0.1 g of liver, magnum, and uterus tissue sample using TRIzol reagent (Thermo Fisher Scientific, Shanghai, China), as per the manufacturer’s instructions, and then reverse-transcribed into single-stranded cDNA using the Thermo First cDNA Synthesis Kit (Promega, Beijing, China). The expressions of nuclear factor E2 related factor 2 (Nrf2), Cu-Zn superoxide dismutase (SOD) 1, Mn superoxide dismutase (SOD) 2, catalase (CAT), glutathione peroxidase 1 (GPX1), and peroxidase 4 (PX4) were determined using qRT-PCR with specific primers (Table 3). An AriaMx Real-Time PCR system (Agilent Technologies, Santa Clara, California, United States) was used for quantitative PCR analysis. After initial denaturation at 95°C for 10 min, 40 cycles of amplification were carried out (95°C for 10 s and 58.2°C for 30 s), followed by the generation of melt curves that could be used to verify the specificity of amplification. The samples were tested in triplicate. The 2^−ΔΔCT (Livak and Schmittgen, 2001) method was used to calculate the relative gene expression levels. Refer to the operation of (Madkour et al., 2021) for specific process.

All statistical analyses were performed using SPSS 22.0 (IBM Corp., Armonk, NY, United States). Data are presented as mean ± standard deviation of the mean (SD). Additionally, polynomial regression analysis was used to test the linear and quadratic nature of the response to the additive PCHM dosage, and Tukey’s multiple comparison tests were used to analyze the differences among various treatments. p < 0.05 was considered significant.

Comparison of the main components of five Chinese herbal medicines is shown in Table 4. Angelica, Astragalus fermentation treatment, Leonurus enzymolysis treatment, Houttuynia, and Epimedium did not have treatment.

The effects of dietary supplementation of PCHM on performance and egg quality are shown in Table 5 and Table 6. PCHM supplementation had no effect on laying rate, feed consumption, yolk color, shell thickness, shape index; PCHM supplementation had a significant effect on feed efficiency, egg weight, albumen height, and eggshell strength (p < 0.05). With increasing dietary supplementation levels of PCHM, the Haugh unit increased linearly (p < 0.05).

The histological analysis was performed to assess the pathological changes in uterine tissues. The results demonstrated that aging resulted in severe injury, including hyperemia, hemorrhage, and many inflammatory cell infiltrations (Figure 1A). However, the aging-induced pathological changes were dramatically improved by PCHM at doses of 0.2 (Figure 1B), 0.4 (Figure 1C), and 0.8% (Figure 1D). These results indicate that PCHM could effectively attenuate aging-induced pathological changes.

The effect of dietary PCHM supplementation on the antioxidant enzyme activity and free radical scavenging capabilities in plasma, liver, magnum, and uterus are depicted in Figures 2–5, respectively. The PCHM supplementation significantly influenced the antioxidant enzyme activity and free radical scavenging capabilities in plasma, liver, magnum, and uterus. In the plasma, a significant (p < 0.05) increased in the protein level of CAT and GSH-Px was noticed in the 0.2% and 0.4% PCHM groups, and CAT and SOD in 0.8% PCHM group supplemented were observed as compared to the other experimental groups. In liver, a significant (p < 0.05) increased in the protein level of GSH-Px and free radical scavenging capabilities was noticed in the PCHM supplementation as compared to the control. Further, in liver a significant (p < 0.05) decreased the content of MDA in the PCHM supplementation as compared to the control. In magnum, a significant (p < 0.05) increased of GSH-Px protein level in the 0.4% PCHM groups, SOD protein level in 0.8% PCHM group, the ability to scavenge superoxide anion in 0.4% and 0.8% PCHM groups were observed as compared to the other experimental groups. Further, in magnum a significant (p < 0.05) decreased the content of MDA in the PCHM supplementation as compared to the control. Similarly, in uterus, a significant (p < 0.05) increased of CAT protein level in the 0.4% and 0.8% PCHM groups, the ability to scavenge hydroxyl radical in 0.2% and 0.4% PCHM group were observed as compared to the other experimental groups. Further, in uterus, significantly (p < 0.05) decreased the content of MDA in the 0.4% PCHM groups were evident as compared to the other experimental groups.

The effect of dietary PCHM supplementation on the expressions of CAT, SOD1, SOD2, GPX1, GPX4, and Nrf2 genes in liver, magnum and uterus are depicted in Figures 6–8, respectively. The PCHM supplementation significantly influenced the expression of these genes in liver, magnum, and uterus. Expression of all the genes was significantly (p < 0.05) upregulated in liver with 0.4% PCHM supplementation as compared to the control. In magnum, a significant (p < 0.05) increase in the expression of CAT and Nrf2 was noticed in the 0.2% and 0.8% PCHM supplemented as compared to the other groups. Further, in magnum, significantly (p < 0.05) greater expression of SOD1 and GPX4 in the 0.2 PCHM groups, and SOD2 and GPX1 in the 0.8% PCHM group were evident as compared to the other experimental groups. Similarly, in uterus, a significant (p < 0.05) upregulated expression of CAT, SOD1 and GPX4 in the 0.2% and 0.4% PCHM groups, SOD2 in 0.2% PCHM group, GPX1 in 0.2%, 0.4%, and 0.8% PCHM groups, and Nrf2 in the 0.4% and 0.8% PCHM groups were observed as compared to the other experimental groups.