Introduction
Taihe silk chicken (Gallus gallus domesticus Brisson) is a unique poultry breed from Taihe County, China, known for its high nutritional value, particularly in egg production. Taihe silk chicken eggs contain higher levels of total cholesterol, phospholipids, vitamins (A and B2), and essential minerals (calcium, magnesium, manganese, iron, zinc, and selenium) compared to standard chicken eggs (Zhang et al. Reference Zhang, XU and LIU2024). Additionally, they have a lower crude fat content, making them a valuable component of a nutritious diet. Long-term consumption of these eggs is believed to promote overall health. However, Taihe silk chicken have problems with slow growth, low egg production rate, short peak laying period, and low feed efficiency (Xiang et al. Reference Xiang, Huang and Wang2022). Additionally, there is relatively little research on the feeding and nutrition of Taihe silk chicken.
The Broussonetia papyrifera (BP), scientifically classified as Broussonetia, is a deciduous tree belonging to the Moraceae family (Penailillo et al. Reference Penailillo, Olivares and Moncada2016). The BP is known for its rapid growth, robust adaptability, extensive distribution, high calorific value, and the ability to be harvested multiple times within a year (Peng et al. Reference Peng, Liu and Chen2019). It boasts high forage value, being rich in nutritional content, including ample proteins, a diverse array of trace elements,and essential amino acids (Si et al. Reference Si, Tao and Zhang2018). BP leaves are rich in polyphenols, flavonoids and alkaloids (Chen et al. Reference Chen, Shui and Chai2020; Hao et al. Reference Hao, Huang and Si2020) and it can be developed and utilized as an unconventional protein feed resource. Indeed, the complex protein structures found in BP pose a challenge for monogastric animals in terms of digestion and absorption. Additionally, its elevated crude fiber content, along with the presence of tannins and other anti-nutritional factors, coupled with concerns like a short storage life and limited palatability, have restricted its utilization within the feed industry (Niu et al. Reference Niu, Wang and Liang2023). Many studies have found that fermentation can increase the content of crude protein, reduce the content of crude fiber, degrade anti-nutrient factors, increase the palatability of feed and improve feed efficiency (Shuai et al. Reference Shuai, Chen and Yu2023; Sugiharto and Ranjitkar Reference Sugiharto and Ranjitkar2019). Research has shown that, compared to unfermented BP, the fermentation of BP with probiotics extends its shelf life and improves its nutritional value by increasing the levels of crude protein, zinc, and amino acids, while reducing the content of crude fiber and tannins (Heres et al. Reference Heres, Engel and Knapen2003). The fermentation process typically involves multiple strains of microorganisms. In this study, Lactiplantibacillus plantarum and Bacillus subtilis were used. These microorganisms play a crucial role not only in fermenting the BP itself but also in enhancing its nutritional profile and bioavailability. The role of these microorganisms includes the breakdown of complex fibers and anti-nutritional factors, the synthesis of beneficial metabolites such as short-chain fatty acids (SCFAs), and the improvement of gut health by modulating the gut microbiota composition. The fermentation process enhances the digestibility and absorption of nutrients in monogastric animals by producing enzymes that degrade complex carbohydrates and fibers into simpler, more digestible forms. Additionally, the probiotics used in fermentation can colonize the gut, promoting a healthy microbiota balance, improving immune response, and potentially reducing pathogen load. Studies have shown that adding fermented BP (FBP) to the diet of Hyline brown laying hens during the laying period can increase average daily feed intake (ADFI), feed conversion rate, yolk color and average egg weight (Niu et al. Reference Niu, Wang and Liang2023). Dietary BP supplementation contributes to improving immune and antioxidant capabilities, improving growth performance and reducing diarrhea incidence of dairy cows and weaned piglets (Chen et al. Reference Chen, Shui and Chai2020; Hao et al. Reference Hao, Huang and Si2020). The addition of 5% FBP to the diet of Taihe silk chicken may have significantly changed the muscle composition (Liao et al. Reference Liao, Shi and Hu2022). However, the effect of FBP on laying performance of Taihe silk chicken has not yet been reported.
Therefore, the aim of this study is to evaluate the effects of FBP on the laying performance, egg quality, egg composition and gut microbiota of Taihe silk chicken during the peak laying period, which provides theoretical reference for the application of FBP in the production of Taihe silk chicken.
Material and methods
Animal ethics statement
All the procedures were approved by the Institutional Animal Care and Use Committee at Zhejiang University.
Preparation of FBP
Lactiplantibacillus plantarum (CICC 21791) used in the present experiment was obtained from China Center of Industrial Culture Collection. Bacillus subtilis HMJZ-B1005S (CCTCC NO: M2020237) used in the present experiment was obtained from Shenzhen HeMin Biotechnology Co., Ltd (Shenzhen, China). The production of FBP was carried out at the Jiangxi Shunjing Biological Technology Co., Ltd (Ji’ an, China). The whole fresh BP plant, which is approximately 100 cm tall, was fully crushed. Then, the crushed fresh BP was proportionally inoculated with cellulase (8000 U/kg), Lactiplantibacillus plantarum (1.2 × 107 CFU/g) and Bacillus subtilis (6.0 × 106 CFU/g). Then, it was stirred well and transfered to a breathing bag with a one-way valve, sealed and fermented at room temperature (temperature of 28 ± 2°C, humidity of 60 ± 5%) for 20 days. The nutritional composition of the BP and FBP, including dry matter, crude protein, trichloroacetic acid–soluble protein (small peptides), crude fiber, neutral detergent fiber, acid detergent fiber, pH, lactic acid, tannin, acid detergent lignin, live Lactiplantibacillus plantarum cells and live Bacillus subtilis cells were analyzed according to the National Standard of the People’s Republic of China and Chinese Agricultural Industry Standards. Specifically, dry matter content was measured by drying the sample at 105°C until a constant weight was achieved. Crude protein and trichloroacetic acid–soluble protein were determined using the Kjeldahl method. Crude fiber, neutral detergent fiber, and acid detergent fiber were measured using the filter bag method. The pH was measured using a pH meter. Lactic acid and tannin contents were determined using high-performance liquid chromatography (HPLC) and spectrophotometry, respectively. Acid detergent lignin content was measured using infrared spectroscopy. The number of live Lactiplantibacillus plantarum cells and live Bacillus subtilis cells was measured using plate count methods. The nutritional composition analysis of the BP and FBP is shown in Table 1.
Table 1. Nutrient composition of Broussonetia papyrifera and fermented Broussonetia papyrifera
BP = Broussonetia papyrifera; FBP = fermented Broussonetia papyrifera; DM = dry matter; CP = crude protein; TCA-SP = trichloroacetic acid–soluble protein (small peptides); TCA-SP:CP ratio = TCA-SP to CP ratio; CF = crude fiber; NDF = neutral detergent fiber; ADF = acid detergent fiber; ADL = acid detergent lignin; LP = Lactiplantibacillus plantarum; BS = Bacillus subtilis.
Experimental design and diets
A total of 240 Taihe silk chicken at 24 weeks of age during the peak laying period were provided by Xichang Fengxiang Poultry Co., Ltd (Ji’ an, China). The chickens were randomly divided into four treatments, with five replicates (12 chickens per replication). The dietary treatments included a basal diet (CON), a basal diet + 2% FBP (T2), a basal diet + 4% FBP (T4), and a basal diet + 8% FBP (T8). The specific percentages of FBP (2%, 4% and 8%) were selected based on previous studies (Niu et al. Reference Niu, Wang and Liang2023) and preliminary experiments that indicated these levels could potentially enhance laying performance and egg quality without negatively affecting feed intake or health. These levels were chosen to evaluate the dose–response effect of FBP supplementation and to determine an optimal inclusion rate for practical application. All experimental diets were formulated to meet the NRC (1994) nutrient requirements and contained similar levels of crude protein and metabolizable energy. The composition and nutritional value of the experimental diets are presented in Table 2. The specific measurement methods are as follows: Metabolizable energy was calculated based on the data provided by the Chinese Table of Feed Composition and Nutritional Values (34th edition, 2023), which did not contain energy values for FBP, so FBP energy was measured by the oxygen bomb calorimeter (6100, PARR, USA). Crude protein content is determined using the Kjeldahl method, in accordance with standard GB/T 6432-2018. Dry matter content is measured by drying the sample at 105°C until a constant weight is achieved, as per standard GB/T 6435-2006. Calcium content is determined using the sodium hydroxide–ethylenediaminetetraacetic acid titration method, following standard GB/T 6436-2018. Non-phytate phosphorus is calculated as the difference between total phosphorus and phytate phosphorus. Total phosphorus is measured using the molybdenum yellow spectrophotometric method described in standard GB/T 6437-2018. Phytate phosphorus is measured using the ferric chloride precipitation method (Rutherfurd et al. Reference Rutherfurd, Chung and Morel2004). Lysine, methionine and cystine contents are measured using an amino acid analyzer, following standard GB/T 18246-2000. The experimental period lasted for 75 days. All chickens were raised in an environment with a temperature of 32 ± 2°C, humidity of 65 ± 5%, and 16 h of light exposure. The housing system used was a three-tiered step cage system, with four chickens per cage, and each replicate had three cages. All chickens had free access to food and water and were fed 2 times (at 8:00 and 17:00) per day. Various production performance data of Taihe silk chicken were recorded at regular intervals.
Table 2. Ingredients and nutrient levels of experimental diets (as-fed basis)
Note: CON = basal diet; T2 = basal diet + 2% fermented Broussonetia papyrifera; T4 = basal diet + 4% fermented Broussonetia papyrifera; T8 = basal diet + 8% fermented Broussonetia papyrifera.
1 Provided the following per kilogram of complete diet: vitamin A, 12000 IU; vitamin E, 49.5 mg; vitamin B1, 3 mg; vitamin B2, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; vitamin D3, 3750 IU; vitamin K3, 6 mg; folic acid, 3 mg; nicotinic acid, 60 mg; pantothenic acid, 18 mg; biotin, 0.3 mg; Mn, 60 mg; Zn, 120 mg; Cu, 10 mg; Fe, 80 mg; I, 1.1 mg; Se, 0.4 mg; Co, 0.2 mg.
2 Enzymic preparations: Xylanase ≥ 22000 U/g; β-glucan ≥ 3000 U/g; β-mannanase ≥ 350 U/g; cellulase ≥ 300 U/g; amylase ≥ 3200 U/g; protease ≥ 2500 U/g.
3 With the exception of metabolizable energy and non-phytate phosphorus, all other nutritional components listed in Table 2 are based on actual measurements, which were analyzed according to the National Standard of the People’s Republic of China.
Laying performance
During the experimental period, daily records were kept for each replicate, including egg production, egg weight, and mortality and the remaining feed to calculate the ADFI, average egg weight, average egg production rate and feed-to-egg ratio.
Sample collection
On days 15, 30, 45, 60, and 75 of the experiment period, 10 eggs (5 replicates per treatment, 2 eggs per replicate) were randomly collected from each treatment group for detection of albumen height, Haugh unit, eggshell strength, yolk color, relative weight of eggshell and eggshell thickness. To analyze yolk nutrient composition, we randomly selected 10 eggs per day from each treatment group (5 replicates per treatment, 2 eggs per replicate) from day 70 to day 75 (6 days in total) of the experiment period. And then, the 12 egg yolks collected in each replicate during the 6 days are mixed together and stored at −80°C, which was used to analyze the content of moisture, protein, fat, vitamin, cholesterol, mineral elements, amino acids and fatty acids in the yolk. Additionally, at the end of the experiment (day 76), 10 laying hens with similar body weights were randomly selected from each group (2 hens per replicate) and were fasted for 12 h before slaughter. Duodenum, jejunum, ileum, and cecum were collected during slaughter and immediately placed in liquid nitrogen. They were stored at −80°C for analysis of gut microbiota. Furthermore, five hens in each group had segments of the intestine fixed in 4% paraformaldehyde for histological examination of the intestinal structure.
Analysis of egg quality
Egg quality was assessed on days 15, 30, 45, 60, and 75 of the experiment. An automated egg quality analyzer (DET-6000, Nabel Co., Ltd., Kyoto, Japan) was used to measure the following egg parameters: egg weight, eggshell strength, albumen height, Haugh unit, and yolk color. An electronic balance (Mettler Toledo, China) was utilized to measure the weight of the eggshell, which represents the eggshell weight. After removing the inner membrane with forceps, a spiral micrometer was used to measure the eggshell thickness at the blunt end, pointed end, and middle portion of the egg, and the average value was calculated as the eggshell thickness.
Analysis of the nutritional composition of egg yolk
The nutritional composition of egg yolk, including moisture content, protein, fat, vitamin A, vitamin B2, folate, cholesterol, mineral elements (copper, zinc, selenium, cutter, magnesium), amino acids and fatty acids were analyzed according to the National Standard of the People’s Republic of China. Specifically, moisture content was determined by drying the sample at 105°C until a constant weight was achieved. Protein content was measured using the Kjeldahl method, and fat content was analyzed by the Soxhlet extraction method. Vitamin A, Vitamin B2, and amino acids were determined using HPLC. Folate content was analyzed using the spectrophotometric method. Cholesterol and fatty acids were measured by gas chromatography. Mineral elements were measured using inductively coupled plasma optical emission spectrometry.
H&E staining and analysis
After removing the duodenum, jejunum, and ileum tissue samples fixed in 4% paraformaldehyde solution, the samples were dehydrated and embedded in paraffin. Then, the paraffin blocks were sectioned into 5-μm-thick slices using a rotary microtome (HistoCore BIOCUT), and the sections were placed on glass slides. The sections were stained with hematoxylin and eosin (H&E) and then dehydrated and coverslipped. Under a microscope (ECLIPSE E100), the sections were observed, and appropriate fields were selected for photography. Subsequently, the K-Viewer software was used to measure villus height and crypt depth. The ratio of villus height to crypt depth was also calculated.
DNA extraction and 16S rRNA gene sequencing analysis
Microbial DNA from the cecum was extracted using the E.Z.N.A. Stool DNA Kit (Omega Bio-Tek, Norcross, GA, USA). The quality of DNA samples was assessed using 1% agarose gel electrophoresis, and the concentration and purity of the samples were measured using the NanoDrop 2000 UV-Visible spectrophotometer (Thermo Scientific, Wilmington, MA, USA). The V3-V4 region of the 16S rRNA gene was amplified using universal primers 341 F (5ʹ-CCTACGGGNGGCWGCAG-3ʹ) and 805 R (5ʹ-GACTACHVGGGTATCTAATCC-3ʹ). The PCR products were purified using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified using Qubit (Invitrogen, USA). The quality of purified PCR products was evaluated using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and the Kapa Biosciences (Woburn, MA, USA) library quantification kit before sequencing on the Illumina NovaSeq PE250 platform.
The paired-end reads obtained from sequencing were merged into longer tags based on the overlap using FLASH (v1.2.8). Sequence quality filtering was performed using fqtrim (v0.94), and chimeric sequences were removed using Vsearch software (v2.3.4). DADA2 was used for length filtering and denoising to obtain ASV (feature) sequence and ASV (feature) abundance table. Alpha-diversity and beta-diversity analyses were conducted using QIIME2 based on these data. ASV (feature) sequences were taxonomically annotated against the SILVA database (Release 138, https://www.arb-silva.de/documentation/release138/) as the NT-16S database. The abundance of each species in each sample was then statistically analyzed based on the ASV (feature) abundance table. Kruskal–Wallis test and linear discriminant analysis effect size (LEfSe) were applied to analyze the significant differences at the genus level between different groups. Functional prediction of the gut microbiota was carried out using PICRUSt2 (V1.0) analysis, and this analysis was conducted using OmicStudio Analysis at https://www.omicstudio.cn/analysis/.
Statistical analysis
The results of egg quality were analyzed for variance considering the interaction of two factors using the general linear model procedure of SPSS version 26.0. (SPSS, Inc., Chicago, IL, USA), and Tukey test was used to conduct significance test. The remaining experimental data were statistically analyzed using One-way ANOVA, and Tukey test in SPSS 26.0 software to used to assess the significance of differences. Orthogonal polynomial contrasts were further used to examine the linear and quadratic effects of the different inclusion levels of dietary FBP. The results of the data analysis are presented as means and standard error of the mean (SEM). A significance level of P < 0.05 was considered to indicate statistically significant differences between groups.
Results
Effect of FBP on laying performance of Taihe silk chicken
The Table 3 showed that the ADFI of Taihe silk chicken increased linearly or quadratically with the increase of the supplemental level of FBP (P < 0.05), the ADFI of 8% FBP group was significantly higher than that of control group, 2% FBP group and 4% FBP group (P < 0.05). At the same time, the average laying rate of Taihe silk chicken increased with the increase of the supplemental level of FBP, but the difference was not significant (P > 0.05), and the average egg production rate in 4% and 8% FBP groups were increased by 1.78% and 7.94%, respectively. In addition, the supplementation of FBP also resulted in a numerical increase in feed-to-egg ratio and average egg weight but no significant difference (P > 0.05).
Table 3. The effect of fermented Broussonetia papyrifera on the laying performance of Taihe silk chicken aged 24–35 weeks
Note: CON = basal diet; T2 = basal diet + 2% fermented Broussonetia papyrifera; T4 = basal diet + 4% fermented Broussonetia papyrifera; T8 = basal diet + 8% fermented Broussonetia papyrifera; ADFI = average daily feed intake.
a–b Within a row, values with different superscripts differ significantly at P < 0.05. Data are expressed as means and SEM, n = 5.
Effect of FBP on egg quality
The effects of different days and supplemental amount of FBP on egg quality of Taihe silk chicken are shown in Table 4. Different days of age had significant effects on albumen height, eggshell strength, yolk color, relative weight of eggshell and eggshell thickness of Taihe silk chicken (P < 0.05) but did not show a regular change. The addition of FBP had a significant effect on the yolk color of Taihe silk chicken. With the increase of FBP content, the yolk color increased linearly or quadratically (P < 0.05), the yolk color of 8% FBP group was significantly increased compared with control group (P < 0.05), but there was no significant difference in yolk color between experimental groups supplemented with different proportions of FBP (P > 0.05). There were no significant effects on albumen height, haugh unit, eggshell strength, relative weight of eggshell and eggshell thickness (P > 0.05). Additionally, there were significant interactions between age and FBP supplementation levels on the relative weight of eggshell and eggshell thickness (P < 0.05). The highest relative weight of eggshell was observed in the 0% FBP group on Day 30, while the lowest was in the 2% FBP group on Day 75. And the thickest eggshells were found in the 8% FBP group on Day 75, whereas the thinnest were in the 4% FBP group on Day 30.
Table 4. The effect of different days and supplemental amounts of fermented Broussonetia papyrifera on egg quality of Taihe silk chicken
Note: CON = basal diet; T2 = basal diet + 2% fermented Broussonetia papyrifera; T4 = basal diet + 4% fermented Broussonetia papyrifera; T8 = basal diet + 8% fermented Broussonetia papyrifera.
a–d Within a row, values with different superscripts differ significantly at P < 0.05. Data are expressed as means and SEM, n = 10.
Table 5. The effect of fermented Broussonetia papyrifera on the egg yolk composition of Taihe silk chicken (wet basis)
Note: CON = basal diet; T2 = basal diet + 2% fermented Broussonetia papyrifera; T4 = basal diet + 4% fermented Broussonetia papyrifera; T8 = basal diet + 8% fermented Broussonetia papyrifera; Cu = copper; Zn = zinc; Se = selenium; Fe = iron; Mg = magnesium. Ten eggs were collected per day from each treatment group (5 replicates per treatment, 2 eggs per replicate) from day 70 to day 75 (6 days in total) of the experiment period. Each replicate consisted of 12 egg yolks mixed together to analyze the nutrient content of the yolk.
a–c Within a row, values with different superscripts differ significantly at P < 0.05. Data are expressed as means and SEM, n = 5.
Effect of FBP on egg yolk nutritional composition
The contents of folate and zinc in egg yolk increased linearly or quadratically with the increase of the supplemental amount of FBP (P < 0.05). Compared with CON, dietary supplementation of 4% and 8% FBP significantly increased the folate content in egg yolk (P < 0.05). Additionally, the folate content in 8% FBP group was significantly increased compared with 2% FBP group and 4% FBP group (P < 0.05). Moreover, the content of zinc in egg yolk was significantly increased by 8% FBP group compared with the CON, 2% and 4% FBP groups (P < 0.05). Table 6 shows that the supplementation of FBP did not yield significantly effected the amino acid content in the egg yolk (P > 0.05). However, the addition of FBP to the diet can affect the fatty acid composition of egg yolk. As shown in Table 7, the contents of C18:2n6c, unsaturated fatty acid (PUFA) and n-6 PUFA in egg yolk decreased linearly with the increase of the supplemental amount of FBP (P < 0.05), and the n-6 PUFA to n-3 PUFA ratio decreased linearly or quadratically (P < 0.05). Compared with CON, the contents of C18:2n6c, PUFA and n-6 PUFA and the n-6 PUFA to n-3 PUFA ratio in egg yolk decreased with the addition of FBP in the diet, and the difference was significant in 8% FBP group compared with the control group (P < 0.05), but there was no significant difference between experimental groups supplemented with different proportions of FBP (P > 0.05).
Table 6. The effect of fermented Broussonetia papyrifera on the amino acid of Taihe silk chicken egg yolk (wet basis)
Note: CON = basal diet; T2 = basal diet + 2% fermented Broussonetia papyrifera; T4 = basal diet + 4% fermented Broussonetia papyrifera; T8 = basal diet + 8% fermented Broussonetia papyrifera; EAA = essential amino acids; NEAA = non-essential amino acids; FAA = flavor amino acids; TAA = total amino acids; FAA = glutamate + aspartate + alanine + arginine + glycine. Ten eggs were collected per day from each treatment group (5 replicates per treatment, 2 eggs per replicate) from day 70 to day 75 (6 days in total) of the experiment period. Each replicate consisted of 12 egg yolks mixed together to analyze the amino acids content of the yolk.
Table 7. The effect of fermented Broussonetia papyrifera on the fatty acid of Taihe silk chicken egg yolk (wet basis)
Note: The measurement result of the fatty acid content in egg yolk is the percentage of a certain fatty acid content in the total fatty acid content.
CON = basal diet; T2 = basal diet + 2% fermented Broussonetia papyrifera; T4 = basal diet + 4% fermented Broussonetia papyrifera; T8 = basal diet + 8% fermented Broussonetia papyrifera; SFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid; SFA = C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0 + C22:0 + C24:0; MUFA = C14:1 + C16:1 + C18:1n9c + C20:1 + C22:1n9 + C24:1 + C18:1n9t; PUFA = C18:2n6c + C18:3n6 + C18:3n3 + C20:2 + C20:3n6 + C20:4n6 + C22:6n3; n-3 PUFA = C18:3n3 + C22:6n3; n-6 PUFA = C18:2n6c + C18:3n6 + C20:3n6 + C20:4n6. Ten eggs were collected per day from each treatment group (5 replicates per treatment, 2 eggs per replicate) from day 70 to day 75 (6 days in total) of the experiment period. Each replicate consisted of 12 egg yolks mixed together to analyze the fatty acids content of the yolk.
a–b Within a row, values with different superscripts differ significantly at P < 0.05. Data are expressed as means and SEM, n = 5.
Effect of FBP on intestinal morphology and gut microbiota
The effects of FBP on the intestinal morphology are shown in Figure 1. By H&E, the small intestine morphology of Taihe silk chicken could be improved by adding FBP to the diet compared with CON. The villus height and crypt depth of duodenum, jejunum and ileum were further counted, and the ratio of villus height to crypt depth was calculated. The results showed that the villus height of duodenum and jejunum of Taihe silk chicken increased linearly or quadratically with the increase of the supplemental amount of FBP (P < 0.05). Compared with CON, the addition of 8% FBP significantly increased the villus height of duodenum and jejunum (P < 0.05). At the same time, the supplementation of FBP all significantly decreased the crypt depth of duodenum, jejunum and ileum (P < 0.05), increased the ratio of villus height to crypt depth (P < 0.05). The crypt depth of each intestinal segment decreased linearly or quadratically with the increase of the supplemental level of FBP (P < 0.05), the ratio of villus height to crypt depth increased linearly or quadratically with the increase of the supplemental level of FBP (P < 0.05).
Figure 1. The effect of fermented Broussonetia papyrifera on the intestinal morphology of Taihe silk chicken. (A) The effect of fermented Broussonetia papyrifera on the villus height, crypt depth, and the ratio of villus height and crypt depth of duodenum, jejunum, and ileum. (B) The effect of fermented Broussonetia papyrifera on the morphology of duodenum, jejunum, and ileum. CON = basal diet; T2 = basal diet + 2% fermented Broussonetia papyrifera; T4 = basal diet + 4% fermented Broussonetia papyrifera; T8 = basal diet + 8% fermented Broussonetia papyrifera. P: P-value for ANOVA. PL: P-value for linear effect. PQ: P-value for quadratic effect. a–d Means with unlike letters are significantly different (P < 0.05). Data are presented as the mean and SEM (n = 5).
The effects of FBP on the intestinal microbial diversity of Taihe silk chicken are shown in Figure 2. The Good’s coverage reached 0.97 in all groups, indicating sufficient sequencing depth for subsequent experimental analysis. Alpha diversity analysis was conducted to assess the abundance and diversity of microbial species. Compared with CON, the addition of FBP in the diet did not significantly affect microbial species diversity and abundance (P > 0.05). The Venn diagram (Figure 2B) showed that there were 10,264 shared ASVs among the four groups, with 1,938, 2,457, 1,927, and 1,588 unique ASVs in the CON, 2% FBP, 4% FBP, and 8% FBP groups, respectively. Beta-diversity analysis was used to evaluate differences in species composition (Figure 2C). Principal coordinate analysis (PCoA) and non-metric multidimensional (NMDS) analysis based on Bray–Curtis distance demonstrated significant separation of sample points between the groups with different supplementation of FBP and the control group, indicating a significant change in the intestinal microbial community structure between the experimental and control groups (P < 0.05).
Figure 2. The effect of fermented Broussonetia papyrifera on gut microbiota of Taihe silk chicken. (A) Alpha diversity of gut microbiota of Taihe silk chicken. (B) The Venn analysis of Taihe silk chicken. (C) PCoA and NMDS analysis of Taihe silk chicken. (D) The relative abundance on phylum level of Taihe silk chicken. (E) The relative abundance on genus level of Taihe silk chicken. (F) The LEfSe analysis of Taihe silk chicken (LDA score > 4). CON = basal diet; T2 = basal diet + 2% fermented Broussonetia papyrifera; T4 = basal diet + 4% fermented Broussonetia papyrifera; T8 = basal diet + 8% fermented Broussonetia papyrifera. P: P-value for ANOVA. PL: P-value for linear effect. PQ: P-value for quadratic effect. a–d Means with unlike letters are significantly different (P < 0.05). All the values contained six repetitions.
Comparisons of the relative abundances of gut microbiota between experimental and control groups are shown in Figure 2D and E. At the phylum level, Firmicutes and Bacteroidota were dominant phyla in the cecal microbiota of Taihe silk chicken. Compared with CON, the relative abundance of Bacteroidota in the cecum was significantly higher with the supplementation of FBP in the diet (P < 0.05), while Firmicutes showed a lower relative abundance (P < 0.05). At the same time, the addition of 8% FBP in the diet also significantly reduced the relative abundance of Actinobacteriota in the cecum (P < 0.05). At the genus level (Figure 2E), Rikenellaceae_RC9_gut_group and Bacteroides were dominant genera in the cecal microbiota of Taihe silk chicken. When FBP was added to the diet, the relative abundance of Bacteroides, Rikenellaceae_RC9_gut_group and Alistipes in cecum increased, while the relative abundance of Olsenella and Ruminococcaceae UCG-005 decreased. The addition of 2% and 4% FBP significantly increased the relative abundance of Rikenellaceae_RC9_gut_group in cecum (P < 0.05); Supplementation with 4% and 8% of FBP significantly increased the relative abundance of Alistipes (P < 0.05); The relative abundance of Bacteroides in cecum was significantly increased by adding different proportions of FBP (P < 0.05), and the relative abundance of Olsenella and Ruminococcaceae UCG-005 were significantly decreased (P < 0.05). The results of LEfSe analysis (Figure 2F) indicated that the dominant species in the control group included Olsenella and Ligilactobacillus. In contrast, the dominant species in the group fed with 4% FBP and 8% FBP were Bacteroides.
Prediction of gut microbiota metabolic functions
Using Phylogentic Investigation of Communities by Reconstruct- ion of Unobserved States (PICRUSt2) for KEGG functional prediction, we assessed the impact of FBP on the metabolic functions of Taihe silk chicken cecal microbiota, and the results are presented in Figure 3. Level 1 functional prediction showed six major functions, namely metabolism, genetic information processing, environmental information processing, cellular processes, human diseases, and organismal systems. Compared with CON, the FBP groups significantly increased the relative abundance of metabolism (P < 0.05), while significantly reducing the relative abundance of environmental information processing (P < 0.05). Results from Level 2 and Level 3 functional predictions demonstrated that, compared with CON, the FBP groups significantly enhanced several functions, including carbohydrate metabolism (citrate cycle), metabolism of cofactors and vitamins (biotin metabolism, folate biosynthesis, vitamin B6 metabolism), energy metabolism (oxidative phosphorylation), amino acid metabolism (phenylalanine metabolism), biosynthesis of other secondary metabolites (penicillin and cephalosporin biosynthesis), and digestive system (protein digestion and absorption) (P < 0.05).
Figure 3. Metabolic function of gut microbiota of Taihe silk chicken. (A) Level 1 metabolic functional prediction. (B) Level 2 metabolic functional prediction. (C) Level 3 significantly different metabolic functional prediction. CON = basal diet; T2 = basal diet + 2% fermented Broussonetia papyrifera; T4 = basal diet + 4% fermented Broussonetia papyrifera; T8 = basal diet + 8% fermented Broussonetia papyrifera. Significant correlation is represented by ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05 respectively. All the values contained six repetitions.
Correlation analysis of differential gut microbiota, laying performance, and egg yolk nutritional composition in Taihe silk chicken
The Spearman correlation analysis results of differential gut microbiota, laying performance, and egg yolk nutritional composition in Taihe silk chicken are presented in Figure 4. The results indicate that in the FBP groups, the significantly enriched Bacteroides are positively correlated with the levels of copper and C18:0 in egg yolk (P< 0.05). They are negatively correlated with protein, C15:0, and vitamin B2 content in the yolk (P< 0.05). In 2% FBP and 4% FBP groups, Rikenellaceae_RC9_gut_group, which is significantly enriched, shows positive correlations with average daily intake, average egg weight, C24:1, and methionine content in the yolk (P < 0.05). In the FBP groups, the relatively higher abundance of Alistipes is positively correlated with folate content in the yolk (P < 0.05) but negatively correlated with moisture content in the yolk (P < 0.05). In the control group, Olsenella and Ruminococcaceae UCG-005 are relatively more abundant. Olsenella has a significant negative correlation with average daily intake and fat content in the yolk (P < 0.05) but a significant positive correlation with moisture content and C16:0 in the yolk (P < 0.05). Ruminococcaceae UCG-005 has a negative correlation with copper content in the yolk (P < 0.05) but a positive correlation with vitamin A content (P < 0.05).
Figure 4. Spearman correlation analysis of gut microbiota, laying performance, and egg composition in Taihe silk chicken. Significant correlation is represented by ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05 respectively. All the values contained six repetitions.
Discussion
The production performance of Taihe silk chicken directly impacts its economic benefits. Numerous research findings have shown that adding FBP to pig diets increases the ADFI of pigs (Song et al. Reference Song, Li and Cheng2017; Zhang et al. Reference Zhang, Yin and Schroyen2021). This is attributed to the improved palatability and digestibility of feed materials after fermentation, leading to an increase in animal feed consumption. Broussonetia papyrifera is rich in polyphenols, flavonoids, and alkaloids (Ma et al. Reference Ma, Xu and LV2019; Pang et al. Reference Pang, Wang and Lin2014). Previous studies have found that the contents of soluble phenolic compounds and flavonoids in guava leaves tea are significantly increased after fermentation. In addition, soluble flavonoids in plants can be produced by the glycosylation process of phytochemicals occurring in the fermentation process (Wang et al. Reference Wang, Luo and Wu2018). Polyphenols possess antibacterial, antioxidant, anti-proliferative, and pro-apoptotic effects (Li et al. Reference Li, Zheng and Zhao2022). Flavonoids are a class of small-molecule compounds with diverse pharmacological effects, including antibacterial, anti-inflammatory, anticancer, and antioxidant properties (Sun et al. Reference Sun, Liu and Zhou2022). In this study, the data results indicate that adding FBP to the diet results in an increase in the ADFI. This may be because FBP contains beneficial nutrients and bioactive compounds that can stimulate appetite and improve gut health, leading to better feed efficiency. The fermentation process also increases the digestibility of nutrients in BP, making it more palatable and easier for the chickens to digest and absorb, thus promoting higher feed intake. Specifically, fermentation can enhance feed palatability by increasing the digestibility of nutrients, reducing the levels of anti-nutritional factors, and promoting the production of beneficial metabolites such as organic acids and microbial peptides (Canibe and Jensen Reference Canibe and Jensen2012; Zhang et al. Reference Zhang, Wei and Yan2022a). Additionally, fermentation improves the texture and physical properties of the feed, making it softer and easier for chickens to consume (Yeh et al. Reference Yeh, Hsieh and Chen2018). These changes result in higher feed consumption and better nutrient utilization. Additionally, flavonoids belong to the category of plant estrogens, with flavonoids being the most common polyphenolic compounds in plant estrogens (Kiyama Reference Kiyama2023). Previous studies have shown that FBP can increase milk yield and the expression of progesterone receptor and estrogen protein receptor in mammary epithelial cells of dairy goats through its rich flavonoid compounds (Zhao et al. Reference Zhao, Lv and HU2022). Therefore, flavonoids can promote follicular development and increase egg production through the reproductive axis. In this study, we observed an upward trend in average egg production rate with increasing FBP supplementation, which might be associated with its potential flavonoid content. However, further research is needed to confirm this relationship.
Egg quality encompasses a broad spectrum of physical, chemical, and nutritional attributes, which constitute vital economic and visual features of eggs. Traditional metrics for assessing egg quality encompass albumen height, haugh unit, shell strength, yolk color, relative eggshell weight, and eggshell thickness. The economic worth of eggs is contingent on their quality. Yolk color, often regarded as the most direct sensory indicator of egg quality, is frequently used by consumers to gauge egg excellence based on the depth of yolk coloration. This coloration is contingent on the type and concentration of carotenoids present in the feed. For instance, a diet rich in lutein results in a yellow yolk, while zeaxanthin dominance imparts a red hue (Papadopoulos et al. Reference Papadopoulos, Lioliopoulou and Ordoudi2022). Previous research has shown that supplementing exogenous pigments such as lutein and zeaxanthin in the diet of laying hens with low carotenoid content can improve yolk color (Wen et al. Reference Wen, Xu and Zhou2022). In this study, adding 1% and 5% FBP to the diet improved yolk color (Niu et al. Reference Niu, Wang and Liang2023), which is consistent with the results of this study.
The yolk color of Taihe silk chicken was significantly affected by the addition of FBP, and the yolk color increased linearly or quadratically with the increase of FBP. These results indicate that carotenoids may exist in FBP, which may affect the color change of egg yolk. Additionally, the age of the Taihe silk chicken also significantly affected yolk color, likely due to the processes of digestion, absorption, and transport of pigments, which require time to achieve optimal yolk pigmentation. It is worth noting that albumen height and Haugh unit serve as indicators of egg white quality. Thinning of egg white is the fundamental reason for the decrease in egg white quality because thin egg white is prone to microbial contamination and spoilage (Omana et al. Reference Omana, Liang and Kav2011). The main cause of egg white thinning is prolonged storage, during which the viscosity of egg white decreases naturally (Liu et al. Reference Liu, Wang and Wang2022b). The protein level in the feed also affects egg white quality, and previous research has found that increasing crude protein levels in the diet from 17% to 21% increases egg white proportion (Keshavarz and Nakajima Reference Keshavarz and Nakajima1995). In this study, supplementation of different proportions of FBP in diets had no significant effects on albumen height and Haugh unit, probably because there was no significant difference in crude protein levels among diets. However, the albumen height of Taihe silk chicken was significantly affected by different days of age. The eggshell serves as an effective barrier to protect the egg white and yolk from contamination and damage. Indicators of eggshell quality include eggshell strength and eggshell thickness. Factors affecting eggshell quality are mainly genetic and external environmental factors (Cheng and Ning Reference Cheng and Ning2023). The formation of eggshell is essentially the nucleation and orderly deposition of calcium carbonate on the outer eggshell membrane, regulated by matrix proteins. Calcium ion and other ion transporter expressions play an integral role in regulating the process (Chiang et al. Reference Chiang, Tseng and Wu2021; Sah and Mishra Reference Sah and Mishra2018). Regulating calcium ions and matrix proteins is key for better eggshell quality. In this study, the supplemental level of FBP had no significant effects on eggshell strength, relative weight of eggshell and eggshell thickness, but different age had significant effects. At the same time, the relative weight of eggshell and eggshell thickness had significant interaction between the age and the supplemental amount of FBP. This suggests that FBP may influence eggshell quality through age-related changes in calcium metabolism. FBP may affect calcium ion absorption and utilization in the intestine, potentially by modulating gut microbiota composition and SCFA production, which are known to influence calcium bioavailability (Trinidad et al. Reference Trinidad, Wolever and Thompson1997). Additionally, FBP may indirectly regulate the expression of key matrix proteins involved in eggshell formation, which play essential roles in calcium carbonate crystallization and eggshell matrix structuring (Fu et al. Reference Fu, Zhou and Schroyen2024; Liang et al. Reference Liang, Liu and Wang2025). However, since our study did not directly assess calcium ion transport mechanisms or matrix protein expression, further research is needed to elucidate the precise pathways through which FBP might interact with eggshell formation.
Taihe silk chicken eggs are nutritionally rich and have a higher nutritional value than regular chicken eggs (Chen et al. Reference Chen, Cai and Su2019). Eggs are abundant in protein, vitamins, cholesterol, trace elements, and amino acids, with a relatively low crude fat content, making them beneficial for health when consumed regularly. Previous research has shown that adding 10% and 15% FBP to grass carp feed significantly reduces the crude fat and crude protein content in the fish muscles (Tang et al. Reference Tang, Bai and Ao2021). In this study, no significant changes were observed in the protein, fat, cholesterol, and amino acid content of the egg yolks. Folate is usually obtained from food, vital for preventing neural tube defects during pregnancy. And it showed an increase in content with rising levels of FBP in the diet (Salamone Reference Salamone2022). The main factors affecting folate enrichment in eggs are the folate content in feed additives and feed ingredients. The folate content in eggs tends to increase first and then saturate as the amount of folate added increases. Additionally, folate produced independently by gut microbes such as Lactobacillus, Bifidobacterium, Streptococcus, Enterobacteriaceae, and Clostridiaceae may also influence the folate content in eggs. In this study, BP was fermented using Lactiplantibacillus plantarum, Bacillus subtilis, and cellulase. Lactiplantibacillus plantarum might produce folate during the fermentation process, thus increasing the folate content in FBP and subsequently affecting the folate content in eggs. However, adding FBP to the diet did not affect the folate-producing microbes in the gut of Taihe silk chicken, and the folate content in the egg yolk increased with the addition of FBP. Specifically, adding 4% and 8% FBP to the diet significantly increased the folate content in the egg yolk. Therefore, it is speculated that this could be due to the high folate content in FBP itself or folate produced by Lactiplantibacillus plantarum during fermentation. It is also possible that the addition of FBP improved the gut morphology of Taihe silk chicken or regulated gut microbes related to folate absorption, thereby enhancing the chickens’ ability to absorb folate from the feed. The specific mechanisms require further research. Zinc, an essential trace element, is critical for proper growth and development, and its deficiency poses a global public health concern (Knez and Stangoulis Reference Knez and Stangoulis2023; Rink and Gabriel Reference Rink and Gabriel2000). In this study, 8% FBP significantly increased folate and zinc content in the egg yolks. In a study examining the effects of adding silage from BP to the diet of Yangzhou geese, it was found that the levels of major PUFAs and monounsaturated fatty acids in the silage group were slightly lower than those in the control group. This difference is attributed to the variations in lipid composition between the diets of the different groups, which may alter the way geese transport fats and energy within their bodies, and exert an influence on lipid metabolism in geese (Wang et al. Reference Wang, Wang and Xiong2024). Similarly, it has been found that the addition of FBP to HY-Line brown hens diet can improve lipid metabolism, significantly reduce the content of total triglycerides, increase the content of high-density lipoprotein cholesterol, and affect the expression of genes related to liver lipid metabolism (Niu et al. Reference Niu, Wang and Liang2023). This is consistent with the results of this study. The supplementation of 8% FBP notably reduced n-6 PUFA and total PUFA levels in the egg yolks, mainly due to a significant decline in C18:2n6c content. And the n-6 PUFA to n-3 PUFA ratio decreased with the rising inclusion of FBP in the diet. Reducing the n-6/n-3 PUFA ratio has significant physiological relevance. Previous research has suggested that a lower n-6 PUFA to n-3 PUFA ratio in the diet may have positive effects on inflammation and myocardial ischemia-reperfusion injury in rats (Ma et al. Reference Ma, Xu and LV2019). It is associated with reduced inflammation, improved cardiovascular health, enhanced egg quality, and better growth and development in poultry. These benefits are achieved by promoting anti-inflammatory eicosanoids, enhancing lipid profiles, improving the fatty acid composition of egg yolks, and supporting neurological development and overall productivity (Calder Reference Calder2006; Cherian Reference Cherian2015; Simopoulos Reference Simopoulos2002; Swanson et al. Reference Swanson, Block and Mousa2012). However, one of the challenges with using BP is its high cellulose content, which acts as an anti-nutritional factor by potentially reducing nutrient absorption and digestion efficiency. Finding a balance between production efficiency and egg nutrition is crucial. The fermentation process can help mitigate this issue by breaking down complex fibers into simpler forms, thereby improving digestibility. Additionally, managing the inclusion levels of FBP in the diet is essential. While higher levels of FBP can enhance certain nutritional aspects such as folate and zinc content, they must be balanced with the potential reduction in nutrient digestibility due to cellulose. Therefore, a moderate supplementation level may provide a good balance, enhancing egg nutritional quality without significantly impairing production efficiency. In summary, proper addition of FBP can improve the nutrient content of egg yolk, notably influencing folate and zinc levels, as well as fatty acid composition.
The small intestine, being the primary site for food digestion and absorption, plays a crucial role in growth performance (Sha et al. Reference Sha, Shang and Miao2021). Intestinal health is closely tied to the digestion and absorption of nutrients, which is influenced by intestinal morphology, particularly villus height and crypt depth. Intestinal villi are protrusions that increase the surface area of the intestine, facilitating nutrient absorption (Kai Reference Kai2021). On the other hand, crypts are tubular glands located at the base of villi, crucial for digestive fluid secretion. Decreased crypt depth leads to enhanced cellular secretory functions and increased digestive fluid secretion (Zhang et al. Reference Zhang, Wang and Su2022b). Consequently, the ratio of villus height to crypt depth serves as a vital indicator for evaluating intestinal digestion and absorption capacity. An increased ratio indicates enhanced intestinal digestion and absorption capacity, contributing to improved growth performance, while a decreased ratio suggests intestinal damage and reduced digestion and absorption capacity. Previous research has shown that adding fermented rice wine residue to the diet of fattening pigs can increase the length of the duodenal villi and reduce crypt depth (Zhang et al. Reference Zhang, Wang and Su2022b). Adding fermented rapeseed meal to broiler diets significantly increased the villus height and the ratio of villus height to crypt depth in duodenum (Wu et al. Reference Wu, Chen and Ahmed2022). However, the addition of fermented grape residue to the diet had no effect on the ileum morphology of broilers (Gungor et al. Reference Gungor, Altop and Erener2021). In the results of this study, the supplementation of FBP influenced the morphological structure of the small intestine. For example, crypt depth decreased, the ratio of villus height to crypt depth increased, and the villus height increased in the duodenum and jejunum. These results indicate that the addition of different proportions of FBP to the diet can improve intestinal morphology and enhance the intestinal digestion and absorption capacity.
The gut microbiota is a complex community of diverse microorganisms that interact with each other and with the host organism (Makki et al. Reference Makki, Deehan and Walter2018). The gut microbiota maintains a dynamic equilibrium, and any disruption to this balance can lead to changes in the gut microbial community, thereby influencing both the gut and the body. These effects can be beneficial to the host or promote inflammatory responses (Hand et al. Reference Hand, Vujkovic-Cvijin and Ridaura2016). Dietary fiber is a key factor in maintaining normal intestinal function, mainly through its impact on gut microbes and thus on gut health. Numerous studies have demonstrated that dietary fiber can regulate intestinal microbiota, promote SCFA – producing bacteria, enhance nutrient digestion and absorption, and improve intestinal integrity, thereby supporting intestinal health and growth performance (Hu et al. Reference Hu, Li and Diao2023). BP is an excellent source of dietary fiber, making it beneficial for gut health. Previous research has indicated that the addition of 4.5% and 9% FBP in cow diets did not result in significant differences at the phylum level, but it decreased the relative abundance of UCG-013 and Tyzzerella-4 at the genus level (Hao et al. Reference Hao, Huang and Si2020). In weaned pig diets supplemented with 150 g/t of BP extract, the alpha-diversity and beta-diversity results showed no significant impact on microbial species diversity and abundance but affected the composition of the microbial community. The addition of BP extract decreased the relative abundance of Firmicutes and Proteobacteria and increased the relative abundance of Bacteroidota and Fusobacteria in the fecal microbiota of weaned pigs. At the genus level, the relative abundance of Lactobacillus was reduced, while the relative abundance of Prevotella-9 increased (Chen et al. Reference Chen, Shui and Chai2020). Consistent with the aforementioned research results, the present study revealed that adding FBP to the diets of Taihe silk chicken had no significant impact on microbial species diversity and abundance. However, it did alter the structure and composition of the gut microbiota. Adding different proportions of FBP to the diets increased the relative abundance of Bacteroidota in the cecum while reducing the relative abundance of Firmicutes. The ratio of Firmicutes to Bacteroidota is an important indicator for assessing the dynamic balance of the gut microbiota (Huttenhower et al. Reference Huttenhower, Gevers and Knight2012). Firmicutes are positively correlated with nutrient and energy absorption, so when the relative abundance of Firmicutes is high, it is conducive to energy absorption, which leads to obesity (Ley et al. Reference Ley, Turnbaugh and Klein2006; Magne et al. Reference Magne, Gotteland and Gauthier2020). On the other hand, Bacteroidota are positively correlated with nutrient digestibility (Jumpertz et al. Reference Jumpertz, Le and Turnbaugh2011). Further analysis of differences at the genus level among the groups revealed that adding different proportions of FBP to the diets increased the relative abundance of Rikenellaceae_RC9_gut_group, Bacteroides, and Alistipes in the cecum while decreasing the relative abundance of Olsenella and Rumenococcaceae UCG-005 in the cecum. Rikenellaceae_RC9_gut_group is known to convert undigested polysaccharides in the gut into SCFAs (Su et al. Reference Su, Tian and Zhang2014). Similarly, Bacteroides can break down complex polysaccharides, starch, and cellulose into simpler compounds, thereby aiding in host digestion and absorption (Lan et al. Reference Lan, Sakamoto and Sakata2006). Our results indicate that adding FBP to the diets may increase the digestibility of nutrients, decrease the absorption of energy, and shifts the gut microbiota toward increased digestion and absorption of polysaccharides, starch, and cellulose, thus affecting the yolk nutrient composition. Previous studies have shown that adding folic acid to the diet of broilers significantly increases the relative abundance of Alistipes in the cecum (Liu et al. Reference Liu, Yang and Liu2023). This is consistent with the results of this study. Adding FBP to the diet increased the content of folate in egg yolk and the relative abundance of Alistipes in the cecum, which may be related to the high content of folate in FBP. Additionally, recent reports indicate that Olsenella can cause lung infections leading to pneumonia (Yan et al. Reference Yan, Li and Li2022). Thus, the inclusion of FBP in the diet may reduce the risk of lung infections in Taihe silk chicken, promoting overall health. On the other hand, the abundance of Rumenococcaceae UCG-005 is positively correlated with the incidence of diarrhea (Liang et al. Reference Liang, Kou and Chen2021). Thus, adding FBP may help reduce the occurrence of diarrhea in Taihe silk chicken, promoting gut health. In summary, the modifications in gut microbiota induced by FBP are associated with improvements in nutrient absorption and overall growth performance by fostering a healthier gut environment. The increase in beneficial bacteria like Bacteroides and Rikenellaceae_RC9_gut_group enhances the breakdown and absorption of nutrients, leading to more efficient feed utilization. Concurrently, the reduction in harmful bacteria contributes to a more stable and less inflammatory gut environment, further promoting the overall health and productivity of the chickens.
The functional prediction of the intestinal microbiota using PICRUSt2 reveals that the inclusion of FBP in the diet significantly enhances the metabolic functions of Taihe silk chickens. The addition of FBP to the diet leads to a notable improvement in carbohydrate metabolism, amino acid metabolism, and digestive functions, indicating an increased rate of carbohydrate and protein digestion in the intestinal tract of Taihe silk chickens. This enhancement is particularly evident in the strengthening of the citrate cycle, phenylalanine metabolism, and protein digestion and absorption. The citrate cycle provides cells with energy by utilizing carbohydrates, fatty acids, amino acids, and acetyl-CoA. It serves as the primary source of cellular energy and is involved in numerous cellular metabolic processes (Kang et al. Reference Kang, Suzuki and Saito2021). Phenylalanine metabolism is critical for the synthesis of tyrosine, an essential amino acid in neonates. When the metabolism of phenylalanine is blocked, the content of phenylpyruvate in plasma increases, which leads to intellectual development disorders (Adams et al. Reference Adams, Ehlting and Edwards2019; Roberts et al. Reference Roberts, Ball and Filler1998). The inclusion of FBP in the diet also enhances energy metabolism, particularly oxidative phosphorylation. Oxidative phosphorylation is the process by which energy generated during the breakdown of carbohydrates, fatty acids, proteins, and other molecules is used to synthesize adenosine triphosphate (ATP) through the respiratory chain. This is the main source of ATP for the body (Nolfi-Donegan et al. Reference Nolfi-Donegan, Braganza and Shiva2020). Moreover, there is a significant increase in biotin metabolism, folate biosynthesis, and vitamin B6 metabolism. Biotin is an essential water-soluble vitamin for the organism and can be synthesized by intestinal microbiota. Folate is primarily sourced from plants, as animals cannot synthesize it themselves, it can only be synthesized in plant mitochondria. A deficiency in folate can lead to anemia, cardiovascular diseases, and cancer. Biotin, folate, and S-adenosylmethionine all play intermediate metabolic roles in one-carbon unit transfer processes (Hanson and Gregory Reference Hanson and Gregory2011; Mcmahon Reference Mcmahon2002). Vitamin B6 serves as a cofactor for various proteins, and its deficiency can lead to epileptic encephalopathy (Bagci et al. Reference Bagci, Zschocke and Hoffmann2008). It is important to note that animals and humans cannot synthesize vitamin B6 on their own and must obtain it from their diet (Rosenberg et al. Reference Rosenberg, Ischebeck and Commichau2017). Furthermore, there is a significant enhancement in the biosynthesis of penicillin and cephalosporin. These antibiotics are synthesized by microorganisms through a series of enzymatic reactions (Aharonowitz et al. Reference Aharonowitz, Cohen and Martin1992). Penicillin is an efficient and low-toxicity antibiotic with a narrow spectrum of antibacterial activity, commonly used in the treatment of pneumonia (Bibian et al. Reference Bibian, Perez-Sanchez and Mejia2020; Llor et al. Reference Llor, Perez and Carandell2019). Cephalosporin is one of the most widely used antibiotics due to its high safety, broad antibacterial spectrum, and strong antibacterial activity (Liu et al. Reference Liu, Chen and Liu2022a). The changes in the metabolome can explain the differences in nutrients in eggs. For example, enhanced biotin metabolism can lead to higher biotin content in eggs, while increased folate biosynthesis can raise folate levels in the egg yolk. Improved protein digestion and absorption may result in higher levels of amino acids in the eggs, contributing to better egg quality. In summary, the functional prediction results indicate that the addition of FBP to the diet can alter the intestinal microbiota, promote metabolic functions, and consequently improve the nutritional composition of eggs.
Spearman correlation analysis revealed significant associations between the intestinal microbiota of Taihe silk chicken and their laying performance as well as the nutritional components in egg yolks. Bacteroides, known for metabolizing polysaccharides and producing beneficial metabolites (Zafar and Saier Reference Zafar and Saier2021), showed significant correlations with key nutritional components, vitamins, trace elements, and fatty acid profiles in egg yolks. Previous studies have indicated significant positive correlations between Rikenellaceae_RC9_gut_group in cattle stomachs and fatty acids (Conte et al. Reference Conte, Dimauro and Daghio2022) as well as amino acids (Pang et al. Reference Pang, Chai and Yang2022). Similarly, in this study, Rikenellaceae_RC9_gut_group was positively correlated with certain fatty acids and methionine content, suggesting its role in influencing egg composition. And Rikenellaceae_RC9_gut_group was significantly positively correlated with average daily intake and average egg weight. While in weaned pig intestines, Rikenellaceae_RC9_gut_group showed no significant correlation with body weight (Liang et al. Reference Liang, Kou and Chen2021). Additionally, a higher relative abundance of Alistipes in the FBP group was positively correlated with folate content in egg yolks, consistent with previous studies on broilers (Liu et al. Reference Liu, Yang and Liu2023). This highlights the potential of FBP to modulate gut microbiota, promoting beneficial bacteria like Alistipes that contribute to improved egg quality. In weaned pig gut microbiota, Olsenella has shown significant positive correlations with fatty acids, amino acids and vitamins (Pang et al. Reference Pang, Chai and Yang2022). In our study, Olsenella exhibited a significant positive correlation with C16:0 content in egg yolks but a significant negative correlation with fat content in egg yolks. The negative correlation of Olsenella with egg fat content further underscores the complex interactions between gut microbiota and host metabolism. Most studies have reported a significant positive correlation between Ruminococcaceae_UCG-005 and diarrhea rates in weaned piglets (Liang et al. Reference Liang, Kou and Chen2021). However, few studies have explored its relationship with nutritional components. In our study, we found that Ruminococcaceae_UCG-005 was positively correlated with vitamin A content in egg yolks but negatively correlated with copper content in egg yolks. In summary, our results suggest that the addition of FBP may improve the laying performance and egg composition of Taihe silk chicken by modulating the relative abundance of intestinal Rikenellaceae_RC9_gut_group and Alistipes, highlighting the importance of gut health in optimizing poultry production.
Conclusion
In conclusion, adding FBP in the diet can improve egg yolk color, increase the contents of folate and zinc in egg yolk, and affect the composition of fatty acids in egg yolk. The addition of FBP can also improve intestinal morphology and significantly increase the ratio of villus height to crypt depth in duodenum, jejunum and ileum. At the same time, the composition of intestinal microbiota was also regulated, which increased the relative abundance of Bacteroides, Rikenellaceae_RC9_gut_group and Alistipes, while decreased the relative abundance of Olsenella and Ruminococcaceae UCG-005. In addition, combined with the results of functional prediction and correlation analysis, it was revealed that the FBP may change the laying performance and nutrient composition of egg yolk by affecting intestinal microbiota. This study provided theoretical basis and technical support for the development and utilization of BP and the healthy breeding and cost reduction and efficiency increase of Taihe silk chicken.
Acknowledgements
This research was supported by the fund from Jiangxi Provincial Key R&D Program Project (20223BBF61006, 20244BDH84006).
Author Contributions
Yang Fu: Conceptualization, Methodology, Investigation, data curation, Writing – original draft. Lutong Zhou, Yutian Shen and Weifa Su: Investigation, Visualization. Wentao Li, Lixia Kai and Wei Wei: Formal analysis, Visualization. Yuanzhi Chen, Fengqin Wang and Yizhen Wang: Writing – review & editing. Jianjun Peng: Provide test site and test animals. Zeqing Lu: Resources, Writing – review and editing, Supervision.
Conflicts of Interest
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Effects of fermented Broussonetia papyrifera on the laying performance, egg quality and gut microbiota of Taihe silk chicken during the peak laying period.”
Open access
