Effect of dietary Flammulina velutipes (Curt.: Fr.) stem waste on ovarian follicles development in laying hens

Abstract

Flammulina velutipes is one of the most consumed mushrooms mainly produced in China but their stem base, which is a waste resource in production usually not used, but has revealed reducing cholesterol effects and preventing fatty liver. Considering the important role of ovarian development in poultry production and the unclear mechanism of dietary nutrition in ovarian development, the effect of F. velutipes stem waste (FVS) on ovarian follicle development in laying hens and its potential mechanism were investigated by adding 2%, 4% and 6% FVS to the diet of laying hens and the production performance, biochemical parameters, hypothalamic-pituitary-ovarian axis hormone level, mRNA and protein changes on laying hens at sexual maturity and peak laying period were evaluated. FVS significantly improved the egg white weight and eggshell strength of laying hens, and markedly reduced the levels of lipid and abdominal fat. In addition to regulate lipid metabolism, FVS also inhibited the ovarian apoptosis pathway, reduced follicular atresia, increased the genes and proteins expression related to follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR) and steroid hormone synthesis signal pathway, and increased the level of serum hormone. FVS could thus promote ovarian development by reducing cholesterol and other lipid levels, stimulating steroid hormone signalling pathway as well as improve the production performance of laying hens, suggesting that FVS could be added to poultry feed as a nutritional supplement.

HIGHLIGHTS

1. Flammulina velutipes stem waste can improve egg white weight and eggshell strength;

2. Flammulina velutipes stem waste reduces ovarian apoptosis and inhibits follicular atresia;

3. Flammulina velutipes stem waste can be used as a feed supplement to improve ovarian health in poultry;

Keywords:

• Flammulina velutipes stem waste
• production performance
• follicular development
• ovary apoptosis
• steroid hormone

Introduction

Flammulina velutipes (Curt.: Fr.) is considered as healthy edible and medicinal fungi in China due to their biological activities and their nutrients richness such as protein, dietary fibre and crude fat (Yeh et al. 2014; Fukushima-Sakuno 2020) but also many bioactive compounds including polysaccharides, phenols, lipids. (Banerjee et al. 2020; Wang and Zhang 2021). Recently, Flammulina velutipes stem waste (FVS) has been revealed to be a feed component in the poultry diet which it could reduce fat deposition and improve performance (Mahfuz et al. 2021). FVS could also improve the antioxidant capacity and production performance of ISA layers (Chen et al. 2020). The development and maturation of ovarian follicles are the key factors affecting animal production performance. Although the mechanism is not clear, it has been found that fibre content affects the hatching ability of chickens (Mohiti-Asli et al. 2012).

The main function of ovary is to regulate and support the reproduction and many evidences have revealed that the content of dietary fibres could have a wide impact on female reproduction. This fibre has been shown to affect ovarian function, steroid biosynthesis, oocyte quality and fertilisation (Willis et al. 2020). The mechanism involved on this female reproduction is usually regulated but partially by hormones, including gonadotropins and steroids (Saez and Drevet 2019).

Many hormones involved in the hypothalamic-pituitary-ovarian axis play a key role in regulating cell proliferation, differentiation and degeneration and cytokine secretion, and determine the follicular development or atresia (Brady et al. 2020; Tan et al. 2021). Follicle stimulating hormone (FSH) promotes the gene expression of cytochrome P450 and up-regulates the concentration of progesterone by stimulating the small yellow follicles (SYF) in poultry (Ma et al. 2020). Otherwise during the steroid hormones synthesis by follicles, the cholesterol passes by blood vessels through Steroidogenic Acute Regulatory (StAR) protein, which is transported from the outer mitochondrial membrane of granulosa cells to the inner membrane, then Cytochrome P450 family member 11A1(CYP11A1) catalyse the formation of pregnenolone, and 3β-Hydroxysteroid dehydrogenase (3β-HSD) catalytic synthesis of progesterone. They play a role in different parts of follicular cells, participate in the synthesis of follicular steroid hormones, and further regulate the follicular development (Miller 2017; Bassi et al. 2021). The availability of cholesterol is determinant for hormone synthesis. Therefore, changes in lipid metabolism may affect the synthesis of steroid hormones and cause different reproductive disorders like polycystic ovary syndrome (PCOS) (Ferrer et al. 2021; Kazemi et al. 2021; Yang et al. 2021).

There is actually no evidence concerning the effect of dietary FVS on the development of reproductive ovaries in laying hens. Thus, this study aimed to evaluate the effects of the addition of FVS in the diet on the development of prehierachical follicles by regulating steroid synthesis signalling pathway and determining the possible mechanism involved in the follicular development.

Material and methods

The chemical composition of FVS

The F. velutipes stem waste (FVS) was supplied by Xue Guo gauficus Biotechnology Co., Ltd. (Changchun, China). This fresh FVS was naturally dried for one week and ground into powder in Hanhong animal husbandry (Jilin, China) for feed preparation. FVS used the same material and had the same composition as Mahfuz et al. (Mahfuz et al. 2019). The results of the component analysis are listed in Table 1.

Table 1. Chemical composition of flammulina velutipes stem waste.

Ingredients	Contents, g/100g
Dry matter	88.40 ± 0.85
Ash	11.60 ± 0.85
Moisture	14.74 ± 0.73
Crude fibre	22.05 ± 1.06
Crude protein	13.75 ± 0.49
Crude fat	2.80 ± 0.14
Ether extract	1.50 ± 0.02
Aspartic acid	1.05 ± 0.08
Calcium	0.36 ± 0.15
Phosphorus	0.88 ± 0.23
Lysine	0.81 ± 0.07
β-Glucan	0.19 ± 0.05
Energy, MJ/kg	1.52 ± 0.03

Note: Randomly weigh 3 equal (100 g) samples for determination. SEM: pooled standard error of the means.

Hens’ management and sample collection

All animals handling procedures strictly complied with the Use and Care of Laboratory Animals and were approved by the Animal Care Review Committee, Jilin Agricultural University (Approval No. GR (J) 19–073). The euthanasia of chickens was performed in full compliance with the laws and regulations of the State Council experimental animal care regulation of the people’s Republic of China (revised 2017). One-day-old Hy-Line Brown female chickens were purchased from Yinong poultry Co., Ltd (Harbin, China) and were housed and fed at the Huiling hens production base (Jilin, China). They were randomly divided into five groups (n = 125 chickens per group). a negative control group (NC), a positive control group (PC, Considering the effect of crude fibre in FVS on laying hens, the basic fibre concentration: 1% inulin group was set as the positive control), and three treatments group (2, 4, and 6% FVS). according to the composition of diets described in Tables 2 and 3, as well as the application of the five iso-energy and iso-nitrogen diets of growth period and laying period. From 0 to 12 weeks, the flat cage feeding was adopted. There were 14 chicks in each cage, each group had three replicates, which were divided into three sub replicates. The size of the cage was 60 × 50 × 40cm. The temperature of the environment was controlled at 27–30 °C, as well as the relative humidity at 60–70%. After 12 weeks, they were moved to ladder cage for feeding, and there were 3 chickens in each cage. The size of the cage was 40 × 45 × 38cm. The temperature was 20–25 °C, while the relative humidity was controlled at 55–65%. At the age of one week, the light was kept 12 h a day, and then gradually decreased to about 10 h at the age of 6–8 weeks, and then increased to 12 h from the 17 weeks age.

Table 2. Ingredients and nutrient composition of experimental diets during growing period (9—17W).

Ingredient, g/kg	NC	PC	2%FVS	4%FVS	6%FVS
Corn	682.5	671.5	675.0	650.0	630.0
Soybean meal (44.2% CP)	170.8	175.7	168.9	165.8	163.0
Wheat bran	94.9	87.0	86.3	91.8	93.2
Calcium phosphate	11.6	11.6	11.6	11.6	11.6
Stone powder	19.0	19.0	19.0	19.0	19.0
Soybean oil	5.0	9.0	3.0	5.6	7.0
Salt	3.4	3.4	3.4	3.4	3.0
Premix^a	10.0	10.0	10.0	10.0	10.0
70% lysine	0.7	0.7	0.7	0.7	0.7
Methionine	1.1	1.1	1.1	1.1	1.1
Choline chloride	1.0	1.0	1.0	1.0	1.0
Inulin		10.0
FVS			20.0	40.0	60.0
Nutrient levels, %
ME, MJ/kg	11.5	11.5	11.50	11.5	11.5
CP	15.0	15.0	15.0	15.0	15.0
Ca	1.0	1.0	1.0	1.0	1.0
AP	0.24	0.24	0.24	0.24	0.24

^aThe premix provided the following per kg of diets: vitamin A 4500 IU, vitaminD3 1200 IU, DL-α-tocopherol acetate 2500 IU, VB1 5000 mg, VB2 20,000 mg, VK 10,000 mg, nicotinic acid 45,000 mg, pantothenic acid 35,000 mg, biotin 1500 mg, folic acid 3000 mg, VB12 40 mg, zinc 45 mg, manganese 50 mg, iron 30 mg, copper 4 mg, cobalt 100 mg μ g. Iodine 1 mg, selenium 100 μ g. FVS: F. velutipes stem waste; NC: negative control, base diet; PC: positive control, add 1% inulin to base diet; ME: metabolic energy; CP: crude protein; AP: available phosphorus.

Table 3. Ingredients and nutrient composition of experimental diets during egg production (18—27W).

Ingredient, g/kg	NC	PC	2%FVS	4%FVS	6%FVS
Corn	616.40	604.00	599.20	582.10	564.90
Soybean meal (44.2%CP)	247.40	249.80	244.60	241.70	238.90
Dicalcium phosphate	20.00	20.00	20.00	20.00	20.00
Limestone	93.00	93.00	93.00	93.00	93.00
Steamed bone meal	10.00	10.00	10.00	10.00	10.00
Soybean oil	7.00	7.00	7.00	7.00	7.00
Salt	1.80	1.80	1.80	1.80	1.80
Choline	1.00	1.00	1.00	1.00	1.00
Premix^a	1.00	1.00	1.00	1.00	1.00
Methionine	1.20	1.20	1.20	1.20	1.20
70% lysine	1.17	1.17	1.17	1.17	1.17
Inulin		10.00
FVS			20.00	40.00	60.00
Nutrient levels, %
ME, MJ/kg	10.80	10.80	10.80	10.80	10.80
CP	17.35	17.35	17.35	17.35	17.35
Ca	3.00	3.00	3.00	3.00	3.00
AP	0.35	0.35	0.35	0.35	0.350

^aThe premix provided the following per kg of diets: vitamin A 4500 IU, vitaminD3 1200 IU, DL-α-tocopherol acetate 2500 IU, VB1 5000 mg, VB2 20000 mg, VK 10000 mg, nicotinic acid 45000 mg, pantothenic acid 35000 mg, biotin 1500 mg, folic acid 3000 mg, VB12 40 mg, zinc 45 mg, manganese 50 mg, iron 30 mg, copper 4 mg, cobalt 100 mg μ g. Iodine 1 mg, selenium 100 μ g. FVS: F. velutipes stem waste; NC: negative control, base diet; PC: positive control, add 1% inulin to base diet; ME: metabolic energy; CP: crude protein; AP: available phosphorus.

Nine laying chickens, fasted for more than 12 h but with free access to water, were randomly selected from each group (3 chickens per replicate) to collect blood, then euthanized by cervical dislocation at sexual maturity (17 weeks) and at the peak of egg production (27 weeks). The blood was stood for 20 min at room temperature and centrifuged at 3000 rpm for 20 min, and the obtained serum was stored at −20 °C. The whole ovary, liver and abdominal fat were quickly removed, weighed and their size was measured. A part of the liver, ovary, large white follicles (4–6 mm in diameter) and small yellow follicles (6–8 mm in diameter) were fixed in 4% paraformaldehyde for histological observation. The remaining portion of those organs were immediately snap frozen in liquid nitrogen and stored in a − 80 °C freezer (Thermo Scientific Co., Ltd, Model: FDE30086FV-ULTS, Massachusetts, USA) until further use.

Production performance and egg quality

For all chickens, the remaining feed and the laying hens’ weight were recorded every week to determine the average daily food intake (ADFI) and average daily gain (ADG). Eggs were collected every day and their laying rate was calculated. At 27 weeks, 30 eggs (6/replicate) were randomly collected from each group to evaluate egg quality parameters (egg weight and height, yolk colour, yolk height and quality, eggshell thickness and strength) according to the American grade. Briefly, egg weight, egg height, yolk colour, yolk height and quality were determined by multifunctional egg quality tester from ORKA (Model EA-01, New York, USA), while eggshell strength was measured by eggshell strength tester from ORKA (Model EFR-01, New York, USA). Eggshell thickness (without inner and outer shell membranes) in blunt, equatorial and sharp zones was measured by Vernier calliper to obtain an average value. The feed conversion rate (FCR) was finally calculated basis from egg production, egg weight, and feed intake.

H&E staining and histological observations

The liver and ovary fixed in formaldehyde were embedded in paraffin and sectioned (3–5 mm). These sections were deparaffinized with xylene, rehydrated with ethanol at different degrees and finally stained with haematoxylin and eosin (H&E). Images were obtained by Leica microscope and at least 10 different regions in each slice were measured, while the number of follicles was evaluated using ImageJ 1.8.0 software analysis system (Schneider et al. 2012; Uslu et al. 2017). The granule cell layer thickness was determined by the morphometric method used for the male testis seminiferous tubules by ImageJ 1.8.0 software analysis (Neves et al. 2002).

Detection of biochemical parameters

The liver tissue was initially homogenised with 75% normal saline at the ratio of 1:10 (w/V) and centrifuged (14,000 rpm x g, 10 min, 4 °C). The supernatant was then collected and used, as well as the serum, to evaluate the levels of Total cholesterol (T-CHO), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C) and triglycerides (TG) purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China) and according to their instructions.

Serum hormone concentrations by ELISA

Chicken Progesterone (PROG), oestradiol (E2), follicle stimulating hormone (FSH) and luteinizing hormone (LH) kits were purchased from Jingmei biotech Co., Ltd (Nanjing, Jiangsu, China) and were used to determine their serum levels by enzyme linked immunosorbent assay (ELISA) according to the manufacturer’s instruction.

Ovarian gene expression by real-time quantitative PCR

Total RNA was isolated from ovaries using Trizol reagent purchased from Takara Biotechnology Co., Ltd (Dalian, Liaoning, China). 3 μL of this RNA was used for the electrophoresis onto 1% agarose gel for 15 min and RNA integrity was assessed. Total RNA concentration as well as the absorbance ratio between 260 and 280 nm were quantified using a microplate analyser. This total RNA was then used for the cDNA synthesis using PrimeScript RT reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd, Dalian, China). PCR was finally performed by a reverse transcriptase Kit with SYBR purchased from genestar (Kangrun Biotechnology Co., Ltd, Beijing, China). The following cycling conditions were used to amplify cDNA: pre denatured at 95 °C for 30 min; denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, extension at 72 °C for 30s, and amplification for 40 cycles. Primer sequences used for real-time PCR are shown in Supplementary Table 1. The relative mRNA expression was calculated following the 2^−ΔΔCt method.

Ovarian protein levels by Western blot analysis

Ovary tissues were homogenised using RIPA lysis buffer from Solarbio life science (Beijing, China), centrifuged at 12,000 rpm for 10 min at 4 °C and their concentration was determined by BCA protein quantitative kit from genestar (Kangrun Biotechnology Co., Ltd, Beijing, China). The collected supernatant was mixed with a loading buffer and denatured at 100 °C for 10 min to obtain proteins for the further western blot analysis. These proteins were subjected to SDS-PAGE, and the bands were transferred onto PVDF membranes, then blocked with 5% skim milk for 2h, followed by incubation overnight at 4 °C with primary mouse antibodies against CYP17A1 (1:500 dilution), CYP19A1 (1:500 dilution), HSD3B1 (1:200 dilution) and LHR (1:200 dilution) purchased from Santa Cruz Biotechnology Co., Ltd (Colorado, USA), against FSHR (1:200 dilution) and Caspase3 (1:200 dilution) purchased from Wanlei biotech Co., Ltd. (Shenyang, Liaoning, China), and against PCNA (1:4000 dilution), Bax (1:1000 dilution) and Bcl2 (1:1000 dilution) purchased from Proteintech, Wuhan SanYing biotechnology Co., Ltd. (Wuhan, China). The expression level of β-actin was used as an internal control. Primary mouse antibody against β-actin (1:4000 dilution) was from cell signalling technology (Beverly, MA, USA). The membranes were then washed and incubated with anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (1:4000 dilution), purchased from Bioss Biotechnology Co., Ltd. (Beijing, China), for 90 min. Positive bands were detected by enhanced chemiluminescence (ECL) substrate (spark jade Biotechnology Co., Ltd., Shandong, China) and quantified with Image J software.

Statistical analysis

The differences among all treatments were reported as means ± standard error of means (SEM) and performed with GraphPad Prism 9 software. For parameters (performance, egg quality, real-time quantitative PCR, Western Blot), data in the completely randomised design experiment were analysed by one-way ANOVA. Tukey’s multiple range test was used to evaluate the differences among groups. For parameters (ELISA) results were tested by non-parametric test (Mann–Withney–Wilcoxon test). The statistical differences were deemed significant when p < 0.05.

Results

Effects of dietary FVS on performance and egg quality of hens

During the experimental period, the effects of dietary supplementation of FVS on laying performance presented in Table 4 have revealed no significant difference in average daily gain, egg heavy and ADFI of hens among the experimental treatments. FVS-fed hens significantly reduced FCR, but improved dramatically egg mass (p < 0.05). compared to NC and PC groups. The average egg laying rate, especially in 4% FVS group was increased in comparison with NC and PC groups. From the beginning to 27 weeks, there were no mortality in 2% and 4% FVS groups while the other groups presented a mortality percentage due to anal prolapse during the feeding process. Concerning the egg quality shown in Table 5, the FVS in diet did not affect egg yolk weight, egg index, egg yolk colour, eggshell thickness, protein height and Haugh unit at 27 weeks. However, FVS significantly improved eggshell strength and egg white weight (p < 0.05).

Table 4. Effect of dietary FVS on performance of laying hens in different stages.

Contents	Stage, week	NC	PC	2% FVS	4% FVS	6% FVS	SEM	p-Value
Average daily gain, g/d	17–27	3.52	4.85	4.15	3.91	3.62	0.221	0.355
Average egg heavy, g	17–27	53.97	57.27	55.40	56.10	57.00	0.663	0.572
Egg mass, kg	17–27	66.38^b	71.01^a	69.25^a	70.13^a	70.11^a	0.451	0.038
Average daily feed intake, ADFI, g/d	17–27	84.15	81.53	83.21	81.59	81.88	0.540	0.481
Feed conversion ratio, FCR, g/g	17–27	1.60^a	1.40^b	1.52^ab	1.46^b	1.43^b	0.243	0.039
Average egg-laying rate, %	17–27	75.56	75.00	73.53	79.63	75.49	0.014	0.063
Mortality, %	0–27	4.55^a	2.27^b	0.00	0.00	4.55^a	0.000	0.000

Data represented the mean value of 125 birds per treatment (n = 125). FVS: F. velutipes stem waste; NC: negative control, base diet; PC: positive control, add 1% inulin to base diet. a, b, means in the same row with different letters are significantly different at p < 0.05. SEM: pooled standard error of the means.

Table 5. Effect of 27-week dietary FVS on egg quality.

Contents	NC	PC	2% FVS	4% FVS	6% FVS	SEM	p-Value
Egg yolk weight/g	14.43	14.24	13.80	14.26	13.71	0.121	0.280
Egg white weight/g	25.81^b	32.24^a	30.96^a	30.27^a	31.94^a	0.364	0.000
Egg index	1.28	1.27	1.28	1.26	1.28	0.000	0.364
Egg yolk colour	5.85	5.68	5.80	5.90	6.05	0.154	0.231
Eggshell thickness/mm	0.39	0.40	0.37	0.40	0.40	0.014	0.054
Eggshell strength/kg cm^−2	33.96^b	41.81^a	37.00^ab	40.46^a	41.80^a	0.663	0.000
Protein height/mm	4.61	5.16	5.13	4.70	4.04	0.180	0.534
Haugh unit	64.45	68.16	64.47	64.14	55.94	1.992	0.628

Data represented the mean value of 20 eggs per treatment (n = 20). FVS: F. velutipes stem waste; NC: negative control, base diet; PC: positive control, add 1% inulin to base diet. a, b, means in the same row with different letters are significantly different at p < 0.05. SEM: pooled standard error of the means.

Impact of dietary FVS on ovarian follicular development in laying hens

In order to evaluate the developmental status of ovary, the basic indexes of ovarian follicles were measured and presented by Table 6, The results revealed that the ovary weight and index had no difference in the FVS groups at 17 weeks. The ovary length in the 4% and 6% FVS group are significantly increased (p < 0.05), while LWFs, SYFs, F1, F2, F3 diameter had no difference (p > 0.05).

Table 6. Effect of FVS on ovarian organ index in different periods.

Contents	Stage, week	NC	PC	2% FVS	4% FVS	6% FVS	SEM	p-Value
Ovary weight, g	17	16.66	16.77	16.65	16.71	16.66	0.020	0.287
	27	35.11^b	39.48^b	51.38^a	34.98^b	35.19^b	2.324	0.005
Ovary index, g/kg	17	12.46	13.16	12.87	12.99	13.26	0.191	0.724
	27	22.83	23.28	31.48	24.96	23.32	1.227	0.112
Ovary length, g	27	2.00^b	2.42^a	2.10^b	2.47^a	2.48^a	0.236	0.011
LWF counts	27	37.33	30.67	37.33	38.33	45.00	2.012	0.082
SYF counts	27	11.33	12.67	13.00	13.00	13.00	1.222	0.559
F1 diameter, cm	27	2.63	2.70	2.90	2.73	3.07	0.056	0.057
F2 diameter, cm	27	2.37	2.50	2.57	2.43	2.77	0.055	0.165
F3 diameter, cm	27	2.10	2.23	2.43	2.03	2.27	0.064	0.340

Data represented the mean value of 9 hens per treatment (n = 125). FVS: F. velutipes stem waste; NC: negative control, base diet; PC: positive control, add 1% inulin to base diet. a, b, means in the same row with different letters are significantly different at p < 0.05. SEM: pooled standard error of the means.

Following Mfoundou et al. (2021), the histological results of the left ovary of Hy-Line brown layer were observed (Mfoundou et al. 2021). H&E staining of ovarian tissue at 17 weeks has shown that yolk was accumulated in primordial, primary follicles and fast-developing follicles with dense round protein particles. The undifferentiated cytoplasmic zone was attached to the perivitelline membrane while lacunar channels were mainly developed around the entire medulla and fast- developing follicles with this medulla rich in blood vessels (Figure 1(A: a–e)). At 27 weeks, granulosa and basal lamina was obvious in the LWFs and SYFs, and the follicles were well developed (Figure 1(A: f–o)). The quantitative analysis of H&E staining revealing the percentage of different kinds of follicles was determined and the results showed that the FDF ratio was significantly increased in the 2% and 4% FVS group compared to the NC group while there was no significant difference in the thickness of granulosa cells for LWF and SYF at 27 weeks (Figure 1(B,C)). These results indicate that FVS dietary supplementation could promote the development of ovarian follicles in laying hens.

Figure 1. Effect of FVS on ovary and liver morphology in different periods. (A) Representative haematoxylin and eosin-stained histological images of (a–e) ovary tissue at 17 weeks, (f–j, LWF) large white follicles and (k–o, SYF) small yellow follicles at 27 weeks, scale:50μm, magnification:10 × 10. (B) The percentage of three kinds of follicles (AF, PF, FDF) in total follicle number, (C) Granulosa lamina thickness of two kinds of follicles (LWF, SYF) in Image J. (D) Morphological observation of liver. Data are expressed as means ± S.E.M., NC: negative control, PC: positive control (n = 9), *p < 0.05, **p < 0.01, vs. the NC group, ^#p < 0.05, ^##p < 0.01, vs. the PC group.

Effects of dietary FVS on ovarian proliferation and apoptosis in laying hens

The analysis of genes and proteins involved on proliferation and apoptosis was assessed to determine the FVS dietary role in the development of ovary. The results showed that the gene expression of PCNA and Bcl2 was significantly increased (p < 0.01, Figure 2(A,D)), while Caspase3, Bax and Bax/Bcl2 were significantly decreased in the FVS groups at 17 and 27 weeks compared to the NC and PC groups (p < 0.01, Figure 2(B,C,E)). Western blot analysis also revealed the similar results (p < 0.01, Figure 2(F–K)) justifying the fact that FVS dietary could regulate the ability of ovarian cell apoptosis in laying hens.

Figure 2. Effects of diet on ovarian proliferation and apoptosis. The mRNA and protein levels of PCNA, Caspase3, Bax, Bcl2 and Bax/Bcl2 in hens ovaries at 17 or 27 weeks by RT-qPCR and Western blot analysis. (A–E) RT-qPCR results, (F) Western blot results, (G–K) Plot of protein expression. β-Actin was used as internal control. Data are expressed as means ± S.E.M., NC: negative control, PC: positive control (n = 9), *p < 0.05, **p < 0.01, vs. the NC group, ^#p < 0.05, ^##p < 0.01, vs. the PC group.

Effects of dietary FVS on gonadotropins and their receptors

Hormone levels are shown in Table 7. Whether it’s 17 weeks or 27 weeks, E2 and PROG levels increased significantly in 4%FVS compared with NC and PC (p < 0.05). At the same time, FSH and LH were significantly increased in 4%FVS at 27 weeks(p < 0.05), but no significant difference was found among all groups at 17 weeks. Otherwise, mRNA and protein expression of FSHR were significantly increased in the 2% and 4% FVS group in comparison to the NC and PC groups, while they were significantly decreased in the 6%FVS group (p < 0.01, Figure 3(A,C,D)). On the other hand, mRNA and protein expressions of LHR were significantly increased in all FVS groups except for the 2% FVS group which was significantly decreased at 27 weeks (p < 0.01, Figure 3(B,C,E)).

Figure 3. Effects of diets on gonadotropins and their receptors. The mRNA and protein levels of FSHR and LHR in hens’ ovaries at 17 or 27 weeks by RT-qPCR and Western blot analysis. (A–B) RT-qPCR results, (C) Western blot results, (D–E) Plot of protein expression. β-Actin was used as internal control. Data are expressed as means ± S.E.M., NC: negative control, PC: positive control (n = 9), *p < 0.05, **p < 0.01, vs. the NC group, ^#p < 0.05, ^##p < 0.01, vs. the PC group.

Table 7. Effect of FVS on serum hormone level in different periods.

Contents	Stage, week	NC	PC	2% FVS	4% FVS	6% FVS	SEM	p-Value
E2, pmol/L	17	50.39^b	49.15^b	50.39^b	60.41^a	54.29^b	6.209	0.003
	27	55.71^b	50.39^b	56.46^b	67.29^a	60.06^ab	7.300	0.010
PROG, pmol/L	17	1716.67^c	1667.92^c	2021.67^a	2130.00^a	1893.33^b	37.938	0.000
	27	1999.17^b	2005.42^b	2282.50^a	2257.92^a	2158.33^a	30.456	0.000
FSH, U/L	17	3.60	3.70	4.57	4.65	3.96	0.165	0.131
	27	4.06^c	5.13^bc	5.90^ab	6.52^a	4.50^c	0.245	0.003
LH, ng/L	17	14.96	16.57	17.20	19.67	17.48	0.621	0.196
	27	19.02^b	18.15^b	20.22^b	28.09^a	21.22^b	1.068	0.016

Data represented the mean value of 9 hens per treatment (n = 125). FVS: F. velutipes stem waste; NC: negative control, base diet; PC: positive control, add 1% inulin to base diet. a, b, means in the same row with different letters are significantly different at p < 0.05. SEM: pooled standard error of the means.

Effects of dietary FVS on cholesterol and other lipid levels of laying hens

Concerning the histological analysis using H&E staining, the liver nucleus and cytoplasm were well distributed and arranged as well as the liver cell membrane was intact (Figure 1(D)). In addition, there was no steatosis, and the tissue structure was normal without any change. revealing that the FVS supplements did not cause any liver lesions.

As shown in Table 8, the change of liver development and fat deposition during FVS dietary was assessed showing that the liver weight was significantly decreased in the PC group (p < 0.05) compared to the NC group at 17 weeks. The abdominal fat ratio was significantly reduced in 4% and 6% FVS groups at 27 weeks compared to the NC group (p < 0.05). On the other hand, T-CHO in serum was significantly decreased in the FVS groups, except for 2% FVS group at 17 weeks. Besides serum TG, HDL-C and LDL-C at 17 weeks which was not affected, TG and LDL-C levels were significantly decreased after FVS diets for 27 weeks, while HDL-C level was significantly elevated (p < 0.05). These results illustrated that FVS dietary could reduce the levels of fat deposition and cholesterol metabolism after a long period of consumption.

Table 8. Effect of FVS on serum lipid and abdominal fat in different periods.

Contents, mmol/L	Stage, week	NC	PC	2% FVS	4% FVS	6% FVS	SEM	p-Value
Abdominal fat rate, g	17	20.58	17.36	16.25	20.19	18.57	2.011	0.703
	27	21.52^a	21.86^ab	29.36^a	10.41^b	19.06^b	4.011	0.009
T-CHO, mmol/L	17	5.53^a	5.00^b	5.46^a	4.87^b	4.85^b	0.358	0.000
	27	4.98^a	3.16^b	3.31^b	3.13^b	3.08^b	0.148	0.000
HDL-C, mmol/L	17	3.01	3.64	3.25	3.37	3.32	0.083	0.202
	27	2.57^b	2.46^b	3.01^a	2.99^a	2.71^b	0.094	0.047
LDL-C, mmol/L	17	0.74	0.60	0.60	0.55	0.63	0.323	0.477
	27	0.61^a	0.40^b	0.43^b	0.40^b	0.49^b	0.324	0.036
TG, mmol/L	17	1.39^a	1.16^b	1.13^b	0.94^b	1.01^b	0.038	0.000
	27	1.37^a	1.06^b	1.05^b	0.87^b	0.94^b	0.035	0.000

Data represented the mean value of 9 hens per treatment (n = 125). FVS: F. velutipes stem waste; NC: negative control, base diet; PC: positive control, add 1% inulin to base diet. a, b, means in the same row with different letters are significantly different at p < 0.05. SEM: pooled standard error of the means.

Effects of dietary FVS on ovarian steroid hormone synthesis signaling pathway

The enzymes and proteins involved in the production of sex hormones were evaluated to explore the capacity of FVS to inhibit steroid hormone production through one or more pathways affecting the transcriptional regulation of steroid biosynthesis related to genes. As shown in Table 7, serum PROG and E2 were improved in the FVS groups compared to the control groups at 17 and 27 weeks (p < 0.05). Furthermore, 3β-HSD mRNA and protein expressions were significantly increased in FVS compared with NC and PC groups (p < 0.01, Figure 4(C,G)). The StAR and CYP11A1 mRNA expression improved in 2% and 4% FVS groups (p < 0.01), but decreased in the 6% FVS group at 17 and 27 weeks (Figure 4(A,B)), while CYP17A1 and CYP19A1 protein expressions were significantly elevated in all FVS groups (p < 0.01, Figure 4(E,F)) showing that FVS dietary could improve the steroid hormone synthesis.

Figure 4. Diets affect steroid synthesis signalling pathway and regulate hormone production. (A–C) The mRNA expression of StAR, CYP11A1 and 3β-HSD in hens ovaries at 17 or 27 weeks by RT-qPCR. The protein levels of CYP17A1, CYP19A1 and 3β-HSD at 17 or 27 weeks in hens’ ovaries by Western blot analysis. (D) Western blot results, (E–G) Plot of protein expression. β-Actin was used as internal control. Data are expressed as means ± S.E.M., NC: negative control, PC: positive control (n = 9), *p < 0.05, **p < 0.01, vs. the NC group, ^#p < 0.05, ^##p < 0.01, vs. the PC group.

Discussion

Many experiments have revealed that the production performance depends largely on the efficient development of ovarian follicles due to the ovary, which is stimulated by sex hormones secreted by the hypothalamic-pituitary-ovarian axis to regulate the ovulation (Brady et al. 2020; Tan et al. 2021). Hormone regulation usually has different signalling pathways, but their mechanism of action remains not clear. Therefore, studies have focussed on the mechanism of diet on the ovarian development finding that bioactive substances can significantly be involved on that hormone regulation.

The results of the present study provided the evidence that the inclusion of FVS improved the egg white weight and eggshell strength. Some trace elements such as calcium, phosphorus, and zinc are known to play a vital role in egg formation and affect the egg quality (Saleh et al. 2020; Dijkslag et al. 2021; Song et al. 2022). The rich content of calcium (0.36%) and phosphorus (0.88%) in FVS may be thus the reason for the improvement of egg quality in addition to the content of lysine in FVS accounted for 0.81%, which is in accordance with Scappaticcio et al. that showed that an increase in lysine improved linearly egg production, egg weight, egg mass (Scappaticcio et al. 2022). Our study found that FVS increased egg mass, which may be due to the rich lysine content in FVS.

The diet can affect the reproductive performance, ovarian follicular development and hormone level in laying hens (Saleh, Ahmed, et al. 2019; Saleh, Kirrella, et al. 2019). At 17 weeks of age, laying hens are in the early stage of laying, and their ovaries are in the stage of sexual maturity, and their developmental status has great influence on subsequent laying. During the sexual maturation, primordial follicles are activated followed by a slow growth process, then develop into fast development follicles within a few weeks (Mfoundou et al. 2021). AF showed a downward trend in 2% and 4% FVS groups, while PF and FDF increased significantly. It is proved that FVS can reduce atresia follicles and increase the development of dominant follicles. Concerning the atretic follicles generally with small diameter and the follicular atresia known as a hormone regulated apoptosis process, the elevated atresia level of primary follicles is characterised by the shrinkage of oocyte nucleus, the decrease of granulosa cell layer, the presence of lipids in cytoplasm, and the further evolution of granulosa cell theca cells into fibrous bodies (Evans 2003; Zhou et al. 2019). It was found that the granulosa cells apoptosis leads to follicle atresia in chicken ovary (Han et al. 2022). In this study, the FVS dietary supplementation significantly regulated genes and proteins expression involved in apoptosis and ovarian proliferation associated with the well follicles’ development. During the process of follicular development in poultry, follicular atresia is very common, but the effect of the number of atretic follicles on egg production is more intuitive. The less atresia will occur, the more follicles could enter the grade follicles for development, and the egg production could increase (Yao et al. 2020). This may indicate that FVS could protect follicles by inhibiting ovarian cell apoptosis to reduce atresia.

Previous studies have revealed that the risk of many metabolic disorders such as fatty liver and abdominal fat deposition in chickens can be involved by high levels of serum TG and LDL (Liu Y et al. 2020). These metabolic disorders could be improved with dietary supplementation of FVS which reduced lipid metabolism in broilers (Mahfuz et al. 2019). This study found that FVS had the same effect on laying hens. F. velutipes polysaccharides have also revealed to be able to reduce obesity and hyperlipidaemia in mice through lipid metabolism (Zhao et al. 2021). In fact, the component analysis showed that FVS is rich in dietary fibres, up to 22.05% (Mahfuz et al. 2019), which is an edible active polysaccharide that can bind to cholesterol or cholic acid by adsorption, reducing the lipid content in the blood (Gill et al. 2021; Liu H et al. 2021). Thus, FVS dietary supplementation for 27 weeks showing the decrease of serum LDL-C and TG levels as well as the abdominal fat deposition and ratio may indicate that FVS, as a fibre source diet, has good ability to reduce blood lipid and could be used as a regulator of lipid disorders. Furthermore, there could have a dose-response relationship between fibre intake and lipid-lowering effect following fibre types due to the different absorption capacity of laying hens at different stages of growth and development (Nie and Luo 2021), the rapid development of ovaries and fallopian tubes with the nutrients absorption required during this period, but also the high concentration of crude fibre in the feed which affect the nutrition in general and particularly their absorption (Mohiti-Asli et al. 2012; Genoveva Gonzalez-Moran 2016). Therefore, an excessive fibre addition is not conducive to the growth and development of laying hens.

It has been found that the transcriptome sequencing of hens at sexual maturity and egg laying peak has shown that different genes are involved in the steroid hormone synthesis and lipid metabolism signalling pathways respectively (Li et al. 2020). Specifically, the cholesterol transport to the site of steroid biosynthesis during the follicular steroidogenesis usually requires a steroidogenesis acute regulatory protein (StAR), while CYP11A1 is a key gene that mediates the conversion of cholesterol to pregnenolone and further to progesterone through 3β-HSD (Nakao et al. 2007; Rytelewska et al. 2020). Otherwise, during the sexual maturity and egg laying peak, others factors could be involved like CYP17A1, which has revealed to inhibit androgen secretion In vivo, and CYP19 which the increase of one of its family member activities (CYP19A1) could exacerbate the sensitivity of follicles to hormones and stimulate the oestrogen secretion inducing a rapid follicular growth, while its deficiency could be responsible for the infertility (Umehara et al. 2017; Secchi et al. 2021). It was thus found that dietary FVS could reduce the cholesterol metabolism as well as improve the genes expression involved in steroid hormone synthesis signalling pathway. However, FVS promoted the secretion of serum PROG and E2 during sexual maturity and egg laying peak affecting the follicular development.

The synthesis of steroid hormones is closely related to the secretion of gonadotropin (Bosch et al. 2021). Gonadotropins (FSH and LH) secreted by pituitary gland are known to play an important role in follicular development and can promote the growth of oocytes and the proliferation of follicular cells (Oduwole et al. 2021). Concerning oestradiol synthesis, FSHR are produced on the membrane surface of granulosa cells (GC), bind to FSH secreted by pituitary gland, activate adenylate cyclase, and then increase the intracellular cAMP level. The activation of this FSH signalling pathway usually include the increased expression of related enzymes like cytochrome P450 family members (CYP11A1 and CYP19A1) and promote the oestradiol synthesis (Lee et al. 2021; Ashry et al. 2022). FSH and LH play a role through their receptors FSHR and LHR on follicular granulosa cells which are almost absent on all follicles of young poultry ovaries. While the poultry age increases, those receptors levels also increase stimulating the hypothalamus to secrete gonadotropin releasing hormone and promote the secretion of the related gonadotropins (FSH and LH) in the anterior pituitary, and then the secretion of progesterone and oestrogen in the ovary, which play a vital role in the follicular development and ovulation. It is reported that FSH can promote the deposition of yolk substances in poultry, increase the follicles weight and volume, and reduce the number of atresia small follicles (Ma et al. 2020). The yolk deposition usually requires the large contribution of lipid, whose the biosynthesis could be up-regulated by the mRNA expression of FSHR in chicken abdominal fat (Cui et al. 2012). FSH could thus affect the egg production wherever, at the peak of egg laying, one follicle is selected every day following its grade to enter the rapid growth stage inducing ovulation (Walzem and Chen 2014). Therefore, it can be inferred that FSH may be closely related to lipids because the FSH signal cutting could inhibit hepatic cholesterol biosynthesis and reduce serum cholesterol levels (Guo et al. 2019). In this study, FVS up-regulated the levels of these gonadotropins as well as their receptors during the two-specific period, while it improved the ovarian weight and the follicles number and diameter at the 27 weeks but there was no correlation between FSH and lipid levels.

Conclusions

In summary, it was found that FVS could regulate the steroid hormone synthesis signalling pathway through the genes and proteins expression related to FSHR and LHR, stimulate the production of sex hormones to promote the development of poultry ovary and improve the egg white weight and eggshell strength. FVS could also inhibit the ovarian apoptosis and reduce the follicular atresia protecting the ovarian proliferation. The beneficial effects were better with 4% FVS suggesting that FVS could be added to the poultry feed as a dietary supplement to regulate the ovarian development of laying hens.

Ethics statement

All animal handling procedures was strictly complied with the Use and Care of Laboratory Animals and were approved by the Animal Care Review Committee, Jilin Agricultural University.

Authors’ contributions

Chang Sun: drafting the manuscript, conception and design. Haoyuan Wu: acquisition of data. Huanwei Xiao: analysis and interpretation of data. Ivan Stève Nguepi Tsopmejio: revising the manuscript. Zhouyu Jin: Given final approval of the version to be published. Hui Song: Agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgements

Thanks to Huiling hens production base for providing laying hens feeding place.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Additional information

Funding

This work was supported by research and development project of safe and efficient new feed products of Jilin Provincial Department of Science and Technology [grant number：20180201019NY].