
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00820-4
10.1016/j.psj.2024.104241
104241
METABOLISM AND NUTRITION
Fermented blueberry pomace supplementation improves egg quality, liver synthesis, and ovary antioxidant capacity of laying hens
Qin Binghua *‡
Li Zhihua *
Azad Md. Abul Kalam ⁎‡
Chen Ting *‡
Cui Yadong †
Lan Wei †
Wang Haoran *‡
Kong Xiangfeng nnkxf@isa.ac.cn
*†‡1
⁎ Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial Key Laboratory of Animal Nutrition Physiology and Metabolic Processes, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
† School of Biology and Food Engineering, Fuyang Normal University, Fuyang 236037, China
‡ College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
1 Corresponding author: nnkxf@isa.ac.cn
22 8 2024
12 2024
22 8 2024
103 12 10424113 5 2024
18 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The present study aimed to investigate the effects of dietary fermented blueberry pomace (FBP) supplementation on production performance, egg quality and nutritional value, plasma biochemical parameters, follicle number, reproductive hormones, lipid metabolism, and antioxidant capacity of laying hens during the late laying period. A total of 320 (345-d-old) Yukou Jingfen No. 8 laying hens were randomly divided into 4 groups, with eight replicates per group and 10 hens per replicate. The birds were fed a basal diet (control group) and a basal diet supplemented with 0.25, 0.5, and 1.0% FBP. The trial lasted 56 d. The results showed that FBP (0.25−1.0%) supplementation increased the egg albumen height and Haugh unit compared with the control group on d 14, while 0.5 to 1.0% FBP increased the eggshell thickness compared with the 0.25% FBP group on d 28 of the trial (P < 0.05). The methionine content in egg white was higher (P < 0.05) in the 1.0% FBP group compared with the 0.25% FBP group. The CAT activity in the ovary was increased (P < 0.05) in the FBP groups compared with the control group, while plasma GSH-PX activity was higher (P < 0.05) in the 1.0% FBP group compared with the 0.25% FBP and 0.5% FBP groups. Dietary FBP supplementation up-regulated (P < 0.05) gene expressions related to lipid metabolism in the liver (ACC, FAS, SCD1, and SREBP1) and yolk precursor synthesis (ESR2 and VTG II). Moreover, CYP11A1 expression in the ovary was up-regulated (P < 0.05) in the FBP groups compared with the control group, as well as in the 0.25% FBP group compared with the 1.0% FBP group. In summary, dietary FBP supplementation improved egg quality and nutritional value, ovarian antioxidant capacity, and yolk precursor synthesis, while 1.0% FBP had better effects than 0.25 and 0.5% doses.

Key words

egg quality
fermented blueberry pomace
laying hen
lipid metabolism
nutritional value
==== Body
pmcINTRODUCTION

Laying hens experience a decline in both production performance and egg quality, which is attributed to ovarian aging and an imbalance in the oxidative system during the late laying period (Liu et al., 2018). The late laying period constitutes over half of the egg-laying cycle for laying hens. Inadequate implementation of nutritional strategies during the late laying period can lead to substantial economic losses in the layer industry. Annual reports indicate that the losses exceed 500 million Chinese yuan due to deteriorating egg quality in the layer industry. Thus, it is imperative to enhance both production performance and egg quality of laying hens during the late laying period.

China has abundant resources of various fruits, yet a significant portion of the by-products from fruit processing, such as peels and pulp, collectively termed pomace, are often discarded due to their high moisture content and anti-nutritional factors, leading to substantial resource wastage and environmental pollution (Fierascu et al., 2020). Extensive research evidence reported that microbial fermentation could mitigate these issues by reducing moisture and degrading anti-nutrient factors of fruit pomace, thereby enhancing its nutritional value (Beaumont, 2002). Previous studies demonstrated the potential utilization of agricultural by-products, such as fruit pomace, as feed additives to enhance the production performance and egg quality of laying hens (Reis et al., 2019; Tufarelli et al., 2022). Blueberries, also known as Vaccinium spp., are deciduous shrubs belonging to the Ericaceae family. Blueberry pomace is rich in functional components such as anthocyanins, phenolic compounds, and dietary fiber (Avendano and Raman, 2021). In addition, blueberry pomace has been found to improve the health status and product quality of poultry as a feed additive. For example, dietary wild blueberry (Vaccinium angustifolium) pomace improved the overall health of broiler chickens by enhancing gut microbiota and blood metabolites (Islam et al., 2019), while dietary 0.5% wild blueberry pomace improved muscle color without compromising meat quality (Xu et al., 2021). Fermentation of blueberry pomace could increase in bioactive substances, including polyphenols, β-carotenoids, and flavonoids (Tian et al., 2023). Previous studies indicated that fermented blueberry pomace (FBP) exhibits potential health benefits, including enhanced intestinal barrier function against high-fat diets, thereby potentially improving human health (Cheng et al., 2020).

Nevertheless, research regarding the impacts of FBP on poultry nutrition remains constrained. Given the potential benefits of FBP, we hypothesized that the inclusion of FBP in laying hens’ diet may enhance production performance and egg quality in laying hens during the late laying period. Thus, to test this hypothesis, the present study aimed to evaluate the impacts of dietary FBP supplementation on production performance, egg quality and nutritional value, plasma biochemical parameters, follicle number, reproductive hormones, lipid metabolism, and antioxidant capacity of laying hens during the late laying period. The findings of this research could potentially contribute to the development of novel feed additives and provide further research direction regarding resource utilization and environmental sustainability.

MATERIALS AND METHODS

Animal Ethics Statement

The animal experiments were reviewed and approved by the protocol management and review committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (No. ISA-2022-056).

Preparation of FBP

The blueberry pomace used in this study was obtained from Anhui Xiuqin Agricultural Technology Co., Ltd. (Fuyang, China). Initially, the blueberry pomace was fermented for 64 hours using a combined starter at 75°C and was subsequently dissolved in oxygen (3–10%) with a relative humidity of 45 to 55%. The combined starter comprised microbial agents, active enzymes, culture medium 1 (artificially cultivated ginseng leaves), culture medium 2 (soybean powder), and sugar, with a mass ratio of 1:1:1:1:1. The microbial agents were provided by Guangdong Microbial Culture Collection Center (Guangzhou, China), and contained Bacillus subtilis (GDMCC 1.182), Bacillus licheniformis (GDMCC 1.648), Lactobacillus plantarum (GDMCC 2.215), and marine red yeast. Subsequently, the combined microbial starter was mixed with the blueberry pomace at a mass ratio of 1:100. The nutrient composition, amino acid profile, and fatty acid content of the FBP are presented in Tables 1 to 2.Table 1 The conventional nutrient content of fermented blueberry pomace (%, air-dried).

Table 1Component	Content	
Dry matter	92.00	
Crude protein	9.52	
Crude fat	5.30	
Crude fiber	21.30	
Crude ash	11.20	
Phosphorus	3.16	
Magnesium	0.29	
Total energy, MJ/kg	16.45	

Table 2 The composition of amino acids and fatty acids in fermented blueberry pomace (%, air-dried).

Table 2Component	Content	
Amino acids		
Alanine	0.46	
Arginine	0.37	
Aspartic acid	0.82	
Cystine	0.14	
Glutamic acid	1.27	
Glycine	0.44	
Histidine	0.16	
Isoleucine	0.34	
Leucine	0.62	
Lysine	0.32	
Methionine	0.12	
Phenylalanine	0.43	
Proline	0.38	
Serine	0.39	
Threonine	0.34	
Tryptophan	0.05	
Tyrosine	0.18	
Valine	0.46	
Total amino acids	7.29	
Fatty acids		
Cis-9-octadecanoic acid	29.59	
Cis-9,12-octadecadienoic acid	42.63	
Cis-9,12,15-octadecatrienoic acid	8.71	
Cis-11-eicosenoic acid	0.26	
Docosanoic acid	0.37	
Eicosanoic acid	1.10	
Hexadecanoic acid	13.89	
Lignoceric acid	0.37	
Octadecanoic acid	3.07	

Birds and Feeding Management

A total of 320 Yukou Jingfen No. 8 laying hens (345-d-old), a cross-breed of Hy-Line Variety Brown and Chicken Wings and Legs with Brown Sauce, were randomly assigned into 4 groups, with eight replicates for each group and ten hens per replicate. The birds in the control group received a basal diet, while the birds in the treatment groups were fed a basal diet supplemented with 0.25, 0.5, or 1.0% FBP, respectively. Prior to the formal feeding trial, all birds had a 7-d adaptation period with the basal diet. The formal experiment lasted 56 d. The experimental birds were housed in wire cages with 5 birds per cage, ensuring free access to feed and water. The experimental birds were reared in a well-ventilated room with a controlled temperature (18−23°C), humidity (55−65%), and light/dark (16/8 h) cycle. The composition and nutrient levels of the basal diet are presented in Table S1.

Sample Collection

On d 14, 28, 42, and 56 of the experiment, 3 eggs per replicate (24 eggs per group) were collected for egg quality analysis. Additionally, on d 56, 6 eggs per group were collected to determine crude protein and amino acid content in egg white, as well as crude fat and fatty acid content in egg yolk. Upon conclusion of the trial (on d 56), one hen per replicate (total 8 hens per group), close to the average BW of the group, was selected for sampling. Blood samples (5 mL) were drawn from the wing veins of the hens into heparin sodium anticoagulant tubes to obtain plasma by centrifuging at 3,000 × g for 10 min at 4°C. The plasma samples were stored at −20°C for subsequent biochemical parameter and reproductive hormone analyses. The ovary and liver samples were harvested and stored at −20°C for further analyses.

Production Performance Analysis

Egg production and egg weight were recorded daily to determine the average laying rate (ALR) and average egg weight (AEW). The feed offered and leftovers were recorded weekly for each replicate to calculate the ADFI and feed-egg ratio (FER).

Egg Quality Analysis

Egg weight, albumen height, yolk color, and Haugh unit were assessed using a multifunction egg quality analyzer (Israel Orka Food Technology Ltd., Ramat Hasharon, Israel). Egg Force Reader 01 (Israel Orka Food Technology Ltd., Ramat Hasharon, Israel) was used to measure eggshell strength. The maximum horizontal and vertical diameters of eggs were measured using an electronic digital Vernier caliper (Shanghai Shenhan Measures Co., Ltd., Shanghai, China) to determine the egg shape index (maximum vertical diameter/maximum horizontal diameter). The eggshell thickness was determined by averaging measurements taken at the blunt, middle, and sharp ends. The weight of egg white, yolk, and eggshell were recorded to calculate the egg white, yolk, and eggshell percentages.

Egg Nutritional Value Analysis

The content of CP in egg white was measured using the KT200 Kjeldahl analyzer (Foss Analytical Co., Ltd., Suzhou, China) in accordance with the guidelines outlined by the National Standard of the People's Republic of China (GB 5009.5-2016). Amino acid contents in egg white were determined using the L-8800 Amino Acid Analyzer (Hitachi Limited, Tokyo, Japan) following the protocols described by the National Standard of the People's Republic of China (GB 5009.124-2016). The crude fat content in yolk was determined following the guidelines provided by the National Standard of the People's Republic of China (GB 5009.6-2016). Fatty acid contents in egg yolk were analyzed using the Nexis GC-2030 gas chromatography (Shimadzu Corporation, Kyoto, Japan) following the National Standard of the People's Republic of China (GB 5009.168-2016) standard protocols.

Plasma Biochemical Parameters Analysis

Plasma biochemical parameters, including albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), calcium (Ca), creatinine (CREA), glucose (GLU), low-density lipoprotein-cholesterol (LDL-C), non-esterified fatty acids (NEFA), total bile acid (TBA), total cholesterol (TC), triglyceride (TG), total protein (TP), and uric acid (UA) were determined using the available commercial kits and Beckman CX4 automatic biochemical analyzer (Cobas c311; Roche Diagnostics GmbH, Mannheim, Germany).

Antioxidant Capacity Analysis

The plasma and ovarian antioxidant indicators, including glutathione (GSH), GSH peroxidase (GSH-PX), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and total antioxidant capability (T-AOC) were assessed using the commercial assay kits (Nanjing Jiancheng Biotechnology Institute, Nanjing, China) on a Microplate Reader (Infinite M200 PRO; Tecan, Männedorf, Switzerland) following the standard procedures described by the manufacture.

Reproductive Hormones Analysis

The levels of plasma and ovary reproductive hormones, including estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH), were measured using the available ELISA kits (Hunan Ruicheng Biotechnology Co., LTD., Changsha, China) on a Microplate Reader (Infinite M200 PRO; Tecan, Männedorf, Switzerland).

Gene Expressions Analysis

The total RNA of the liver and ovary was extracted with the AG RNAex Pro reagent (Accurate Biology, Changsha, China) following the manufacturer's instructions. The purity and concentration of the extracted RNA were determined using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Then, the extracted total RNA was reverse-transcribed into cDNA using an Evo M-MLV RT kit (Accurate Biology, Changsha, China). The real-time quantitative PCR (RT-qPCR) analysis was performed on a LightCycler 480II Real-Time PCR System (Roche, Basel, Switzerland) with SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Changsha, China). The RT-qPCR was performed in a total reaction volume of 10 µL, including 1 µL cDNA, 0.4 µL forward primer, 0.4 µL reverse primer, 5 µL SYBR Green Premix (Accurate Biology, Changsha, China), and 3.2 µL ddH2O. The RT-qPCR cycling conditions were as follows: pre-denaturation at 95 °C for 5 min, then denaturation at 95 °C for 10 s and annealing at 53°C for 10 s, finally extending at 72°C for 20 s, which includes a total of 45 cycles. The primer sequences are presented in Table S2. The relative mRNA expression for each gene was calculated using the 2−ΔΔCt method (Schmittgen and Livak, 2008).

Statistical Analysis

Statistical analyses were conducted using the IBM SPSS 26.0 software (IBM Inc., Chicago, IL). One-way ANOVA and Tukey's post-hoc test were performed to analyze significant differences among the groups. The linear and best-fit quadratic models for dietary FBP supplementation dose were determined by regressions of estimation curves. Data are presented as means and SEM. Data are considered statistically significant if P < 0.05, and a trend if 0.05 ≤ P < 0.10.

RESULTS

Effects of FBP on Production Performance of Laying Hens

The effects of dietary FBP on the production performance of laying hens are presented in Table 3. Dietary 0.5% FBP supplementation exhibited an increasing trend (P = 0.088) on ADFI of laying hens during d 43−56 of the trial compared with the control group. Dietary FBP supplementation had significant quadratic effects on ADFI during d 28−42 (P = 0.035, model: Y = 100.680 + 13.786X − 12.755X2, estimated optimal dose required was 0.54% FBP) and 43−56 (P = 0.033, model: Y = 97.297 + 9.634X − 8.493X2, estimated optimal dose required was 0.57% FBP) of the trial. However, there were no significant differences (P > 0.05) in the ALR, AEW, ADFI, and FER between the control and FBP-supplemented groups throughout the trial.Table 3 Effects of dietary fermented blueberry pomace (FBP) on production performance of laying hens.

Table 3Item	Treatments	SEM	P-values	
Control	0.25% FBP	0.5% FBP	1.0% FBP	FBP	Linear	Quadratic	
D 1−14 of the trial			
 ALR, %	71.79	69.33	71.58	69.20	0.986	0.699	0.547	0.985	
 AEW, g	51.10	51.37	51.45	51.39	0.178	0.912	0.572	0.658	
 ADFI, g/hen/d	95.31	93.54	94.66	95.33	0.616	0.728	0.841	0.344	
 FER, g/g	2.60	2.65	2.58	2.70	0.033	0.614	0.492	0.573	
D 15−28 of the trial			
 ALR, %	69.91	70.57	68.34	72.05	1.289	0.802	0.727	0.571	
 AEW, g	52.67	51.70	51.32	51.22	0.267	0.206	0.051	0.405	
 ADFI, g/hen/d	91.74	89.49	90.17	88.19	0.598	0.208	0.066	0.904	
 FER, g/g	2.51	2.48	2.60	2.40	0.041	0.397	0.598	0.285	
D 29−42 of the trial			
 ALR, %	73.21	70.82	70.64	72.32	1.217	0.870	0.802	0.428	
 AEW, g	51.54	51.82	51.68	51.60	0.193	0.967	0.978	0.666	
 ADFI, g/hen/d	100.75	103.14	104.52	101.69	0.618	0.142	0.434	0.035	
 FER, g/g	2.69	2.83	2.89	2.74	0.043	0.339	0.589	0.091	
D 43−56 of the trial			
 ALR, %	71.43	70.33	72.72	72.23	1.281	0.927	0.691	0.909	
 AEW, g	51.01	51.93	51.83	52.04	0.225	0.364	0.147	0.429	
 ADFI, g/hen/d	97.37	98.99	100.13	98.41	0.396	0.088	0.208	0.033	
 FER, g/g	2.71	2.72	2.70	2.63	0.047	0.906	0.540	0.684	
D 1−56 of the trial			
 ALR, %	71.58	70.25	70.79	71.45	0.926	0.958	0.987	0.609	
 AEW, g	51.57	51.70	51.56	51.57	0.156	0.989	0.926	0.845	
 ADFI, g/hen/d	96.29	96.27	97.32	95.90	0.384	0.615	0.974	0.378	
 FER, g/g	2.62	2.66	2.68	2.61	0.029	0.797	0.990	0.339	
Data are expressed as means with their SEM (n = 8). ADFI, average daily feed intake; ALR, average laying rate; AEW, average egg weight; FER, feed-egg ratio.

Effects of FBP on Egg Quality of Laying Hens

The effects of dietary FBP on the egg quality of laying hens are shown in Table 4. Dietary FBP (0.25−1.0%) supplementation increased (P < 0.05) albumen height and Haugh unit compared to the control group on d 14 of the trial. The eggshell thickness was higher (P < 0.05) in the control, 0.5% FBP, and 1.0% FBP groups compared with the 0.25% FBP group on d 28 of the trial. Dietary FBP supplementation had significant linear and quadratic effects on albumen height (P < 0.001, linear model: Y = 3.797 + 1.131X; P = 0.028, quadratic model: Y = 3.516 + 3.460X − 2.248X2, estimated optimal dose required was 0.77% FBP) and Haugh unit (P < 0.001, linear model: Y = 59.110 + 11.306X; P = 0.049, quadratic model: Y = 56.199 + 35.425X − 23.287X2, estimated optimal dose required was 0.76% FBP) on d 14 of the trial. Additionally, dietary FBP supplementation had significant effects on egg yolk color (P = 0.023, linear model: Y = 13.954 − 0.550X) and eggshell thickness (P = 0.008, quadratic model: Y = 0.332 − 0.031X − 0.037X2, estimated optimal dose required was 0.42% FBP) on d 28 of the trial, and egg shape index on d 42 (P = 0.031, quadratic model: Y = 1.357 − 0.140X − 0.126X2, estimated optimal dose required was 0.56% FBP) of the trial. However, there were no significant differences (P > 0.05) in egg shape index, egg white percent, egg yolk color, eggshell percent, and eggshell strength between the control and FBP-supplemented groups throughout the trial.Table 4 Effects of dietary fermented blueberry pomace (FBP) on egg quality of laying hens.

Table 4Item	Treatments	SEM	P-values	
Control	0.25% FBP	0.5% FBP	1.0% FBP	FBP	Linear	Quadratic	
D 14 of the trial			
 Albumen height, mm	3.38b	4.61a	4.41a	4.77a	0.132	<0.001	<0.001	0.028	
 Egg shape index	1.33	1.34	1.32	1.34	0.006	0.700	0.965	0.684	
 Egg white percent, %	53.88	54.24	54.72	54.47	0.268	0.743	0.372	0.584	
 Egg yolk color	13.67	13.88	13.67	13.92	0.112	0.801	0.604	0.929	
 Egg yolk percent, %	32.72	32.60	32.24	32.29	0.229	0.865	0.444	0.863	
 Eggshell percent, %	13.34	13.15	13.14	13.24	0.095	0.879	0.724	0.468	
 Eggshell strength, N	38.72	39.67	40.19	40.63	0.967	0.919	0.495	0.901	
 Eggshell thickness, mm	0.34	0.35	0.34	0.35	0.003	0.230	0.220	0.504	
 Haugh Unit	55.04b	66.70a	65.77a	68.72a	1.387	<0.001	<0.001	0.049	
D 28 of the trial			
 Albumen height, mm	5.17	4.02	4.78	4.79	0.173	0.111	0.805	0.085	
 Egg shape index	1.32	1.35	1.31	1.31	0.006	0.114	0.263	0.197	
 Egg white percent, %	56.29	55.47	55.87	55.50	0.332	0.814	0.527	0.746	
 Egg yolk color	13.96	13.83	13.65	13.42	0.088	0.145	0.023	0.760	
 Egg yolk percent, %	30.48	31.43	31.36	31.47	0.322	0.674	0.332	0.530	
 Eggshell percent, %	13.24	13.10	12.77	13.03	0.108	0.510	0.341	0.381	
 Eggshell strength, N	40.74	40.12	37.10	42.27	0.831	0.162	0.830	0.081	
 Eggshell thickness, mm	0.34a	0.32b	0.33a	0.34a	0.002	0.004	0.251	0.008	
 Haugh Unit	68.70	60.11	68.45	67.67	1.459	0.109	0.673	0.168	
D 42 of the trial			
 Albumen height, mm	4.21	3.96	4.43	4.32	0.106	0.457	0.421	0.736	
 Egg shape index	1.36	1.33	1.32	1.34	0.006	0.122	0.296	0.031	
 Egg white percent, %	55.54	58.45	56.17	57.44	0.533	0.219	0.466	0.439	
 Egg yolk color	13.75	13.79	14.33	13.58	0.120	0.133	0.968	0.094	
 Egg yolk percent, %	31.34	28.33	31.02	29.35	0.575	0.208	0.518	0.552	
 Eggshell percent, %	13.12	13.23	12.81	13.20	0.100	0.453	0.860	0.490	
 Eggshell strength, N	39.81	43.36	37.64	40.67	0.818	0.091	0.651	0.866	
 Eggshell thickness, mm	0.34	0.34	0.33	0.34	0.003	0.506	0.571	0.427	
 Haugh Unit	63.86	60.53	65.28	64.44	1.112	0.470	0.522	0.582	
D 56 of the trial			
 Albumen height, mm	5.18	4.83	4.80	5.34	0.131	0.398	0.706	0.101	
 Egg shape index	1.32	1.35	1.32	1.32	0.007	0.230	0.497	0.266	
 Egg white percent, %	55.70	56.13	56.02	56.34	0.275	0.884	0.487	0.924	
 Egg yolk color	14.04	14.00	13.96	14.25	0.088	0.670	0.473	0.360	
 Egg yolk percent, %	31.25	30.66	30.75	30.58	0.239	0.775	0.394	0.672	
 Eggshell percent, %	13.05	13.21	13.24	13.08	0.121	0.940	0.923	0.540	
 Eggshell strength, N	40.14	38.50	39.43	41.81	0.811	0.547	0.427	0.232	
 Eggshell thickness, mm	0.33	0.34	0.33	0.34	0.003	0.760	0.570	0.720	
 Haugh Unit	71.25	67.42	68.43	73.08	1.373	0.462	0.604	0.136	
Data are expressed as means with their SEM (n = 8).

a,b Mean values with different superscript letters represent a significant difference (P < 0.05).

Effects of FBP on Egg Nutritional Value of Laying Hens

Egg nutritional values of laying hens are presented in Tables 5 to 7. The methione (Met) content in egg white was higher (P < 0.05) in the 1.0% FBP group compared with the 0.25% FBP group. Dietary 0.5% FBP supplementation decreased the proline (Pro; P < 0.05) and valine (Val; P = 0.060) contents in the egg white compared with the control group (Table 5). Regression analysis showed that dietary FBP supplementation had significant linear and quadratic effects on Met (P = 0.042, linear model: Y = 0.471 + 0.124X; P = 0.044, quadratic model: Y = 0.488 − 0.287X + 0.822X2), while had quadratic effects on alanine (Ala; P = 0.044, model: Y = 0.612 − 0.346X + 0.635X2), glutamate (Glu; P = 0.044, model: Y = 1.355 − 0.743X + 1.354X2), glycine (Gly; P = 0.040, model: Y = 0.357 − 0.215X + 0.401X2), Pro (P = 0.031, model: Y = 0.335 − 0.192X + 0.327X2), serine (Ser; P = 0.026, model: Y = 0.693 − 0.395X + 0.781X2), tyrosine (Tyr; P = 0.040, model: Y = 0.385 − 0.168X + 0.347X2), and nonessential amino acid (NEAA; P = 0.037, model: Y = 4.787 − 2.690X + 5.006X2).Table 5 Effects of dietary fermented blueberry pomace (FBP) on crude protein and amino acid contents in egg white of laying hens.

Table 5Item, g/100 g	Treatments	SEM	P-values	
Control	0.25% FBP	0.5% FBP	1.0% FBP	FBP	Linear	Quadratic	
Crude protein	9.93	9.73	9.39	9.62	0.112	0.412	0.219	0.360	
Ala	0.61	0.59	0.57	0.60	0.007	0.128	0.239	0.044	
Arg	0.57	0.55	0.53	0.55	0.007	0.418	0.354	0.181	
Asp	1.06	1.00	0.98	1.02	0.013	0.134	0.177	0.054	
Glu	1.37	1.29	1.26	1.32	0.016	0.134	0.226	0.044	
Gly	0.36	0.34	0.33	0.35	0.004	0.133	0.244	0.040	
His	0.23	0.22	0.22	0.23	0.003	0.234	0.244	0.103	
Ile	0.53	0.51	0.48	0.51	0.007	0.102	0.138	0.079	
Leu	0.86	0.83	0.80	0.84	0.011	0.164	0.366	0.053	
Lys	0.65	0.62	0.61	0.63	0.009	0.489	0.467	0.187	
Met	0.49ab	0.46b	0.50ab	0.55a	0.011	0.035	0.026	0.049	
Phe	0.62	0.60	0.57	0.61	0.008	0.137	0.345	0.063	
Pro	0.34a	0.32ab	0.31b	0.32ab	0.004	0.044	0.055	0.031	
Ser	0.70	0.66	0.66	0.69	0.009	0.144	0.621	0.026	
Thr	0.46	0.44	0.44	0.45	0.006	0.330	0.447	0.096	
Tyr	0.39	0.38	0.36	0.39	0.005	0.147	0.932	0.040	
Val	0.69	0.66	0.62	0.66	0.009	0.060	0.062	0.081	
EAA	5.11	4.90	4.78	5.04	0.063	0.254	0.533	0.070	
NEAA	4.83	4.57	4.47	4.68	0.057	0.130	0.274	0.037	
FAA	3.97	3.76	3.67	3.83	0.047	0.155	0.226	0.055	
BCAA	2.08	2.00	1.91	2.01	0.026	0.107	0.164	0.065	
TAA	9.94	9.47	9.24	9.72	0.119	0.187	0.396	0.051	
Data are expressed as means with their SEM (n = 6).

a,b Values with different superscript letters represent a significant difference (P < 0.05). Ala, alanine; Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Tyr, tyrosine; Val, valine; EAA, essential amino acid; NEAA, nonessential amino acid; FAA, flavor amino acids; BCAA, branched chain amino acid; TAA, total amino acids. EAA include Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, and Pro; NEAA include Ala, Asp, Cys, Glu, Gly, Ser, and Tyr; FAA include Asp, Glu, Gly, Ala, and Arg; BCAA include Ile, Leu, and Val.

The effects of dietary FBP on fatty acid contents are presented in Table 6. Dietary FBP supplementation displayed an increasing trend in the C18:3n3 (P = 0.087) content while had a decreasing trend in the C18:1n9c (P = 0.065) content in the yolk of the 0.25% FBP group (Table 6). Moreover, saturated fatty acids (SFA) content (P = 0.091) in the 0.25 to 1.0% FBP groups and monounsaturated fatty acids (MUFA) content (P = 0.087) in the 0.25% FBP group displayed a decreasing trend compared with the control group (Table 7). However, there were no significant differences (P > 0.05) in CP and crude fat contents among different groups (Table 5, Table 6).Table 6 Effects of dietary fermented blueberry pomace (FBP) on crude fat and fatty acid contents in egg yolk of laying hens.

Table 6Item	Treatments	SEM	P-values	
Control	0.25% FBP	0.5% FBP	1.0% FBP	FBP	Linear	Quadratic	
Crude fat, g/100g	33.89	35.25	34.36	34.64	0.313	0.511	0.634	0.410	
C14:0, %	0.40	0.41	0.38	0.37	0.009	0.329	0.129	0.694	
C16:0, %	25.78	25.39	24.85	25.22	0.165	0.254	0.135	0.248	
C16:1n7c, %	3.12	3.03	2.75	2.80	0.074	0.251	0.073	0.630	
C17:0, %	0.14	0.16	0.15	0.15	0.004	0.255	0.278	0.305	
C18:0, %	8.26	7.74	7.93	7.93	0.092	0.268	0.328	0.172	
C18:1n9c, %	42.90	41.00	44.39	42.51	0.460	0.065	0.556	0.994	
C18:2n6c, %	16.26	18.96	16.49	17.78	0.464	0.133	0.594	0.425	
C18:3n6, %	0.12	0.14	0.11	0.12	0.004	0.321	0.422	0.812	
C20:1n9c, %	0.22	0.22	0.23	0.21	0.003	0.390	0.558	0.217	
C18:3n3, %	0.65	0.81	0.66	0.72	0.025	0.087	0.785	0.290	
C20:2n6, %	0.13	0.16	0.13	0.14	0.005	0.109	0.799	0.538	
C20:3n6, %	0.14	0.14	0.12	0.13	0.003	0.117	0.104	0.473	
C20:4n6, %	1.29	1.27	1.23	1.27	0.014	0.530	0.372	0.301	
C22:6n3, %	0.60	0.58	0.61	0.65	0.011	0.121	0.064	0.140	
Data are expressed as means with their SEM (n = 6).

Table 7 Effects of dietary fermented blueberry pomace (FBP) on lipid health index in egg yolk of laying hens.

Table 7Item	Treatments	SEM	P-values	
Control	0.25% FBP	0.5% FBP	1.0% FBP	FBP	Linear	Quadratic	
SFA, %	34.58	33.70	33.30	33.67	0.188	0.091	0.055	0.087	
MUFA, %	46.23	44.25	47.36	45.52	0.450	0.087	0.793	0.933	
PUFA, %	19.19	22.05	19.35	20.81	0.505	0.145	0.616	0.471	
PUFA/SFA	0.56	0.66	0.58	0.62	0.017	0.161	0.408	0.336	
n-3 PUFA, %	1.25	1.39	1.27	1.37	0.031	0.311	0.378	0.773	
n-6 PUFA, %	17.94	20.66	18.08	19.44	0.480	0.144	0.639	0.460	
n6/n3 PUFA	14.32	14.87	14.22	14.22	0.210	0.679	0.626	0.534	
AI	0.42	0.41	0.40	0.40	0.004	0.181	0.084	0.222	
TI	0.96	0.91	0.91	0.91	0.009	0.106	0.057	0.109	
H/H	2.36	2.43	2.52	2.47	0.025	0.142	0.062	0.214	
HPI	2.39	2.45	2.54	2.49	0.024	0.160	0.067	0.240	
DFA, %	73.68	74.04	74.65	74.26	0.168	0.235	0.125	0.265	
Data are expressed as means with their SEM (n = 6). SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acid; AI, atherosclerosis index; TI, thrombosis index; H/H, hypocholesterolemia to hypercholesterolemia ratio; HPI, health promotion index; DFA, desirable fatty acids. AI = (4 × C14:0 + C16:0)/UFA, TI = (C14:0 + C16:0 + C18:0)/(0.5 × MUFA + 0.5 × n-6 PUFA + 3 × n-3 PUFA + n-3/ n-6 PUFA), H/H ratio = (C18:1n-9 + C18:2n-6 + C20:4n-6 + C18:3n-3 + C20:5n-3 + C22:5n-3+C22:6n-3)/(C14:0 + C16:0), HPI = (MUFA + PUFA)/(C12:0 + 4 × C14:0 +C16:0), DFA = C18:0 + MUFA + PUFA.

Effects of FBP on Plasma Biochemical Parameters of Laying Hens

The effects of dietary FBP on the plasma biochemical parameters of laying hens are presented in Table 8. No significant differences in plasma biochemical parameters were observed between the control and FBP-supplemented groups (P > 0.05).Table 8 Effects of dietary fermented blueberry pomace (FBP) on plasma biochemical parameters of laying hens.

Table 8Item	Treatments	SEM	P -values	
Control	0.25% FBP	0.5% FBP	1.0% FBP	FBP	Linear	Quadratic	
ALB, g/L	21.13	20.48	20.25	20.18	0.483	0.907	0.500	0.777	
ALT, U/L	146.44	155.24	151.02	146.99	3.853	0.852	0.943	0.429	
AST, U/L	170.88	163.25	182.75	173.63	4.794	0.570	0.529	0.939	
Ca, mmol/L	6.52	6.58	6.44	6.71	0.143	0.931	0.756	0.721	
CREA, umol/L	10.56	11.13	12.50	9.75	0.471	0.213	0.797	0.081	
GLU, mmol/L	14.53	14.98	15.24	15.03	0.131	0.280	0.138	0.210	
LDL-C, mmol/L	0.35	0.32	0.43	0.28	0.022	0.110	0.687	0.178	
NEFA, mmol/L	0.30	0.29	0.29	0.30	0.009	0.896	0.932	0.449	
TBA, μmol/L	34.81	35.08	41.71	43.36	2.558	0.541	0.175	0.894	
TC, mmol/L	4.07	3.91	4.19	4.00	0.220	0.980	0.972	0.970	
TG, mmol/L	12.83	11.85	12.51	12.27	0.262	0.621	0.673	0.497	
TP, g/L	58.51	58.16	62.13	58.84	1.166	0.625	0.646	0.542	
UA, mg/dL	5.12	4.95	4.94	4.26	0.258	0.677	0.285	0.636	
Data are expressed as means with their SEM (n = 8). ALB, albumin; ALT, alanine aminotransferase; AST, aspartate transaminase; Ca, calcium; CREA, creatinine; GLU, glucose; LDL-C, low-density lipoprotein-cholesterol; NEFA, non-esterified fatty acids; TBA, total bile acid; TC, total cholesterol; TG, triglyceride; TP, total protein; UA, uric acid.

Effects of FBP on the Number of Ovarian Follicles in Laying Hens

The effects of dietary FBP on ovarian follicle count in laying hens are depicted in Table 9. Laying hens fed with a diet supplemented with 1.0% FBP exhibited an increasing trend in hierarchical follicle (HF) count (P = 0.091) compared to the control group. Regression analysis showed that dietary FBP supplementation had significant linear effects on large white follicles (LWF; P = 0.042, model: Y = 19.325 + 7.328X) while had quadratic effects on HF (P = 0.019, model: Y = 4.384 − 2.261X − 2.499X2, estimated optimal dose required was 0.45% FBP). However, there were no significant differences in ovarian follicle count between the control and FBP-supplemented groups (P > 0.05).Table 9 Effects of dietary fermented blueberry pomace (FBP) on follicle numbers of laying hens.

Table 9Item	Treatments	SEM	P-values	
Control	0.25% FBP	0.5% FBP	1.0% FBP	FBP	Linear	Quadratic	
LWF	20.00	19.75	23.86	26.63	1.314	0.184	0.042	0.542	
SYF	5.00	3.38	4.57	4.88	0.359	0.360	0.799	0.172	
LYF	0.63	1.25	0.86	0.88	0.117	0.295	0.722	0.179	
HF	4.38	4.00	3.86	4.63	0.120	0.091	0.514	0.019	
Data are expressed as means with their SEM (n = 8). LWF, large white follicles (3–5 mm); SYF, small yellow follicle (5–8 mm); LYF, large yellow follicle (8–12 mm); HF, hierarchy follicle (> 12 mm).

Effects of FBP on Reproductive Hormones of Laying Hens

The effects of dietary FBP on the reproductive hormones of laying hens are presented in Table 10. The 0.25% FBP group had an increasing trend (P = 0.075) in the LH level in the ovary compared with the control group. Regression analysis showed that the LH level in the ovary was significantly increased (P = 0.025) and fitted a quadratic model: Y = 1.442 + 5.184X − 4.568X2, indicating that 0.57% FBP was the optimal level of supplementation. However, different FBP levels had no significant impacts on the levels of E2, FSH, and LH in the plasma and ovary of laying hens (P > 0.05).Table 10 Effects of dietary fermented blueberry pomace (FBP) on reproductive hormones in plasma and ovarian of laying hens.

Table 10Item	Treatments	SEM	P-values	
Control	0.25% FBP	0.5% FBP	1.0% FBP	FBP	Linear	Quadratic	
Plasma									
 E2, pmol/L	78.36	78.17	82.44	91.35	3.000	0.380	0.117	0.453	
 FSH, mIU/mL	26.10	29.11	27.89	26.05	0.837	0.520	0.859	0.163	
 LH, mIU/mL	12.87	12.95	13.09	12.83	0.477	0.998	0.996	0.866	
Ovarian									
 E2, pmol/L	9.75	13.55	12.24	11.49	0.840	0.463	0.610	0.189	
 FSH, mIU/mL	3.27	4.29	4.63	3.69	0.257	0.252	0.488	0.063	
 LH, mIU/mL	1.30	2.82	2.62	2.10	0.227	0.075	0.255	0.023	
Data are expressed as means with their SEM (n = 8). E2, estradiol; FSH, follicle stimulating hormone; LH, luteinizing hormone.

Effects of FBP on the Plasma and Ovarian Antioxidant Capacity of Laying Hens

The effects of dietary FBP on the plasma and ovarian antioxidant capacity of laying hens are presented in Table 11. The plasma GSH-PX activity was higher (P < 0.05) in the 1.0% FBP group compared with the 0.25% FBP and 0.5% FBP groups. In the ovary, the CAT activity was higher (P < 0.05) in the 0.5% FBP group compared with the control group. Moreover, FBP supplementation (0.25%−1.0%) displayed an increasing trend (P = 0.072) in the ovary GSH-PX activity compared with the control group. Regression analysis showed that dietary FBP supplementation had significant linear and quadratic effects on plasma GSH-PX activity (P = 0.009, linear model: Y = 1005.036 + 7.328X; P < 0.001, quadratic model: Y = 1151.182 − 859.780X + 1169.170X2) and ovary CAT activity (P = 0.013, quadratic model: Y = 241.695 + 721.681X − 631.512X2, estimated optimal dose required was 0.57% FBP). Dietary FBP had no significant effects on the SOD activity and MDA, GSH, and T-AOC concentrations in the plasma and ovary of laying hens (P > 0.05).Table 11 Effects of dietary fermented blueberry pomace (FBP) on antioxidant capacity in plasma and ovarian of laying hens.

Table 11Item	Treatments	SEM	P-values	
Control	0.25% FBP	0.5% FBP	1.0% FBP	FBP	Linear	Quadratic	
Plasma									
 GSH, U/mL	0.61	1.02	0.64	0.63	0.081	0.214	0.676	0.193	
 GSH-PX, U/mL	1167.38ab	966.12b	1045.98b	1455.17a	48.985	0.001	0.009	<0.001	
 MDA, nmol/mL	9.82	8.54	9.92	7.52	0.601	0.458	0.316	0.648	
 SOD, U/mL	327.48	353.82	333.05	319.30	10.988	0.737	0.658	0.383	
 T-AOC, mM/mL	0.56	0.55	0.60	0.60	0.061	0.988	0.755	0.955	
Ovary									
 CAT, U/mg prot	255.65b	345.43ab	472.57a	327.21ab	25.408	0.016	0.097	0.013	
 GSH, U/mg prot	1.57	1.70	1.76	1.78	0.054	0.535	0.169	0.629	
 GSH-PX, U/mg prot	8.57	13.45	14.96	13.30	0.896	0.072	0.056	0.060	
 MDA, nmol/mg prot	1.05	1.42	1.56	1.51	0.126	0.491	0.191	0.417	
 SOD, U/mL	12.45	14.16	16.01	13.51	0.828	0.505	0.508	0.219	
 T-AOC, mM/mg	0.24	0.27	0.32	0.28	0.016	0.450	0.292	0.319	
Data are expressed as means with their SEM (n = 8).

a,b Mean values with different superscript letters represent a significant difference (P < 0.05). CAT, catalase; GSH, glutathione; GSH-PX, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase; T-AOC, total antioxidant capacity.

Effects of FBP on Gene Expressions Related to Hormone Synthesis and Hormone Receptors in the Ovary of Laying Hens

The effects of dietary FBP on gene expressions associated with hormone synthesis and hormone receptors in the ovary of laying hens are presented in Figure 1A. Dietary 0.25% FBP supplementation up-regulated the cytochrome P450 family 11 subfamily A member 1 (CYP11A1) expression, whereas it down-regulated the estrogen receptor 2 (ESR2) expression in the ovary of laying hens compared with the control group (P < 0.05). Dietary FBP supplementation had significant quadratic effects on CYP11A1 expression (P = 0.008, quadratic model: Y = 1.076 + 0.702X − 0.909X2, estimated optimal dose required was 0.39% FBP) in the ovary of laying hens. No significant differences were observed in other gene expressions related to hormone synthesis and hormone receptors between the control and FBP-supplemented groups (P > 0.05).Figure 1 Effects of dietary fermented blueberry pomace (FBP) on the ovarian and liver functions of laying hens during the late laying period. Gene expressions related to hormone synthesis and hormone receptors in the ovary (A), lipid metabolism in the liver (B), yolk precursor synthesis in the liver and ovary (C), and antioxidant capacity in the ovary (D) of laying hens. Mean values with different lowercase letters represent a significant difference (P < 0.05). Abbreviations: ACC, acetyl-CoA carboxylase; ApoB, apolipoprotein B; ApoVLDL II, apo very low density lipoprotein II; CAT, catalase; CYP11A1, cytochrome P450 family 11 subfamily A member 1; CYP17A1, cytochrome P450 family 17 subfamily A member 1; CYP19A1, cytochrome P450 family 19 subfamily A member 1; ESR, estrogen receptor; FAS, fatty acid synthase; FSHR, follicle stimulating hormone receptor; GPX1, glutathione peroxidase 1; HO-1, heme oxygenase-1; HSD17B1, 17β-hydroxysteroid dehydrogenase 1; HSD3B1, 3β-hydroxysteroid dehydrogenase 1; Keap1, kelch-like ECH-associated protein 1; LHCGR, Luteinizing hormone/chorionic gonadotropin receptor; MTTP, microsomal triglyceride transfer protein; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, Nuclear factor erythroid 2-related factor 2; oVLDLR, ovarian very low density lipoprotein receptor; PPAR, peroxisome proliferator activated receptor; SCD1, stearoyl-CoA desaturase 1; SOD, superoxide dismutase; SREBP1, sterol regulatory element binding protein 1; VLDLR, very low density lipoprotein receptor; VTG II, vitellogenin 2.

Figure 1

Effects of FBP on Gene Expressions Related to Lipid Metabolism in the Liver of Laying Hens

The effects of dietary FBP on gene expressions related to lipid metabolism in the liver of laying hens are presented in Figure 1B. Dietary 0.25% FBP and 1.0% FBP supplementation up-regulated (P < 0.05) the acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) expressions compared with the control group. The stearoyl-CoA desaturase 1 (SCD1) expression in the FBP-supplemented groups and sterol regulatory element binding protein 1 (SREBP1) expression in the 0.25% FBP group were up-regulated compared with the control group (P < 0.05). Regression analysis showed that dietary FBP supplementation had significant linear and quadratic effects on the expressions of ACC (P = 0.022, linear model: Y = 1.411 + 0.674X), FAS (P < 0.001, linear model: Y = 1.469 + 1.364X), SCD1 (P < 0.001, linear model: Y = 1.920 + 1.357X; P < 0.001, quadratic model: Y = 1.270 + 6.738X − 5.195X2, estimated optimal dose required was 0.65% FBP).

Effects of FBP on Gene Expressions Related to Yolk Precursor Synthesis in the Liver of Laying Hens

The effects of dietary FBP on gene expressions related to yolk precursor synthesis in the liver of laying hens are presented in Figure 1C. Dietary 0.25% FBP and 1.0% FBP supplementation up-regulated (P < 0.05) the ESR2 expression, while 0.25% FBP up-regulated (P < 0.05) the vitellogenin II (VTG II) expression in the liver of laying hens compared with the control group. Furthermore, regression analysis showed that dietary FBP supplementation had significant linear and quadratic effects on apolipoprotein B (ApoB; P = 0.043, quadratic model: Y = 0.9822 + 1.555X − 1.455X2, estimated optimal dose required was 0.53% FBP), apolipoprotein very low-density lipoprotein II (ApoVLDL II; P = 0.008, quadratic model: Y = 1.025 + 1.174X − 1.189X2, estimated optimal dose required was 0.49% FBP), ESR2 (P = 0.025, quadratic model: Y = 1.106 + 1.351X − 1.047X2, estimated optimal dose required was 0.65% FBP), VTG II (P = 0.001, quadratic model: Y = 1.167 + 2.560X − 2.345X2, estimated optimal dose required was 0.55% FBP). Dietary FBP supplementation had no significant impacts on the ESR1, ApoVLDL II, ApoB, and very low-density lipoprotein receptor (VLDLR) expressions in the liver of laying hens (P > 0.05).

Effects of FBP on Gene Expressions Related to Antioxidant Capacity in the Ovary of Laying Hens

The effects of dietary FBP on gene expressions related to the antioxidant capacity in the ovary of laying hens are presented in Figure 1D. The kelch-like ECH-associated protein 1 (Keap1) expression in the 0.5% FBP group and NAD(P)H quinone oxidoreductase 1 (NQO1) expression in the 0.25% FBP and 1.0% FBP groups were down-regulated (P < 0.05) in the ovary of laying hens compared with the control group. Regression analysis revealed that dietary FBP supplementation had significant linear and quadratic effects on the expressions of Keap1 (P = 0.005, quadratic model: Y = 0.994 − 1.071X + 1.004X2) and NQO1 (P = 0.002, linear model: Y = 0.918 − 0.270X). Antioxidant capacity-related other gene expressions, including nuclear factor erythroid2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), CAT, GPX1, SOD1, and SOD2, did not significantly change between the control and FBP-supplemented groups (P > 0.05).

DISCUSSION

Developing novel feed additives to improve the production performance and egg quality of hens during the late laying period, thereby enhancing the economic benefit, has gained more interest in the past few years. To explore whether FBP can be applied as a novel feed additive in the layer industry, the present study investigated the effects of different levels of FBP on production performance, egg quality and nutritional value, plasma biochemical parameters, follicle number, reproductive hormones, lipid metabolism, and antioxidant capacity of laying hens during the late laying period. The cost of FBP is relatively lower than conventional poultry feed. Adding FBP as a feed additive in laying hens’ diet during the late laying period improved egg quality and nutritional value of laying hens, thus enhancing breeding efficiency. Moreover, agricultural by-products as feed additives in poultry production practices are also associated with environmental protection by eliminating pollution control measures, as well as saving financial resources for waste management. Therefore, utilization of agricultural by-products as feed additives would be economical and beneficial to improve the productivity of the poultry industry.

Maintaining a higher egg production performance is essential for achieving high economic benefits for the layer industry. The ALR, AER, FER, and egg quality are the main indicators to measure the production performance of laying hens. In the present study, although dietary FBP had no significant impacts on the egg production performance and ALR; however, there was an increasing trend with the 0.5% FBP supplementation in the ADFI during d 43−56 of the trial. Previous studies also found that diets supplemented with various pomace (e.g., grape, raspberry, black currant, and black chokeberry pomace) had no significant effects on the production performance of laying hens (Kara et al., 2016; Sosnówka-Czajka and Skomorucha, 2021). The increased ADFI might be related to the distinct fruit flavor of the FBP, which might increase the palatability of the diet. A previous study also showed that the egg production performance of laying hens could be influenced by dietary protein and energy levels (Heijmans et al., 2021). However, as the bioactive compounds present in FBP primarily pertain to antioxidant properties, they do not affect protein and energy levels within the diet. This might be one of the possible reasons that dietary FBP had no significant effect on egg production performance.

Egg quality, encompassing both external and internal attributes, significantly influences the egg's nutritional value and consumers’ acceptability. External quality includes egg weight, egg shape index, eggshell strength, etc. (Sadaf et al., 2021). Internal quality includes protein quality, such as albumen height; yolk quality such as yolk color; and other indicators, such as blood spots (Lokapirnasari et al., 2019). The Haugh unit is a crucial metric for evaluating egg quality, with a higher value indicating better egg white consistency and overall quality. In the present study, dietary FBP supplementation (0.25−1.0%) notably increased egg albumen height and the Haugh unit on d 14 of the trial, indicating FBP improved egg quality. These findings are consistent with previous studies that reported that dietary supplementation of plant-derived mixtures containing fermented Schisandra pomace and grape pomace to laying hens increased the Haugh unit values of eggs (Moon et al., 2021; Romero et al., 2022). Additionally, a previous study also highlighted that dietary supplementation of tea polyphenols rather than tea catechins during the late laying period improved the Haugh unit of eggs (Wang et al., 2018). The phenolic acids and flavonoid contents were increased in blueberry pomace after fermentation, which led to a greater increase in antioxidant activity in FBP (Tian et al., 2023). Therefore, the improvements in the Haugh unit may be attributed to the bioactive substances, such as polyphenols present in FBP. These findings also suggested that the Haugh unit of eggs was optimized at 0.76% of FBP. Overall, dietary FBP supplementation enhanced egg quality, with a notable impact observed with a 1.0% FBP inclusion level.

The nutritional value of eggs primarily relies on CP content, amino acid composition, and fatty acid contents. Met, an essential amino acid, serves as the first limiting amino acid in the diet of laying hens and plays a vital role in DNA methylation and contributes to flavor provision (Wang et al., 2012). Previous research indicated that dietary flaxseed supplemented with Met enhanced the production performance of laying hens compared to diets without Met supplementation (Beheshti Moghadam et al., 2021). In the present study, 1.0% FBP supplementation increased the Met content in the egg white compared to 0.25% FBP, suggesting that higher FBP levels altered the amino acid profile in egg white. Currently, there are no relevant reports on the effects of FBP on the nutritional composition of eggs, which warrants further research to elucidate the specific mechanism.

Lipid health indices, including PUFA/SFA ratio and n6/n3 PUFA ratio, serve as crucial parameters for assessing the nutritional value of egg yolks. Egg yolk contains various PUFAs essential for human health, such as α-linolenic acid (C18:3n3, ALA), linoleic acid (LA), etc. Among them, ALA is a type of omega-3 fatty acid and is the primary precursor of other omega-3 fatty acids that have attracted wide attention due to their potential to prevent diseases such as heart disease and dementia (Dajnowska et al., 2023). In the present study, 0.25% and 1.0% FBP supplementation increased the content of C18:3n3 (a PUFA) and decreased the contents of C18:1n9c (MUFA), total SFA, and MUFA in the yolk, indicating that FBP altered the fatty acid profile in the yolk. Previous studies reported that dietary grape pomace supplementation decreased the contents of SFA and MUFA and increased the content of PUFA (Romero et al., 2022; Selim et al., 2023), which is consistent with the findings in the present study. Additionally, Moghadam et al. (2017) demonstrated that dietary Met could increase ALA and n-3 fatty acids contents of broilers by altering lipid metabolism in the liver. Therefore, FBP may increase ALA content by elevating Met content and modulating lipid metabolism in the liver.

Moreover, atherosclerosis index (AI), thrombosis index (TI), hypocholesterolemia to hypercholesterolemia ratio (H/H), health promotion index (HPI), and desirable fatty acids (DFA) are also the common indicators to evaluate the nutritional value of eggs. AI and TI indicate the thrombotic potential of fatty acids, H/H signifies the impact of specific fatty acids on cholesterol metabolism, and HPI assesses the nutritional value of fatty acids and their influence on cardiovascular diseases (Chen and Liu, 2020; Attia et al., 2024). Regarding the nutriology, the relatively lower AI and TI values and higher H/H, HPI, and DFA values have the potential to maintain the physical health of human beings (Chen and Liu, 2020). For foods recommended for human consumption, AI should be below 0.5 and TI below 1.0 (Wołoszyn et al., 2020). In the present study, AI and TI indices were below the recommended thresholds, indicating that dietary FBP did not affect the lipid health indices of eggs. Overall, dietary FBP supplementation improved egg nutrients, of which 1.0% was the optimal additive dose.

Follicle development encompasses primordial follicle assembly, recruitment, growth, maturation, ovulation, and atresia. Ovarian follicles in poultry are classified based on size into HF (diameter > 12 mm), large yellow follicles (LYF, diameter 8–12 mm), small yellow follicles (SYF, diameter 5–8 mm), and LWF (diameter 3–5 mm) (Li et al., 2020). The HFs are categorized as F1, F2, F3, F4, F5, and F6 in descending order. During ovulation, static primordial follicles enter the small white follicle (SWF) pool before being recruited into the SYF pool. Most of the follicles in the SYF pool are atresia, and only a small part of the follicles can grow into LYFs. After F1 ovulation, the LYF replaces the previous F6. The selected follicle quickly deposits yolk material, increases in diameter, and eventually achieves ovulation (Johnson, 2015). In the present study, an increasing trend in the HF number was observed with the 1.0% FBP supplementation, and regression analysis also showed that the HF number was optimized at 0.45% FBP. Therefore, these findings suggest that 0.45 to 1.0% FBP improved the development of follicles in laying hens.

Gonadal hormones, including FSH, LH, and E2, regulate the development of mature follicles. When gonadotropin levels decrease in the body, the hypothalamus is stimulated to produce gonadotropin-releasing hormone, which acts on the pituitary gland. Consequently, the pituitary gland produces FSH and LH, stimulating ovulation in the ovary (Li et al., 2023). In the present study, the LH concentration in the ovary was higher in the FBP-supplemented groups, indicating that dietary FBP improved the synthesis of gonadotropins in laying hens. However, there was no significant difference in ALR in the present study. Senthilkumaran et al., 2006 evaluated LH level changes in the plasma of laying hens at different ages within 24 h using vascular access port continuous bleeding technology. They found no significant differences in plasma LH level among laying hens with different ALRs. The basic level and amplitude of LH in the plasma of aged laying hens did not significantly differ from that of laying hens at the peak of laying. It is speculated that gonadotropins only play a regulatory role or that the concentrations of gonadotropins have not reached the threshold required to increase ALR.

Androgens, estrogens, and progesterone are steroid hormones closely associated with ovarian ovulation. CYP11A1, cytochrome P450 family 17 subfamily A member 1 (CYP17A1), cytochrome P450 family 19 subfamily A member 1 (CYP19A1), 3β-hydroxysteroid dehydrogenase 1 (HSD3B1), and 17β-hydroxysteroid dehydrogenase 1 (HSD17B1) are the main genes regulating steroid hormone synthesis in poultry. CYP11A1 is the primary rate-limiting enzyme, and LH level in granulosa cells can positively regulate the transportation of steroidogenic acute regulatory protein (StAR), thereby promoting steroid hormone synthesis (Thomas et al., 2003). In the present study, FBP supplementation increased the ovarian LH level, facilitating enhanced transport of StAR protein and promoting steroid hormone synthesis. Additionally, ovarian CYP11A1 expression was up-regulated in the 0.25% FBP group, further promoting steroid hormone synthesis. These findings suggest that dietary FBP could enhance steroid hormone synthesis and expression of hormone receptor-related genes, consequently elevating ovarian steroid hormone levels.

The liver is the primary metabolic organ of laying hens, crucial for lipid and yolk precursor synthesis, as well as the transportation and breakdown of lipids (Li et al., 2015). ACC, SREBP1, FAS, and SCD1 are involved in fatty acid synthesis and fat deposition in the liver. Furthermore, carnitine palmitoyl transferase (CPT1A), MTTP, and peroxisome proliferator-activated receptor-gamma (PPAR-γ) are involved in fatty acid degradation and transportation, being localized on the inner and outer membranes of mitochondria (Houten et al., 2016; Bougarne et al., 2018). The present study exhibited up-regulation of genes associated with fat synthesis, including ACC and FAS in the 0.25% FBP and 1.0% FBP groups, SREBP1 in the 0.25% FBP group, and SCD1 in the FBP-supplemented groups compared to the control group. These findings suggest that FBP enhanced lipid metabolism in the liver of laying hens. Previously, Chai et al. (2022) demonstrated that obese mice fed FBP prepared with a mixture of Lactobacillus rhamnosus and Lactobacillus plantarum improved lipid metabolism by up-regulating PPAR-α and down-regulating SREBP1 and FAS expressions, thus mitigating obesity. Nevertheless, discrepancies between our findings and those of Chai et al. (2022) may be the variations in the nutritional composition, nutrient levels, and bioactive substances of the FBP used (different compound starters and microbial agents were employed during fermentation) in the present study. Additionally, the microorganisms contained in FBP may also have positive impacts on laying hens, which may be another possible reason; thus, further in-depth studies are needed to elucidate the impacts of FBP on the microbiota composition and their specific mechanisms. Moreover, different experimental animals might have different utilization abilities and utilization degrees of FBP.

During egg yolk formation, E2 first acts on estrogen receptors ERα and ERβ, regulating the synthesis of VTGⅡ, ApoVLDLⅡ, and ApoB, thus promoting the production of VTG and VLDL (Walzem et al., 1999). The VTG and VLDL are the 2 major yolk precursors (Schneider, 1992). Subsequently, VLDL and VTG are transported by ApoVLDL II and ApoB into the ovary through blood and then transported into oocytes through VLDLR-mediated endocytosis. Triglycerides and total cholesterol carried by VLDL and VTG provide energy for follicular development in oocytes to form egg yolks (Pan et al., 2002; Lin et al., 2019). In the present study, up-regulation of ESR2 expression in the 0.25% and 1.0% FBP groups, along with VTG II expression in the 0.25% FBP group, suggested an enhancement of yolk formation with FBP supplementation. Therefore, the findings of regression analysis suggested that the egg yolk formation was optimized at 0.49 to 0.55% FBP.

Oxidative stress is the main cause of decreased ovarian function in laying hens during the late laying period (Yang et al., 2021). MDA, T-AOC, SOD, GSH, CAT, and GSH-PX are the primary indicators reflecting the degree of oxidative stress in the body. MDA, a product of lipid peroxidation during oxidative stress, mirrors the extent of cell damage (Gęgotek and Skrzydlewska, 2019). SOD, GSH, and CAT are endogenous antioxidant enzymes that scavenge free radicals generated during oxidative stress, indicating the body's antioxidant capacity. In the present study, dietary supplementation with 0.5% FBP resulted in elevated CAT and GSH-PX activities in the ovary, signifying improved antioxidant capacity in laying hens. These findings are consistent with a previous study, which demonstrated that dietary FBP improved the antioxidant capacity of high-fat mice by increasing serum SOD, GSH-PX, CAT, and T-AOC (Chai et al., 2022).

The Nrf2 can bind to several antioxidant response elements (ARE) and plays an important role in the transcriptional induction of phase II enzymes and synergistic transcriptional regulation of phase II genes (Itoh et al., 1997). The degradation of hemachrome catalyzed by HO-1 not only prevents the pro-oxidation effect, but also its by-product bilirubin has the activity of clearing ROS, and HO-1 expression and protein will be activated and up-regulated after oxidative stress. Moreover, Jin et al., 2016 demonstrated that activation of Nrf2 can induce HO-1 expression. Previous studies have also reported that anthocyanins can reduce ROS levels and up-regulate the Nrf2/HO-1 pathway by regulating the phosphorylation of the PI3K/Akt/GSK3β pathway, thereby alleviating oxidative stress (Ali et al., 2018). In the present study, the Keap1 expression was down-regulated in the 0.5% FBP group, while NQO1 expression was down-regulated in the 0.25% and 1.0% FBP groups in the ovary of laying hens. The presence of anthocyanin in FBP is hypothesized to inhibit Keap1 function and increase Nrf2 activity, potentially inducing HO-1 expression and thereby enhancing ovarian antioxidant capacity.

CONCLUSIONS

Dietary FBP supplementation during the late laying period improved egg quality and nutritional values by increasing albumen height, Haugh unit, and eggshell thickness, which may be partly mediated by up-regulating expressions of genes related to yolk precursor synthesis (ESR2 and VTG II). Additionally, FBP supplementation improved antioxidant capacity in the ovary of laying hens by increasing CAT and GSH-PX activities. Based on the regression analysis, the optimal dose of FBP was 0.39 to 0.76% in the laying hen's diet during the late laying period. These findings provide a guiding significance for the application of dietary FBP in the layer industry, and dietary FBP would be a potential feed additive during the late laying period.

DISCLOSURES

The authors declare no conflict of interest.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

Funding: This study was supported by the Future Partner Special Fund of Chinese Academy of Sciences (092GJHZ2022044FN ) and City-School Cooperation Project of the Special Funds of Science and Technology in Fuyang City undertaken by Fuyang Normal University (SXHZ2020007 ).

Author contributions: Binghua Qin: Formal analysis, Investigation, Methodology, data analysis, Visualization, Writing−original draft, Writing−review and editing. Md. Abul Kalam Azad: Formal analysis, Investigation, Visualization, Validation, Funding acquisition, Writing−original draft, Writing−review and editing. Zhihua Li: Formal analysis, Investigation, Methodology, Writing−review and editing. Ting Chen: Formal analysis, Investigation, Methodology, data analysis. Yadong Cui: Formal analysis, Investigation, Methodology, data analysis, Visualization, Writing−review and editing. Wei Lan: Formal analysis, Investigation, Methodology, data analysis, Visualization, Writing−review and editing. Haoran Wang: Formal analysis, Investigation, Methodology, data analysis, Visualization, Writing−original draft. Xiangfeng Kong: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing−review and editing. All authors contributed to the article and approved the submitted version.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104241.
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