Effects of Fagopyrum dibotrys Rhizoma Meal Supplementation on Productive Performance, Egg Quality, Egg Nutritional Value and Serum Biochemical Parameters of Shanma Laying Ducks

1. Introduction

Laying duck industry serves as a pivotal pillar of China’s agricultural economy. In the context of its rapid development, key industry concerns now revolve around enhancing production performance, optimizing egg quality, improving the nutritional value of duck eggs, and achieving green and sustainable development. With rapid economic development and continuous improvement in living standards, consumers are placing higher demands on the nutritional and health-promoting qualities of egg products, making it imperative to enhance the nutritional and functional value of duck eggs [1]. Recent studies have revealed that Chinese herbal medicine or plant extracts are rich in bioactive compounds such as disease-resistant alkaloids, antioxidant flavonoids, and phenolics. These substances can effectively improve the nutritional value of eggs by enhancing antioxidant capacity, boosting immune function, and optimizing gut microbiota structure in poultry, demonstrating significant potential for practical applications [2,3,4,5,6,7,8,9].

Fagopyrum dibotrys (F. dibotrys) (D. Don) Hara, a perennial herbaceous plant belonging to the Polygonaceae family and Fagopyrum genus, possesses significant medicinal and edible value, and has been officially included in China’s Feed Materials Directory [10]. Its rhizomata, stems, leaves, and flowers contain trace mineral elements essential for livestock and poultry, including copper (Cu), iron (Fe), and zinc (Zn), etc, as well as 17 amino acids such as methionine (Met), arginine (Arg), and lysine (Lys), etc, along with various vitamins including vitamin B₁, vitamin B₂, and vitamin E, etc. [11]。The rhizoma of F. dibotrys (FDR) has a long history of application in traditional Chinese medicine. It contains various bioactive compounds including flavonoids, phenolics, triterpenes, and tannins, etc. [12,13,14], and exhibits multiple biological properties such as antioxidant, antibacterial, anti-inflammatory, and immunomodulatory effects [15,16,17]. Currently, its stems and leaves are widely used in pharmaceuticals, health foods, beverages, and forage, while research on its rhizomata in livestock production remains limited. As a forage crop, F. dibotrys exhibits strong adaptability, high propagation efficiency, and substantial biomass yield (up to 112,500 kg·hm^⁻2). Its stems and leaves are rich in crude protein (12.28% DM) with low crude fiber (24.06% DM), neutral detergent fiber (39.74% DM), and acid detergent fiber (30.18% DM), making it a potential unconventional feed ingredient to replace part of swine diets and alleviate feed shortages [11,18,19,20]. Studies show that supplementing 10% fresh F. dibotrys in laying hens diets deepened yolk color and improved amino acids composition in Changshun green-shell eggs [20]. Furthermore, adding 400-800 mg/kg of F. dibotrys stem-leaf extract not only enhanced egg production, quality, and nutritional value but also improved immunity by modulating serum biochemical parameters [21]. Additionally, dietary inclusion of 1-2% F. dibotrys rhizoma meal (FDRM) in broilers alleviated oxidative stress induced by oxidized oil, thereby improving poultry health [16].

Nevertheless, based on our knowledge, researches on Fagopyrum dibotrys has primarily focused on its stems and leaves, with studies conducted on growing-finishing pigs [9], sows [19], mice [17], and laying hens [20,21], demonstrating certain improvements in animal growth performance and farming efficiency. However, studies on the application of its rhizomata in poultry are very scarce. It is currently unclear whether dietary supplementation with FDRM can exert similarly positive effects on intensive laying duck production as its stem and leaf derivatives. Consequently, this study aims to investigate the effects of FDRM on productive performance, egg quality, nutritional value of eggs, and serum biochemical parameters, thereby providing a scientific basis for utilizing FDRM as an unconventional feed resource in Shanma laying duck production.

2. Materials and Methods

The experimental use of animals and related procedures were performed according to the Chinese Guidelines for Animal Welfare and approved by the Institutional Animal Care and Use Committee of Jiangxi Academy of Agricultural Sciences (Ethical Committee Number: 2025-JXAAS-XM-18).

2.1. Experimental Materials

The Fagopyrum dibotrys rhizoma meal (FDRM) used in this study was provided by the Institute of Animal Husbandry and Veterinary Medicine, Jiangxi Academy of Agricultural Sciences. Fresh F. dibotrys rhizomata were collected, crushed and passed through the 80-mesh screen to prepare FDRM. The main bioactive compounds of FDRM are total flavonoids and polyphenols, quantified using a UV spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan) at the Jiangxi Province Key Laboratory of Animal Green and Healthy Breeding, which the contents are 19.6 mg/g and 63.85 mg/g respectively. Moreover, the nutritional compositions of FDRM in this experiment are shown in Table 1. The contents of gross energy, dry matter, crude protein, crude fat, crude fiber, crude ash, calcium, and total phosphorus in FDRM were determined according to the methods described in Feed Analysis and Feed Quality Testing Technology [22].

2.2. Ducks, Experimental Design and Treatments

This study was conducted on 32-weeks-old Longyan Shanma laying ducks for a 49-day period with a completely randomized design. A total of 512 laying ducks with similar productive performance (80.88 ± 5.17 %) and body weight (1.24 ± 0.02 kg) were used in this experiment. Ducks were randomly allocated to 4 groups with 8 replicates per group and 16 ducks per replicate (128 laying ducks per group). The control group (F0) received the basal diet, while the treatment groups were provided with diets containing 1% (F1), 2% (F2), and 3% (F3) FDRM supplementation. All diets were nutritionally balanced and formulated to meet identical nutritional specifications.

2.3. Diets and Management

This trial was carried out at the test field of laying ducks in Gaoan, Institute of Animal Husbandry and Veterinary Science, Jiangxi Academy of Agricultural Sciences, PR China. The basal diet fed animals was maize-soybean meal diet, which was formulated based on the China's national standard “nutrient requirements for egg duck” (GB/T 41189-2021) to meet the nutrient requirements of Longyan Shanma ducks. Table 2 presents the composition and nutrient levels of experimental diets. The experimental laying ducks were raised in three-layer three-dimensional netting, consisted of 4 adjacent cages (40 × 38 × 38 cm; length × width × height) with 2 animals per cage, providing 28,880 cm³ per animal in closed fully automated duck house. Each replicate was raised on the upper and middle floors and each group was guaranteed to be equal in the number of distributed upper and middle layers. During the period of study, the housing temperature and relative humidity were 23.0 ± 2 ℃ and 55 to 75 %, respectively. Furthermore, the photoperiod was set at 16L:8D through a 49-d experimental period. Animals were kept with ad libitum access to feed and water during the entire experimental period.

2.4. Productive Performance

Throughout the trial, the ducks' egg production and egg weight were monitored daily, and feed consumption was meticulously recorded on a replicate basis at weekly intervals using an electronic balance (HLD-5003, Youheng Weighing Equipment Co., Ltd., Hangzhou, China). At the end of the feeding trial, these values processed using Excel 2016 (Microsoft Corp., USA) were used to analyze the daily egg weight (DEW), average egg weight (AEW), laying rate (LR), average daily feed intake (ADFI) and the ratio of feed to egg (F/E) of the ducks for the 49-d feeding period.

2.5. Egg Quality

On the final day of the experiment, a random subset of eight freshly laid eggs (a total of 64 eggs from each treatment) were collected for each replicate, which were used for conventional egg quality analysis (within 48 h after laying), including shape index, shell strength, shell thickness, haugh unit, yolk color, vitellus proportion, albumen proportion and shell proportion. The shape index was measured with a precision caliper marked at 0.01mm intervals and was represented by the formula shape index (SI) = (egg length/egg width) [1]. Shell strength was assessed along the vertical axis with a compression tester (EFG-0503, Robotmation, Tokyo, Japan). The shell thickness was determined (excluding shell membrane) using a micrometer with the least count of 0.01 mm and was expressed by the mean of measurements taken at three points (air cell, equator, and sharp end) of the egg. Haugh unit, yolk color and albumen height were measured using an Egg Multi-tester (EMT-5200, Robotmation, Tokyo, Japan). The vitellus, albumen, and shell were separated, weighed, and expressed as a percentage of total egg weight.

2.6. Egg Nutritional Value

After measuring the egg physical parameters, the vitelluses were sampled, lyophilized, and subsequently analyzed for nutrient composition, amino acid composition and fatty acids profile. Nutrient composition included moisture, crude protein (CP), crude fat, cholesterol and Ca were determined in accordance with AOAC methods [23]. Crude protein content was estimated by measuring nitrogen content (Kjeldahl method) with an automatic Kjeldahl nitrogen analyzer (SKD-200, Shanghai Peiou Analysis Instruments Co.,Ltd., Shanghai, China) and applying a 6.25 conversion factor. Ether extract was measured using the Soxhlet method with petroleum ether extraction in a Hanon Automatic Soxhlet Extractor (SZF-06A, Shanghai Lichen Instruments Technology Co., Ltd., Shanghai, China). The method for the Ca and cholesterol determination were used by an UV spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan) and commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) as described by Zhang et al. [24]. According to the methods reported by Cullere et al. [25] and Xu et al. [26], the amino acids content and fatty acids profile of the eggs were analyzed. The determination of amino acids content in egg yolk was determined by ion-exchange chromatography with the following procedure: approximately 0.1 g of egg yolk powder sample was weighed and digested with 5 mL of 6 mol/L HCl solution at 105°C in an oven for 24 h. After digestion, the solution was diluted to 50 mL in a volumetric flask with deionized water and filtered through a 0.22 μm aqueous-phase filter into a centrifuge tube. Then, 2 mL of the filtrate was evaporated in an evaporating dish in a 60°C water bath, followed by the addition of 4 mL of 0.02 mol/L HCl solution for dissolution. Once fully dissolved, the sample was stored at 4°C for analysis using an ion-exchange amino acid analyzer (L8900, Hitachi, Tokyo, Japan). A total of 17 amino acids were determined, as detailed in Table 6. While the fatty acids profile was performed using gas chromatography (GC), the detailed procedure was as follows: Approximately 20 g of egg yolk sample was dried at 105°C for 1 h, followed by accurate weighing of 1 g dried sample for petroleum ether extraction (3 h). Subsequently, 60 mg of extracted fat was dissolved in 4 mL of isooctane, followed by sequential addition of 200 μL potassium hydroxide-methanol solution and 1 g sodium bisulfate. After salting-out, the upper layer containing fatty acid methyl esters was collected and stored at 4°C. Prior to GC analysis (Agilent 7890 A, Agilent Technologies, CA, USA), all samples were filtered through a 0.22 μm membrane. Quantitative analysis of fatty acids was conducted using GC ChemStation B.04.03 software (Agilent Technologies, CA, USA). A total of 15 fatty acids were determined, as detailed in Table 7.

2.7. Serum Biochemical Parameters

At the end of the 8 weeks, two laying ducks with close to the average weight were randomly selected from each replicate for collection of 5 mL of fasting blood samples from the wing vein using a black 7-gauge needle and vacuum coagulation tubes. The blood samples were centrifuged at 1,006 × g for 10 minutes to separate serum within 2 hours after blood collection using a centrifuge (LC-LX-L50C, Shanghai LiChen Instrument Technology, Ltd., Shanghai, China) at the Jiangxi Province Key Laboratory of Animal Green and Healthy Breeding, and the serum was separated and stored at -20°C for further use. The serum levels of triglycerides (TG, Cat no, A110-2-1), total cholesterol (TC, Cat no, A111-2-1), high-density lipoprotein (HDL, Cat no, A112-2-1), and low-density lipoprotein (LDL, Cat no, A113-2-1), total protein (TP, Cat no, A045-2-2), albumin (ALB, Cat no, A028-2-1), blood urea nitrogen (BUN, Cat no, C013-2-1), alkaline phosphatase (AKP, Cat no, A059-2-2), and calcium (Ca, Cat no, C004-2-1) were measured by an Automatic Biochemistry Instrument (BS-420, Shenzhen Myriad Bio-Medical Electronics Co., Ltd., Shenzhen, China) using commercial assay kits. All assay kits were provided by the Nanjing Jiancheng Bioengineering Institute.

2.8. Statistical Analyses

All data were organized using Excel 2013, and then subjected to a one-way analysis of variance (One-Way ANOVA) using the Bonferroni method in SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA) to test for multiple comparisons. The experimental results were presented as mean and pooled SEM. A value of P<0.05 was considered statistically significant, while a value of 0.05<P<0.10 indicated a trend towards an increase or decrease.

3. Results

3.1. Productive Performance

The effects of dietary FDRM supplementation on productive performance of laying ducks are shown in Table 3. During the entire period, there were no significant effects (P>0.05) by adding FDRM in laying ducks diet, regardless of the supplementation levels, on DEW, AEW, DEN, LR, ADFI and F/E.

3.2. Egg Quality

The egg quality parameters of laying ducks are depicted in Table 4. Compared with the F0 group, the shell strength and yolk color in F2 and F3 groups were significantly increased (P<0.05). Furthermore, the yolk color in F1 group and the shell proportion in F3 group were significantly (P<0.05) higher than that in F0 group. No significant differences were observed in shape index, shell thickness, albumen height, haugh unit, vitellus proportion and albumen proportion (P>0.05), in response to dietary FDRM supplementation levels.

3.3. Egg Nutritional Value

3.3.1. Nutrient Composition of Egg

For the conventional nutrient levels of the vitellus in laying ducks, no significant dietary effects were observed across the measured parameters (moisture; crude protein; ether extract; cholesterol and Ca) (Table 5). Nevertheless, compared with the F0 group, the Ca content in F2 and F3 groups tend to improve as the inclusion level of FDRM increased(P=0.073).

3.3.2. Amino Acids Composition

As can be seen from Table 6, the contents of Thr, Val, Ile, Leu, Asp, Glu, Gly, Cys, total EAAs, total AAs, and umami AAs in the vitellus showed significant response to the increasing FDRM supplement levels (P<0.05). Compared with the control group (F0), there was no difference in the amino acid profile in F1 group (P>0.05), dietary supplementation with 2% FDRM (F2) group could markedly increase the contents of Thr, Leu, Asp, Glu, Gly, Cys, total EAAs, total AAs, and umami AAs (P<0.05), while the Thr and Asp concentrations in the group supplemented with 3% FDRM (F3) were statistically heighted (P<0.05), with no dietary effect on other amino acids contents (P>0.05). Furthermore, compared with the the F1 group, the F2 group significantly increase the content of Val, Ile, Leu, Gly, Cys, and total EAAs (P<0.05), without significantly affecting the contents of other amino acids (P>0.05).

3.3.3. Fatty Acids Profile

Table 7 showed the effect of dietary FDRM addition on the fatty acid profile in the vitellus of laying ducks. The levels of C16:0 and total SFAs in the F1 and F2 groups exhibited a significant decrease compared to the F0 group (P<0.05), while the UFAs:SFAs ratio increased significantly (P<0.05). Moreover, the levels of C14:0 significantly decreased in the F2 and F3 groups (P<0.05), the F3 group statistically heighted the contents of C20:1 and C20:3, markedly reduced the contents of total SFAs (P<0.05). Compared with the F0 group, the PUFAs:SFAs ratio in the vitellus from the F1 group displayed a remarkable enhancement (P<0.05).

3.4. Serum Biochemical Parameters

For serum biochemical parameters, the levels of TP and BUN along with the concentration of Ca in serum of laying ducks showed significant responses to increased dietary FDRM supplement levels (Table 8, P<0.05). The serum levels of TP in F2 group and BUN in F3 group were significantly (P<0.01) higher than that in F0 group. Additionally, in comparison to the F2 group, the F1 and F3 group dramatically lowered the Ca concentration in serum of laying ducks.

4. Discussion

In general, compared to Fagopyrum esculentum (common buckwheat) and Fagopyrum tataricum (tartary buckwheat), F. dibotrys (golden buckwheat), as a member of the Fagopyrum genus, contains a greater variety of flavonoids and phenolic acids, exhibits stronger antimicrobial activity, and demonstrates intermediate antioxidant capacity [14,27]. Additionally, Chinese herbal medicine rich in flavonoids and phenolic acids have been shown to improve poultry health status and egg production performance [28,29]. This experiment aimed to investigate the effects of dietary supplementation with FDRM on laying performance, egg quality, egg nutritional value and serum biochemical indicators of Shanma laying ducks. Our findings demonstrated that dietary inclusion of FDRM had no significant impact on daily egg weight, average egg weight, daily egg number, laying rate, average daily feed intake, and the ratio of feed to egg in laying ducks, but markedly enhanced shell strength and yolk coloration. This aligns with previous reports where supplementation of Mulberry leaf extract (rich in flavonoids such as rutin and phenolic acids like chlorogenic acid) in Lohmann Silber layers diet showed no adverse effects on production performance, yet significantly improved yolk pigmentation [24]. In contrast, Chen, et al. [1] and Feng, et al. [30] reported that dietary supplementation with honeycomb extracts (rich in flavonoids such as quercetin and phenolic acids like caffeic acid.) and Eucommia ulmoides leaf powder (rich in phenolic acids such as chlorogenic acid) in laying ducks have no significant improvements in laying performance and egg quality. Similarly, in the study by Iskender et al. [31], it was observed that no significant differences in laying performance and eggshell quality, in response to dietary supplementation with hesperidin, naringin and quercetin (All belong to the flavonoids). These discrepancies might be attributed to variations in extracts types, poultry breeds and diets. Given the limited existing research on FDRM in laying duck nutrition, further mechanistic investigations are warranted to elucidate its functional properties.

Duck eggs, containing abundant protein and amino acids, fatty acids, minerals and vitamins, serve as an excellent source of essential nutrients for human food and health. The primary indicators for assessing their nutritional value and sensory quality typically encompass amino acid composition and fatty acid profiles [1,21]. As fundamental building blocks of life, essential amino acids such as lysine, methionine, threonine, and phenylalanine not only play critical roles in regulating lipid and protein metabolism but also constitute indispensable nutrients that cannot be endogenously synthesized by animals and must be supplemented through dietary intake [32]. This study revealed that compared to the control group, the 2% FDRM-supplemented group significantly increased the contents of total amino acids (by 9.27%), total essential amino acids (by 9.34%), and umami amino acids (by 7.62%) in egg yolks, confirming the beneficial effect of FDRM on the nutritional value of duck eggs. The potential underlying mechanisms may involve enhancing antioxidant capacity and modulating the expression of genes related to amino acid metabolism [33]. Notably, Yao et al. [34] also reported that sea buckthorn extract rich in flavonoid such as quercetin significantly improved the contents of total amino acids, essential amino acids, and umami amino acids in eggs through a similar mechanism. Nevertheless, current research remains insufficient in identifying the specific bioactive components within FDRM and their molecular targets, which represents a critical focus for future investigations.

Accumulating evidence highlights the dual implications of fatty acid intake on human health. Scientific evidence indicated that high intake of saturated fatty acids is associated with elevated risks of type 2 diabetes and cardiovascular disorders, while monounsaturated and polyunsaturated fatty acids demonstrate various protective health effects, including anti-inflammatory effects, regulation of glucose and lipid metabolism, and promotion of muscle growth [26,35]. In the present trial, adding FDRM in laying duck diet led to an increase in the ratio of unsaturated to saturated fatty acids (UFAs:SFAs) and a decrease in total SFAs in egg yolks, with the 2% FDRM group showing a pronounced decrease in total SFAs. Researches conducted by Zhang et al. [20]and Zhang et al. [21] revealed that supplementing laying hen diets with Fagopyrum dibotrys stems and leaves or their extracts could improve the amino acid composition and content of whole eggs while significantly increasing the levels of C20:4 and C22:6 in egg yolks. Furthermore, Chen et al. [1] also reported that the contents of total unsaturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids in duck eggs showed an increasing trend with the dietary supplementation level of honeycomb extracts, while the total saturated fatty acids content decreased significantly. Our findings are in full accordance with these previously established conclusions. The potential mechanism is that the polyphenols in FDRM scavenge ROS, protecting PUFAs from oxidative degradation and thereby reducing lipid peroxidation [36]. In addition, the flavonoids in Fagopyrum dibotrys enhance fatty acid elongation, thereby increasing the deposition of PUFAs in egg yolks [30].

Serum biochemical parameters serve as critical indicators for assessing metabolic status and health conditions in animals, primarily encompassing serum enzymes, protein and lipid metabolites. Serum TP, composed of ALB and GLB, reflects protein absorption and metabolism in the body. Elevated TP levels indicate enhanced protein metabolism and immune competence [37,38,39]. The experimental data confirmed that FDRM supplementation led to a marked rise in serum TP concentration, aligning with findings reported by Chen, et al. [38] and Zhang, et al. [21]. Serum BUN levels serve as an indicator of protein and amino acid utilization, with decreased concentrations suggesting favorable amino acids balance [40]. Tan, et al. [39] discovered that dietary Fagopyrum dibotrys supplementation with 1% in broilers decreased the serum BUN levels. However, this study revealed that the 3% FDRM supplementation group significantly elevated the serum BUN content compared to the control group, diverging from the aforementioned findings. This discrepancy suggested that dietary FDRM supplementation should not exceed 3%, as higher levels may compromise protein utilization efficiency.

5. Conclusions

This study found that adding 2% FDRM to the diet of Shanma laying ducks could improve the shell strength, yolk color and shell thickness in duck egg. Additionally, it improved the fatty acid profile, increased the levels of total amino acids, essential amino acids and umami amino acids in egg yolks. Concurrently, elevated the serum total protein levels indicated augmented physiological processes related to protein synthesis. These modifications suggested that 2% FDRM had a potential improvement in egg quality and egg nutritional value, with no negative impact on laying performance and health status of Shanma laying ducks. Under the conditions of this experiment, FDRM could be effectively utilized as a phytogenic feed additive in Shanma laying duck diets.