Effects of marine-derived Enterococcus durans YQ-6 on growth performance, intestinal health and microbial community of Leizhou black ducks

Abstract

Under the dual influence of antibiotic restriction policies in animal husbandry, marine-derived Enterococcus durans YQ-6 has been considered a promising alternative feed additive. A total of 180 one-day-old male Leizhou black ducks were randomly allocated into five groups: a blank control, three YQ-6 dose groups (1 × 10⁷, 1 × 10⁸, 1 × 10⁹ CFU/kg), and a Lactobacillus acidophilus control group, with six replicates per group. The trial lasted 42 days. The results showed that YQ-6 could significantly increase the body weight and average daily gain of Leizhou black ducks (P < 0.05) and reduce the feed conversion ratio. The high dose group (M3) significantly increased the dressing percentage, breast muscle rate and leg muscle rate (P < 0.05), decreased the sheer force and b value of breast muscle and optimized the amino acid composition of muscle, the contents of l-aspartic Acid, alanine and l-Phenylalanine were significantly increased (P < 0.05). In addition, YQ-6 could improve intestinal morphology, increase the ratio of villus height to crypt depth, enhance digestion and absorption capacity and regulate the structure of intestinal flora, such as increasing the relative abundance and proportion of Firmicutes and Bacteroides. In conclusion, dietary YQ-6 supplementation, especially at a dose of 1 × 10⁹ CFU/kg, can effectively promote the growth, improve meat quality and enhance intestinal health of Leizhou black ducks.

Introduction

The enforcement of international regulations restricting antibiotic use in livestock and poultry necessitates the advancement of sustainable alternatives aimed at enhancing production efficiency while maintaining food safety and promoting animal welfare (Abouelezz et al., 2019). Probiotics, as functional microbial agents, form a biological barrier within the host gastrointestinal tract via a dose-dependent colonization process. Their advantageous effects extend to various health outcomes, such as modulation of the intestinal microbiota, prevention of infectious diseases and preservation of mucosal barrier integrity (Suez et al., 2019; Williams, 2010). The mechanism of action of probiotics mainly includes reducing the pH value of the intestinal lumen, competing with harmful bacteria for nutrient resources, secreting antibacterial substances (such as bacteriocins), inhibiting the adhesion of harmful bacteria and epithelial cell shedding, so as to effectively maintain the balance of intestinal flora (Indira et al., 2019; Yan and Polk, 2020). In the domain of animal husbandry, probiotics-defined as live microorganisms that confer health benefits to the host-have emerged as a significant alternative to antibiotics and a means to enhance production efficiency in animal farming. Through the modulation of intestinal microecological equilibrium and metabolic activities, these agents exhibit dual advantages in enhancing both the yield and quality of animal-derived products (Kogut and Arsenault, 2016). Lactobacillus acidophilus, recognized is one of the most extensively studied and widely used probiotics, recognized as GRAS by the FDA and multiple international agencies (Gibson et al., 2017). When evaluating the probiotic potential or safety of a new strain (such as Enterococcus), Lactobacillus acidophilus is often used as the benchmark for comparison (Ghatani et al., 2022). As a mature feed microorganism, it effectively promotes animal growth and development, enhances immune function, and regulates the diversity of the intestinal microbiota (Wu et al., 2021).

Enterococcus is a Gram-positive facultative anaerobic bacterium commonly found throughout the gastrointestinal tracts of both humans and animals (Ramakrishnan et al., 2015). In the practice of livestock production, it is regarded as a potential alternative to antibiotics and can be used in the research and development of feed additives. By optimizing the structure of intestinal microflora, Enterococcus preparation can improve the digestion and absorption efficiency of nutrients, maintain the biological effect of the stability of intestinal microbial community structure and ultimately improve the growth performance of animals (Dempsey and Corr, 2022). Enterococcus durans YQ-6, a probiotic strain exhibiting high activity, was isolated from the intestinal tract of the yellow croaker (Larimichthys polyactis) inhabiting the South China Sea. Its distinctive adaptability to marine environments endows it with robust acid tolerance and resistance to bile salts, indicating considerable potential for modulating intestinal health in livestock and poultry (Prichula et al., 2021). YQ-6 also exhibits antibacterial properties. Li et al. (Li et al., 2023a) isolated a novel class IId bacteriocin, CAMT6, from YQ-6. The culture filtrate of this strain demonstrates a broad spectrum of activity, targeting both Gram-positive and Gram-negative bacteria while also inhibiting fungi. Studies have demonstrated that YQ-6 significantly enhances egg production rates and egg quality in poultry. This improvement is attributed to its ability to modulate the intestinal microbiota balance, increase nutrient absorption efficiency and promote the secretion of digestive enzymes and antimicrobial peptides. It also markedly enhances growth performance in meat poultry, decreases the feed-to-meat conversion ratio and optimizes muscle protein accumulation and fat distribution, thereby ultimately improving the flavor and nutritional quality of the meat (Ben Braïek and Smaoui, 2019; Hussain et al., 2025; Li et al., 2023a). The utilization of this strain offers novel microbial resources for environmentally sustainable livestock farming, playing a crucial role in decreasing reliance on antibiotics and promoting sustainable production practices.

In the rearing of meat ducks, key growth indicators such as body weight, daily gain, feed intake and feed conversion rate are important basis for evaluating the growth process and regularity, the slaughter rate, breast muscle rate, leg muscle rate and other slaughter performance indicators directly reflect the quality of carcass, which is the key basis for breeding (Bai et al., 2022; Kokoszyński et al., 2019). Meat quality assessment covers multiple dimensions such as meat color, pH, water holding capacity and shear force, which are essential to measure the economic value of meat ducks. The nutritional composition of meat mainly includes moisture, protein, fat and collagen and the content and proportion of amino acids in muscle are important indicators to evaluate the nutritional value and flavor contribution of poultry meat (Huo et al., 2021). The absorption of nutrients mainly occurs in the small intestine. Villi and microvilli greatly increase the absorption surface area and crypts are responsible for secreting digestive enzymes to promote food decomposition. Villi height and crypt depth complement each other, together they affect and ensure the overall absorption efficiency of the small intestine. In addition, the diversity of the intestinal microbial community is an important indicator to measure the stability of the microbiota and Enterococcus can also improve the intestinal barrier function and nutrient metabolism efficiency, significantly improve animal products (such as meat, eggs, etc.) output indicators (Anee et al., 2021; Cani et al., 2019). In meat production, it promotes weight gain and muscle protein deposition. Elke Albrecht et al. (Albrecht et al., 2022) fed Cobb 500 roosters Enterococcus faecium AL41 and found robust weight and pectoral muscle gain during the experiment. In terms of egg quality, Uchechukwu Edna Obianwuna et al. (Obianwuna et al., 2022)found that supplementation of poultry diets with probiotics enhanced eggshell structural integrity and improved egg white quality. As a green and safe natural feed additive, probiotics can realize the synergistic improvement of animal product yield and nutritional value.

Enterococcus has garnered significant interest owing to its advantageous attributes, including the promotion of growth, enhancement of immune function and regulation of intestinal microecological homeostasis in animals. However, investigations into the utilization of marine-derived Enterococcus durans strains within meat duck production systems are still comparatively limited. This study aims to investigate the probiotic functional potential of the isolate YQ-6, and its systematic safety assessment will be a critical step in subsequent translational research. The Leizhou black duck, a regionally distinctive breed of waterfowl, is highly regarded by consumers due to its tender meat and distinctive flavor (Zhen et al., 2022). Consequently, this study utilized the Leizhou black duck as the experimental model to systematically examine the overall impact of the marine-derived Enterococcus durans YQ-6 on the growth performance and intestinal health of Leizhou black ducks. The findings aim to establish a scientific foundation for the application of Enterococcus durans in poultry production. This initiative is anticipated to enhance both the safety and quality of livestock products, while simultaneously advancing the development of local specialty agricultural sectors.

Materials and methods

Experimental strain

The experimental strain employed in this study was Enterococcus durans YQ-6, a marine-derived isolate obtained from yellow croaker specimens collected in the South China Sea. This strain has been deposited at the Guangdong Microbial Culture Collection Center (GDMCC66265). YQ-6 was inoculated into the culture medium (1% yeast extract, 2% glucose, 2% peptone, 2% sodium chloride) and cultured at 37°C on a shaker at 120 rpm for 48 hours. Collect the culture, centrifuge (conditions: 4°C, 10,000 rpm, 10 min), discard the supernatant, wash twice with sterile physiological saline, and prepare a bacterial suspension. Determine the viable cell count of the suspension using the dilute plate method, then adjust to the required concentration as needed for the experiment.

Lactobacillus acidophilus (BNCC185342) was obtained from Beina Biotechnology Co., Ltd. and served as the positive control in this experiment. The dosage of Lactobacillus acidophilus is 1 × 10⁹ CFU/kg. This dosage was established based on the national standard GB 7300.504-2023 approved by the Ministry of Agriculture and Rural Affairs, as well as multiple benchmark studies (Liu et al., 2021) (Hatami et al., 2022). The research team previously established a positive control system using a 1 × 10⁹ CFU/kg dosage of Lactobacillus acidophilus (Wu et al., 2025). According to probiotic research methodologies, the effective starting dose of Enterococcus probiotics demonstrates direct comparability and safety. Lactobacillus acidophilus was inoculated into MRS medium and anaerobically cultured at 37°C on a shaker at 180 rpm for 24 hours. Collect the culture, centrifuge (conditions: 4°C, 8,000 rpm, 10 min), discard the supernatant, resuspend in sterile physiological saline, wash twice, and prepare a bacterial suspension. Calculate the viable cell count using the dilution plate method, then adjust to the required concentration as needed for the experiment.

Experimental animals and grouping methodology

The study involved 180 healthy male Lei Zhou black ducks, each one day old and of comparable weight. These ducks were randomly assigned to five groups, with each group comprising six replicates and each replicate containing six Lei Zhou black ducks. The control group received a basal diet, whereas the experimental groups were administered the basal diet supplemented with durable Enterococcus YQ-6 at concentrations of 1 × 10⁷ CFU/kg, 1 × 10⁸ CFU/kg and 1 × 10⁹ CFU/kg, respectively. These groups were designated as the Control group, M1 (1 × 10⁷ CFU/kg), M2 (1 × 10⁸ CFU/kg) and M3 (1 × 10⁹ CFU/kg). Furthermore, the positive control group received a basal diet supplemented with Lactobacillus acidophilus at a concentration of 1 × 10⁹ CFU/kg, designated as the LA (1 × 10⁹ CFU/kg LA) group, for a duration of 42 days.

Nutritional management and fundamental feed components

Before the experiment, the duck house and its related instruments were thoroughly cleaned and disinfected, followed by ventilation and air purification to ensure the suitability of the test environment. During the experiment, Leizhou Black Ducks were reared in the indoor grid pattern and fed with mixed feed. The Ducks ate and drank freely. At the same time, the temperature, humidity and light conditions in the duck house were strictly controlled. The foundational diet is formulated in accordance with the Chinese national guideline “Feeding Standards for Meat Ducks” (NY/T 2122-2012), with its composition and nutritional specifications detailed in Table 1.

Tablet 1.

Composition and nutrient levels of basal diets (air-dry basis) %.

Items	1-21 days	22-42 days	Items	1-21 days	22-42 days
Ingredients			Nutrition levels
Corn	61.85	64.60	ME/(MJ/kg)	12.24	12.35
Soybean meal	28.33	25.30	CP	20.70	18.6
Wheat bran	3.25	3.14	Ca	0.98	0.93
Fishmeal	2.50	2.80	AP	0.45	0.40
NaCl	0.30	0.30	Lys	1.10	0.86
Limestone	1.30	1.29	Met	0.50	0.45
CaHPO4	1.28	1.26
DL-Met	0.19	0.21
Premix1	1.00	1.10
Total	100.00	100.00

¹The premix supplied the following nutrients per kilogram of starter feed: Vitamin A (8, 000 IU), Vitamin B (12 mg), Vitamin D (2, 000 IU), Vitamin E (20 IU), Vitamin K3 (2 mg), Nicotinic acid (40 mg), d-pantothenic acid (15 mg), Folic acid (1 mg), Iron (60 mg), Copper (8 mg), Zinc (60 mg), Manganese (100 mg) and Selenium (0. 35 mg).

Growth performance evaluation

On the mornings of days 1, 7, 14, 21, 28, 35 and 42 during the formal experimental period, the Leizhou black ducks, following a fasting protocol, were weighed and their body weights documented. Concurrently, daily feed intake was recorded. Subsequently, the average body weight (BW), average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) were calculated.

$$\text{BW}\left(\mathrm{g}\right)=\text{Total}\text{Weight}/\text{Number}\text{of}\text{Ducks}$$

$$\text{ADFI}\left(\mathrm{g}/\mathrm{d}\right)=\text{Material}\text{consumption}\text{during}\text{the}\text{test}\text{period}/\text{Number}\text{of}\text{test}\text{days}$$

$$\text{ADG}\left(\mathrm{g}/\mathrm{d}\right)=\left(\text{Final}\text{average}\text{weight}-\text{Initial}\text{average}\text{weight}\right)/\text{Number}\text{of}\text{days}\text{in}\text{the}\text{trial}\text{period}$$

$$\text{FCR}=\text{ADFI}/\text{ADG}$$

Slaughtering performance

Subject the animals to a 12-hour fasting period prior to slaughter. On day 42 of the experimental timeline, select two Leizhou black ducks exhibiting comparable body weight and health conditions from each replicate group to record their pre-slaughter weights. The experimental ducks were subsequently exsanguinated through the jugular vein, weighed to determine carcass weight and dissected. Bilateral breast and leg muscles were carefully isolated and collected. In accordance with the agricultural industry standard NY/T 823-202, the following parameters were measured and calculated: slaughter yield, semi-eviscerated yield, fully eviscerated yield, breast muscle yield and leg muscle yield.

$$\text{Slaughter}\text{yield}(\%)=\text{Slaughter}\text{weight}/\text{Pre}-\text{slaughter}\text{weight}\times 100$$

$$\text{Semi}-\text{trimming}\text{rate}(\%)=\text{Semi}-\text{trimming}\text{weight}/\text{Pre}-\text{slaughter}\text{weight}\times 100$$

$$\text{Total}\text{Net}\text{Carcass}\text{Yield}(\%)=\text{Total}\text{Net}\text{Carcass}\text{Weight}/\text{Pre}-\text{slaughter}\text{Body}\text{Weight}\times 100$$

$$\text{Chest}\text{Muscle}\text{Percentage}(\%)=\text{Weight}\text{of}\text{Both}\text{Chest}\text{Muscles}/\text{Total}\text{Cleaned}\text{Carcass}\text{Weight}\times 100$$

$$\text{Leg}\text{Muscle}\text{Percentage}(\%)=\text{Weight}\text{of}\text{Both}\text{Leg}\text{Muscles}/\text{Total}\text{Net}\text{Carcass}\text{Weight}\times 100$$

Assessment of meat quality parameters

Samples of the left breast and thigh muscles were collected from 42-day-old Leizhou black ducks. Subsequently, measurements were conducted to assess pH, meat color, water loss rate, cooking loss rate and shear force. The specific testing parameters are detailed as follows:

• Meat Color: The colorimeter probe should be placed perpendicularly on the cross-sectional surface of the muscle to determine the lightness (L*), redness (a*) and yellowness (b*) values. Measurements should be recorded after stabilization of the readings, with each measurement repeated three times to ensure accuracy.

• pH45min: At 45 to 60 minutes post-mortem, collect samples from three separate sites on both the breast and leg muscles. Insert the pH probe into each sample to obtain pH measurements, recording the value once it reaches stabilization. Compute the mean pH value from the three measurements to determine the final pH at 45 minutes.

• pH24h: Meat samples should be stored at a refrigeration temperature of 4°C. After 24 hours post-slaughter, the samples are to be retrieved and their pH values at 24 hours (pH24h) should be measured employing the same methodology utilized for determining pH at 45 minutes post-slaughter (pH45min).

• Water loss rate: Utilizing a sharp circular sampler, obtain two meat specimens, each measuring 1 cm in thickness and approximately 2.5 cm in diameter, from the breast and leg muscles. Weigh these samples and document the initial mass as m1. Subsequently, position the meat specimens on a compression platform, ensuring that each is sandwiched between 16 layers of qualitative filter paper both above and below. Apply a pressure of 35 kg for duration of 5 minutes. Following compression, reweigh the samples and record the resulting mass as m2.

  $$\text{Water}\text{loss}\text{rate}(\%)=\left(\mathrm{m}1-\mathrm{m}2\right)/\mathrm{m}1\times 100\%.$$
• Cooking loss: Determine the cooking loss of breast and leg muscle samples stored at 4°C for a duration of up to 24 hours post-mortem. Prepare the meat specimens by trimming them into rectangular prisms aligned with the muscle fiber orientation and record the initial weight as m3. Subsequently, enclose each sample in a resalable plastic bag and immerse it in a water bath maintained at 80°C, heating until the internal temperature attains 75°C After heating, remove the samples, allow them to cool to ambient temperature, gently blot surface moisture using filter paper and weigh the samples again, documenting this final weight as m4.

  $$\text{Steaming}\text{loss}(\%)=\left(\mathrm{m}3-\mathrm{m}4\right)/\mathrm{m}3\times 100\%$$

• Shear force: The sheer force of each cooked meat sample was measured using a meat tenderness tester. Each sample underwent two measurements and the mean value was subsequently calculated.

Evaluation of the conventional nutrient profile in muscle tissue

Nutrient Composition of Breast and Leg Muscles in Leizhou black ducks Following 42 Days of Surface Fat and Fascia Removal: Analysis of Moisture, Crude Ash, Crude Fat and Crude Protein Levels. Moisture content shall be measured employing the direct drying method outlined in the national standard GB 5009.3-2016. Crude fat content shall be assessed using the Soxhlet extraction technique as prescribed by the national standard GB 5009.6-2016. Crude protein content shall be quantified via the Kjeldahl method in accordance with the national standard GB 5009.5-2016. Crude ash content shall be determined following the procedures specified in the national standard GB 5009.4-2016.

Analysis of amino acid composition in muscle tissue

Following the homogenization of muscle tissue from 42-day-old Leizhou black ducks into a paste, the fatty acid and amino acid compositions of the duck muscle were analyzed. The amino acid quantification procedure adhered to the national standard GB 5009.124-2016. Amino acid content in the muscle samples was determined using a fully automated amino acid analyzer. Concentrations of individual amino acids were assessed by comparing the retention times and peak areas with those of established amino acid reference standards.

Histological analysis of intestinal tissue morphology

Intestinal segments obtained at 42 days were fixed in a 4% paraformaldehyde solution for a minimum duration of 72 hours. Subsequently, using a surgical blade, the segments were trimmed into lengths of approximately 2–3 mm, ensuring that both ends of each cross-section were parallel and smooth. The tissue samples then underwent pre-embedding processing and were placed into embedding cassettes. Following completion of the embedding procedure, paraffin blocks were sectioned at a consistent speed using a microtome, producing sections with a thickness of 5 μm for hematoxylin and eosin (H&E) staining. Post-staining, the slides were subjected to drying, hardening and mounting prior to microscopic examination. Villus height (VH) and crypt depth (CD) were measured and the villus height to crypt depth ratio (V/C) was subsequently calculated.

Analysis of intestinal microbiota structure

On the 42nd day of the experimental period, two Leizhou black ducks with comparable body weights were chosen from each replicate group for euthanasia. The cecal contents were aseptically harvested and transferred into sterile centrifuge tubes, promptly flash-frozen using liquid nitrogen and subsequently stored at −80°C for further analysis.

The procedure commenced with the extraction of total DNA from the microbiome, followed by PCR amplification of the target fragments. Subsequently, the amplification products were recovered and purified and quantitative fluorescent analysis was conducted. Sequencing was then carried out utilizing the PacBio Sequel platform. For sequence denoising, the QIIME2 dada2 analysis pipeline was applied. Sequences with abundance below five were excluded, resulting in the final amplicon sequence variants (ASVs) and feature table, which were used to analyze the microbial community present in the cecal contents of Leizhou black ducks.

Data analysis

A univariate analysis of variance was conducted utilizing SPSS version 26.0 statistical software, subsequently followed by Duncan's multiple range test. Statistical significance was defined at a threshold of P < 0.05, while results with P < 0.01 were regarded as highly significant. Results are expressed as mean±standard deviation, with graphical representations generated using GraphPad Prism 8 software.

To explore the correlation between intestinal microbiota (taxonomic level from phylum to genus) and the growth performance, meat quality, flavor compounds and intestinal health of Leizhou Black Duck, the study was conducted; Spearman's rank correlation analysis was conducted utilizing the OmicStudio online platform (https://www.omicstudio.cn/tool). Statistical significance was defined as P < 0.05, while values within the range of 0.05≤P < 0.10 were considered indicative of a notable trend.

Result

Impact of marine-derived enterococcus durans YQ-6 on the growth performance and slaughter characteristics of Leizhou black ducks

At different developmental time points (14,21,28,35,42d), as shown in Table 2, the average body weight of each treatment group (M1, M2, M3, LA) was significantly higher than that of the control group. Especially on the 35th and 42nd day, the average body weight of M3 group was significantly higher than that of other groups (P < 0.05). No statistically significant difference was observed in the average daily feed intake between the treatment groups and the control group (P > 0.05). The mean daily weight gain observed across all treatment groups exceeded that of the control group, with this difference being especially pronounced during the intervals spanning 21 to 28 days and 28 to 35 days. The M3 group exhibited a significantly greater weight gain compared to the other groups (P < 0.05). During the intervals of 7–14 days and 21–28 days, the feed conversion ratio in the M3 group was significantly lower than that observed in the control group and the other treatment groups (P < 0.05). At all other time points, no significant differences in feed conversion ratios were detected among the treatment groups relative to the control group (P > 0.05).

Table 2.

Impact of the Marine-Derived Enterococcus durans YQ-6 on the growth performance of Leizhou black ducks.

	Control			M1		M2	M3		LA
BW (g)
14d	701. 33±19. 62			723. 33±20. 53		749. 24±22. 45	766. 07±23. 04		747. 08±16. 72
21d	856. 67±26. 63b			905. 46±33. 29a		911. 35±22. 01ab	968. 19±15. 72a		915. 24±27. 62ab
28d	1070. 58±22. 25b			1183. 62±42. 88a		1197. 14±41. 94a	1257. 39±40. 89a		1211. 32±46. 42a
35d	1438. 59±18. 90c			1491. 01±40. 36bc		1537. 62±40. 72bc	1710. 16±47. 39a		1583. 64±36. 97b
42d	1940. 15±38. 05c			1982. 67±44. 84bc		2058. 43±48. 38b	2200. 51±38. 69a		2041. 29±56. 45bc
ADFI (g/d)
7-14d	88. 76±1. 90ab			84. 47±1. 68a		87. 03±2. 88abc	89. 99±2. 73a		84. 72±1. 22bc
14-21d	111. 98±3. 36b			117. 20±2. 21ab		114. 62±2. 96ab	112. 22±2. 52b		118. 92±1. 04a
21-28d	193. 34±3. 08a			186. 35±4. 31ab		188. 78±3. 33ab	191. 66±3. 55ab		185. 99±3. 68b
28-35d	228. 60±4. 18			230. 02±4. 97		234. 90±2. 34	231. 20±3. 82		233. 99±4. 87
35-42d	234. 11±2. 94			238. 43±3. 86		237. 91±5. 68	242. 23±3. 12		236. 15±5. 39
ADG (g/d)
7-14d	19. 86±0. 89			19. 77±1. 23		20. 29±2. 23	21. 68±1. 26		22. 43±1. 62
14-21d	22. 33±1. 44b			25. 32±1. 68b		24. 38±1. 44b	28. 85±1. 99a		24. 76±1. 93b
21-28d	35. 24±1. 49d			36. 67±1. 64cd		38. 71±1. 86bc	42. 29±1. 19a		39. 52±2. 46b
28-35d	52. 57±1. 94c			54. 19±2. 52bc		55. 24±3. 06bc	64. 81±1. 21a		56. 57±1. 3b
35-42d	65. 05±2. 42c			68. 67±1. 54bc		70. 24±1. 05ab	74. 43±3. 79a		71. 24±1. 72ab
FCR
7-14d	4. 47±0. 13a			4. 39±0. 56b		4. 19±0. 39bc	3. 88±0. 15c		4. 16±0. 16bc
14-21d	5. 03±0. 38a			4. 97±0. 27a		4. 90±0. 41a	3. 90±0. 18b		4. 97±0. 12a
21-28d	5. 49±0. 16a			5. 01±0. 21b		4. 87±0. 34bc	4. 53±0. 28c		4. 76±0. 28abc
28-35d	4. 35±0. 14a			4. 33±0. 25a		4. 17±0. 29a	3. 57±0. 13b		4. 16±0. 17a
35-42d	3. 60±0. 12a			3. 44±0. 10ab		3. 40±0. 09ab	3. 26±0. 14b		3. 34±0. 16b

Values accompanied by different lowercase letters on the shoulder scale denote statistically significant differences (P < 0.05), whereas those with different uppercase letters indicate highly significant differences (P < 0.01). Identical or absent lowercase letters signify non-significant differences (P > 0.05). This notation applies consistently to the tables below.

This study evaluated multiple slaughter performance parameters of Leizhou black ducks, with the findings presented in Table 3. Compared to the control group, the M3 group exhibited a statistically significant increase in dressing percentage (P < 0.05). Additionally, both the semi-gutted and fully gutted rates in the M3 group were significantly higher than those observed in the control group (P < 0.05). Furthermore, the breast muscle rate and leg muscle rate of the M3 group were also significantly elevated relative to the control group (P < 0.05).

Table 3.

Impact of Marine-Derived Enterococcus durans YQ-6 on the slaughter performance of Leizhou black ducks (%).

	Control	M1	M2	M3	LA
Slaughter yield (%)	80. 81±1. 96c	83. 30±1. 01b	83. 86±0. 95b	86. 18±1. 17a	83. 40±1. 35b
Semi-trimming rate (%)	71. 63±0. 53b	76. 23±1. 31ab	76. 81±1. 22ab	78. 69±0. 86a	76. 28±0. 78ab
Total Net Carcass Yield (%)	66. 27±0. 90b	71. 09±0. 93a	71. 19±0. 83a	72. 30±0. 71a	71. 26±1. 19a
Chest Muscle Percentage (%)	6. 96±0. 64b	7. 24±0. 58	7. 58±0. 75	8. 64±0. 38a	7. 90±0. 61
Leg Muscle Percentage (%)	9. 12±0. 34b	9. 40±0. 35	9. 49±0. 62	10. 84±0. 64a	9. 34±0. 64b

Impact of marine-derived enterococcus durans YQ-6 on the muscle quality, conventional nutritional components and muscle amino acids of Leizhou black ducks

The impact of YQ-6 on muscle quality and nutritional composition was evaluated by assessing key parameters in the breast and leg muscles of Leizhou black ducks. The findings, presented in Table 4, indicate that, relative to the control group, the b-value of the breast muscle was significantly decreased in the four experimental groups (P < 0.05). Furthermore, the M3 group exhibited a significant reduction in both shear force and fat content in the breast and leg muscles (P < 0.05). Although an increasing supplementation level of the probiotic Enterococcus YQ-6 corresponded with a downward trend in the dehydration rate, this change did not reach statistical significance.

Table 4.

Impact of Marine-Derived Enterococcus durans YQ-6 on the muscle quality and nutritional composition of Leizhou black ducks.

	Control	M1	M2	M3	LA
Chest muscle
pH45min	6. 03±0. 03	6. 40±0. 10	6. 09±0. 12	6. 22±0. 04	6. 21±0. 04
pH24h	5. 95±0. 11	5. 86±0. 05	6. 07±0. 06	5. 9 ± 0. 02	5. 88±0. 06
L*	48. 80±0. 49	47. 93±0. 54	47. 20±0. 95	48. 02±0. 32	47. 57±0. 61
a*	12. 05±0. 36	10. 57±0. 67	11. 37±1. 18	10. 57±0. 38	11. 07±0. 49
b*	15. 49±0. 40a	12. 40±1. 27b	11. 83±0. 37b	11. 52±0. 89b	11. 9 ± 0. 90b
Water loss rate (%)	31. 13±2. 23	30. 50±0. 59	31. 57±1. 07	28. 75±1. 02	29. 19±0. 82
Cooking loss (%)	68. 40±1. 67	69. 96±4. 05	67. 40±2. 72	75. 97±3. 38	73. 88±3. 69
Shear force	38. 36±0. 98a	34. 34±0. 18b	34. 37±1. 92b	32. 59±0. 90b	36. 07±0. 57ab
moisture	71. 06±1. 48	69. 07±2. 91	72. 75±0. 97	72. 79±0. 40	72. 28±1. 10
crude ash	1. 27±0. 31	1. 23±0. 19	1. 28±0. 36	1. 15±0. 24	1. 41±0. 06
crude fat	15. 57±0. 43a	15. 25±0. 39ab	15. 53±049bc	12. 59±0. 70c	14. 85±0. 76ab
crude protein	18. 52±0. 44	18. 40±0. 82	18. 84±0. 97	17. 53±1. 69	15. 53±1. 41
Leg muscle
pH45min	6. 16±0. 04	6. 20±0. 09	6. 26±0. 05	6. 17±0. 03	6. 06±0. 06
pH24h	5. 98±0. 09	6. 05±0. 12	6. 21±0. 07	6. 11±0. 03	6. 05±0. 14
L*	45. 57±0. 77	47. 23±1. 51	45. 31±0. 36	46. 79±0. 60	46. 2 ± 0. 59
a*	11. 83±0. 28	10. 69±0. 47	11. 41±0. 75	10. 57±0. 35	10. 57±0. 58
b*	13. 53±0. 26	14. 60±0. 59	13. 94±0. 46	14. 11±0. 46	14. 23±0. 28
Water loss rate (%)	32. 00±2. 26	29. 59±1. 07	28. 98±0. 80	29. 04±0. 74	27. 93±0. 92
Cooking loss (%)	77. 03±4. 85	69. 80±1. 41	73. 51±0. 56	76. 29±4. 6	74. 67±1. 71
Shear force	44. 01±0. 83a	42. 05±0. 71ab	40. 69±0. 44b	40. 10±0. 64b	42. 63±0. 98ab
moisture	65. 72±1. 44	69. 70±1. 70	69. 39±0. 89	68. 94±1. 12	68. 86±1. 59
crude ash	1. 46±0. 25	1. 24±0. 48	1. 16±0. 10	1. 23±0. 51	1. 15±0. 36
crude fat	15. 57±0. 43a	15. 25±0. 39ab	15. 53±049bc	12. 59±0. 70c	14. 85±0. 76ab
crude protein	15. 92±1. 60	15. 39±1. 02	17. 11±1. 23	17. 60±0. 90	18. 05±2. 14

A fully automated amino acid analyzer was employed to determine the amino acid composition in the muscle tissue of Leizhou black ducks, with the findings presented in Table 5 Compared to the control group, the M3 group demonstrated significantly elevated concentrations of aspartic acid, alanine, phenylalanine, essential amino acids and total amino acids in the thigh muscle (P < 0.05). Furthermore, although the levels of both umami amino acids (UAA) and essential amino acids (EAA) were higher across all M treatment groups relative to the control, these differences did not reach statistical significance (P > 0.05).

Table 5.

Impact of the Marine-Derived enterococcus durans YQ-6 on amino acid composition in the muscle tissue of Leizhou black ducks (%).

	Control	M1	M2	M3	LA
Asp1	1. 89±0. 07b	1. 91±0. 08b	1. 95±0. 08b	2. 21±0. 12a	2. 05±0. 04ab
Thr2	0. 98±0. 02	0. 90±0. 06	0. 87±0. 02	0. 99±0. 02	0. 91±0. 06
Ser	0. 87±0. 01	0. 81±0. 04	0. 80±0. 02	0. 87±0. 03	0. 80±0. 05
Glu1,2	3. 15±0. 06	3. 32±0. 27	3. 42±0. 13	3. 52±0. 05	3. 32±0. 30
Pro	3. 42±0. 13	3. 32±0. 27	3. 25±0. 06	3. 72±0. 05	3. 32±0. 30
Gly1,2	0. 77±0. 02	0. 79±0. 03	0. 81±0. 03	0. 92±0. 02	0. 86±0. 08
Ala1,2	1. 10±0. 03b	1. 27±0. 03b	1. 38±0. 05ab	1. 42±0. 01a	1. 31±0. 06ab
Val2	1. 08±0. 01ab	0. 95±0. 01c	0. 98±0. 03bc	1. 10±0. 05a	1. 01±0. 03abc
Met2	0. 55±0. 06	0. 46±0. 01	0. 49±0. 03	0. 51±0. 07	0. 55±0. 07
Ile2	0. 92±0. 03	0. 91±0. 03	0. 93±0. 02	0. 98±0. 03	0. 94±0. 01
Leu2	1. 62±0. 03	1. 60±0. 04	1. 63±0. 05	1. 86±0. 09	1. 68±0. 06
Tyr1	0. 86±0. 03	0. 75±0. 02	0. 81±0. 04	0. 91±0. 05	0. 80±0. 03
Phe1,2	0. 84±0. 01b	0. 96±0. 05b	0. 98±0. 02ab	1. 06±0. 07a	0. 97±0. 03ab
His	0. 98±0. 04	0. 85±0. 03	0. 85±0. 05	0. 94±0. 04	0. 85±0. 01
Lys2	1. 67±0. 03	1. 65±0. 04	1. 65±0. 07	1. 88±0. 08	1. 72±0. 02
Arg	1. 18±0. 03	1. 01±0. 02	1. 00±0. 02	1. 18±0. 08	1. 12±0. 03
UAA	8. 61±0. 22	9. 01±0. 18	9. 31±0. 20	10. 04±0. 09	9. 35±0. 33
EAA	11. 91±0. 11c	12. 02±0. 15b	12. 33±0. 23bc	13. 22±0. 22a	12. 41±0. 14ab
TFAA	21. 88±0. 66b	21. 46±0. 43b	21. 80±0. 20ab	24. 07±0. 81a	22. 21±0. 85ab

¹Umami amino acids.

²Essential amino acids.

Impact of the Marine-Derived Enterococcus durans YQ-6 on the intestinal architecture of Leizhou black ducks

Morphological examination of the intestinal structure as depicted in Fig. 1 reveals that the duodenum, jejunum and ileum across all groups’ exhibit intact villous architecture with well-defined layers and clearly distinguishable crypts, with no evident pathological alterations observed. Relative to the control group, administration of the marine-derived Enterococcus durans YQ-6 was associated with a denser villus arrangement and more well-preserved villus margins in the intestines of Leizhou black ducks, thereby contributing to enhanced preservation of intestinal morphological integrity.

Fig. 1.

Histomorphological analysis of the duodenum, jejunum and ileum in Leizhou black ducks.

The villus height (VH) and crypt depth (CD) of intestinal tissues in Leizhou black ducks were measured and the villus-to-crypt ratio (V/C) was calculated to assess the impact of dietary supplementation with marine-derived Enterococcus durans YQ-6, as shown in Table 6. Compared to the control group, supplementation with Enterococcus durans YQ-6 enhanced VH in both the duodenum and ileum, increased the V/C ratio across all intestinal segments and significantly decreased CD. The most pronounced effects (P < 0.01) were observed at a supplementation level of 1 × 10⁹ CFU/kg.

Table 6.

Impact of Marine-Derived Enterococcus durans YQ-6 on the intestinal architecture of Leizhou black ducks.

	Control	M1	M2	M3	LA
Duodenum
VH/μm	762. 00±10. 84c	777. 03±19. 60c	844. 71±17. 41b	889. 26±12. 29a	850. 18±14. 77ab
CD/μm	280. 15±5. 68a	248. 35±10. 14b	243. 06±8. 65b	240. 19±7. 89b	241. 25±12. 10b
V/C	2. 72±0. 12d	3. 13±0. 05c	3. 48±0. 06b	3. 71±0. 11a	3. 53±0. 07ab
Ileum
VH/μm	1018. 61±23. 26c	1100. 51±15. 97b	1149. 26±15. 18b	1241. 16±28. 06a	1114. 65±15. 89b
CD/μm	311. 88±12. 92a	299. 48±7. 52a	275. 93±16. 77b	252. 08±10. 35c	276. 03±9. 90b
V/C	3. 27±0. 13d	3. 68±0. 10c	4. 18±0. 18b	4. 93±0. 17a	4. 04±0. 14bc
Jejunum
VH/μm	949. 20±19. 64	934. 18±13. 07	975. 68±14. 57	980. 01±24. 77	998. 71±26. 14
CD/μm	323. 65±9. 13	313. 29±9. 56	301. 35±11. 73	297. 51±14. 81	309. 08±9. 32
V/C	2. 93±0. 06	2. 98±0. 05	3. 11±0. 07	3. 29±0. 07	3. 22±0. 09

Impact of the Marine-Derived enterococcus durans YQ-6 on the intestinal microbiota of Leizhou black ducks

The figure illustrates the quantity of applicant sequence variants (ASVs) that are common to, as well as distinct within, the five sample groups. Specifically, Fig. 2 indicates that 128 ASVs are shared across all five groups. Additionally, the control group, M1 group, M2 group, M3 group and LA group contain 252, 478, 500, 634 and 549 unique ASVs, respectively.

Fig. 2.

Venn diagram illustrating the cecal microbiota composition in Leizhou black ducks.

ASV refers to Amplicon Sequence Variant. Each colored segment in the Fig. denotes a distinct grouping. The intersecting regions between segments represent ASVs that are common to the respective groups. The numerical value displayed on each segment corresponds to the quantity of ASVs encompassed within that grouping.

The Shannon index serves as a measure of species diversity within a given sample, with higher values denoting increased diversity. As illustrated in Fig. 3A, no significant differences were observed in the Shannon indices across the five sample groups, suggesting comparable species diversity among them. Notably, the M3 group demonstrated the highest Shannon index, indicating the greatest species diversity within this cohort. As illustrated in Fig. 3B, the Chao1 index, which quantifies species richness, showed no significant variation among the five groups, implying that species richness was consistent across samples. The M3 group also exhibited the highest Chao1 index, further indicating that this group harbored the greatest species richness.

Fig. 3.

Analysis of alpha and beta diversity of cecal microbiota in Leizhou black ducks.

Fig. 3 illustrates the microbial community characteristics within the cecum of Leizhou black ducks. Panel A depicts the Shannon diversity index; Panel B presents the Chao1 richness estimator; and Panel C displays the results of the beta diversity analysis.

Principal coordinate analysis (PCoA) based on Bray-Curtis distance metrics, accompanied by scatter plot visualization, was employed to assess the β-diversity of bacterial communities. As illustrated in Fig. 3C, the composition of bacterial communities across the five groups demonstrated significant intergroup variation (R² = 0.1743, P = 0.004). Notably, there was no overlap observed between the LA group, the Control group and the M2/M3 groups, indicating distinct differences in bacterial community composition among these groups. Conversely, partial overlap was detected among the M1, M2 and M3 groups, suggesting a degree of similarity in their bacterial community structures. The Control group was positioned more distantly relative to the other groups, further underscoring its markedly different bacterial community composition. Statistical analysis of the scatter plots revealed significant differences in bacterial communities between the Control group and both the M2 and M3 groups (P < 0.05), as well as between the LA group and the M2 and M3 groups (P < 0.05).

Phylum-level, genus-level and current discriminant analysis of intestinal microbiota

The results of the 16S amplicon sequencing, as illustrated in Fig. 4, indicate that the duck intestinal microbiota comprises 3,549 amplicon sequence variants (ASVs) representing 12 distinct phyla. For visualization purposes, the phyla with the top 11 relative abundances in species composition were selected, while the remaining phyla were collectively categorized as “Other.” As illustrated in Fig. 4A, among others, the ten most abundant phyla identified were Firmicutes, Bacteroidota, Campylobacterota, Desulfobacterota, Proteobacteria, Deferribacterota, Actinobacteria, Spirochaetota, Cyanobacteria and Synergistota. As illustrated in Fig. 4B, relative to the control group, the relative abundance of Cyanobacteria was significantly elevated in the M2 and M3 groups (P < 0.05). Among these phyla, Firmicutes was identified as the predominant phylum within the intestinal microbiota of ducks.

Fig. 4.

Taxonomic composition and relative abundance at the phylum level in Leizhou black ducks.

The top 11 genera based on relative species abundance were selected for visualization in Fig. 5, while all other genera were grouped under the category “Other.” Among others, the ten genera with the highest relative abundance comprised Bacteroides, Faecalibacterium, Helicobacter, Streptococcus, Desulfovibrio, Uncultured, Prevotellaceae_UCG-001, Phascolarctobacterium, Clostridia_UCG-014 and Unclassified. As illustrated in Fig. 5B and 5C, compared to the Control group, the relative abundance of Desulfovibrio exhibited an increasing trend across all RM groups (P > 0.05), whereas the relative abundance of Streptococcus demonstrated a significant decreasing trend in all RM groups (P < 0.05).

Fig. 5.

Taxonomic composition and relative abundance at the genus level in Leizhou black ducks.

Linear Discriminant Analysis Effect Size (LEfSe) is a specialized analytical method developed to investigate and interpret variations in biological features-such as taxonomic classifications, metabolic pathways and genes-within complex, high-dimensional datasets. This approach facilitates comparative analyses across two or more groups and enables detailed examination of differences among subgroups within a given group. Consequently, LEfSe allows for the precise identification of biomarkers that demonstrate statistically significant differences between groups. In the present study, linear discriminant analysis was applied to genus-level gut microbiota data from five distinct groups to identify biomarkers capable of discriminating among these groups. Concurrently, bar charts were utilized to visualize classification units exhibiting Linear Discriminant Analysis (LDA) scores exceeding 2. The Linear Discriminant Analysis (LDA) score is employed to quantify the extent to which a taxonomic unit contributes to differences in gut microbiota composition between groups. A higher LDA score signifies a more substantial role of the respective taxonomic unit in distinguishing intergroup microbial variations. As illustrated in Fig. 6, in this study, significant differences were observed across taxonomic levels, including 1 phylum, 2 classes, 3 orders, 7 families, 10 genera and 4 species. These taxa were designated as biomarkers for group differentiation, as depicted in the accompanying figure. Specifically, within the M3 group, the following taxa were identified: 1 order (Monoglobales), 3 families (Monoglobaceae, Eggerthellaceae, Oxalobacteraceae), 5 genera (CHKCI001, NK4A214_group, Monoglobus, Eisenbergiella, Oxalobacter) and 1 species (Oxalobacter formigenes). In the M2 group, the taxa included 1 phylum (Deferribacterota), 1 class (Deferribacteres), 1 order (Deferribacterales), 2 families (Deferribacteraceae, Marinifilaceae) and 1 genus (Mucispirillum). The M1 group was characterized by 2 genera: CHKCI002 and Subdoligranulum. In the Control group, 1 genus and 1 species were identified: Enterococcus columbae and Butyricimonas, respectively. Lastly, the LA group exhibited 1 phylum (Spirochaetota), 1 class (Spirochaetia), 1 order (Spirochaetales), 2 families (Spirochaetaceae, Barnesiellaceae), 1 genus (Sphaerochaeta) and 1 species (Bacteroides sp.).

Figure. 6.

Multilevel discriminant analysis using LEfSe to identify species differences in Leizhou black ducks.

In summary, the M3 group exhibited the greatest quantity of identified biomarkers, whereas the Control group demonstrated the presence of only two biomarkers.

An analysis of the relationship between gut microbiota, meat quality, intestinal health, meat quality and flavor compounds in Leizhou black ducks

This study utilized OmicStudio to perform Spearman correlation analyses between biomarkers at the phylum and genus levels and various indicators of meat quality and gut health in Leizhou black ducks. The analysis identified a significant positive correlation between the phylum Bacteroidetes and crude protein content in the leg muscle, with a correlation coefficient of 0.584 (P < 0.05). Furthermore, species within the genus Campylobacter demonstrated a significant positive correlation with crude fat content in the breast muscle, exhibiting a correlation coefficient of 0.694 (P < 0.05) (Fig. 7A). At the genus level, Bacteroides was significantly positively correlated with crude protein in the leg muscle (r = 0.761, P < 0.05), while Helicobacter pylori showed a significant positive correlation with crude fat content in the breast muscle (r = 0.694, P < 0.05) (Fig. 7B). Notably, biomarkers at the portal level did not exhibit significant correlations with the crypt ratio in the duodenum, jejunum, or ileum (P > 0.05) (Fig. 7C). Additionally, the Prevotellaceae family member UCG-001 was significantly positively correlated with the jejunal crypt ratio, with a correlation coefficient of 0. 480 (P < 0.05) (Fig. 7D).

Fig. 7.

Spearman correlation analysis of intestinal microorganisms and meat performance and intestinal health status of Leizhou black duck at the phylum (A, C) and genus (B, D) levels. Meat performance: phylum (A) and genus (B); intestinal health status: phylum (C) and genus (D).

In the figure, JWH-V/C denotes the jejuni wool/hidden ratio (V/C) of the jejunum at 42 days, ILWH-V/C denotes the ileum wool/hidden ratio (V/C) of the ileum at 42 days and DVCR-V/C denotes the duodenal velvet/crypto-ratio (V/C) of the duodenum at 42 days.

Spearman correlation analysis demonstrated a statistically significant positive association between the phylum Phyllotectrum and the pH measured at 24 hours postmortem in the pectoral muscle, with a correlation coefficient of 0.674 (P < 0.05). Similarly, the phylum Actinobacteria exhibited a significant positive correlation with the 24-hour pH of the pectoral muscle, yielding a correlation coefficient of 0.9623 (P < 0.05). Additionally, the genus Campylobacter was significantly positively correlated with the water loss rate of the pectoral muscle, with a correlation coefficient of 0.858 (P < 0.05) (Fig. 8A). The genus Streptococcus showed significant positive correlations with the a* and b* color values of the leg muscle, with correlation coefficients of 0.777 and 0.722, respectively (P < 0.05) and an extremely significant positive correlation with the L* value, with a coefficient of 0.980 (P < 0.01). Furthermore, Helicobacter pylori were significantly positively correlated with the water loss rate of the pectoral muscle, exhibiting a correlation coefficient of 0.858 (P < 0.05) (Fig. 8B).

Fig. 8.

Spearman correlation analysis between intestinal microorganisms and meat quality of Leizhou black duck at the phylum (A) and genus (B) levels.

M in the Fig. denotes muscle.

Spearman correlation analysis indicated that there was no statistically significant relationship between the door-level biomarker and the three flavor amino acids (P > 0.05) (Fig. 9A). Notably, Clostridium_UCG-014 demonstrated a significant positive correlation with threonine, with a correlation coefficient of 0.742 (P < 0.05). Additionally, other bacterial genera exhibited a highly significant positive correlation with threonine, reflected by a correlation coefficient of 0.500 (P < 0.01) (Fig. 9B).

Fig. 9.

Spearman correlation analysis between intestinal microorganisms and flavor substances in Leizhou black duck at the phylum (A) and genus (B) levels.

Discussion

Effects of Marine-Derived enterococcus durans YQ-6 on growth performance and slaughter characteristics of Leizhou black ducks

Growth performance serves as the primary indicator for evaluating the effectiveness of feeding regimens. The results of this study indicate that supplementing feed with marine-derived Enterococcus durans YQ-6 significantly increased the average daily gain and reduced the feed conversion ratio in Leizhou black ducks, with the most pronounced effect observed at a dosage of 1 × 10⁹ CFU/kg (M3 group). This finding aligns with widely reported evidence demonstrating the growth-promoting effects of probiotics in poultry (Nasr et al., 2022) (Hu and Kim, 2022) (Yu et al., 2022) Its growth-promoting mechanism may involve multiple aspects: YQ-6 colonizes and proliferates in the gut, potentially stimulating the secretion of host digestive enzymes to enhance the breakdown and absorption of nutrients (Bai et al., 2017) (Patnala et al., 2016). Additionally, as probiotics, they may inhibit the growth of potential pathogens through competitive exclusion, thereby maintaining intestinal microecological balance and allowing more nutrients to be utilized for growth (Ramos et al., 2020). The “optimal dose” of 1 × 10⁹ CFU/kg determined in this trial is limited to the tested range of 1 × 10⁷ to 1 × 10⁹ CFU/kg. Future studies should explore a broader (including higher) dose gradient to establish a complete dose-response curve.

Slaughter performance directly affects the economic value of meat ducks (Chen et al., 2022). In this study, the M3 group significantly improved the dressing percentage, semi-gutted rate, fully gutted rate, breast muscle percentage, and leg muscle percentage of Leizhou Black Ducks. This finding aligns with the results reported by Xin Zhu et al. (Zhu et al., 2025), who demonstrated that a composite probiotic formulation enhanced the dressing percentage of broiler chickens. The mechanism by which YQ-6 improves slaughter performance may be related to its enhancement of overall growth performance and protein deposition efficiency. Probiotics optimize intestinal health, thereby increasing the absorption and utilization of nutrients such as amino acids. This promotes muscle tissue synthesis, ultimately manifesting as improved slaughter yield (Hu et al., 2024).

Effects of Marine-Derived enterococcus durans YQ-6 on muscle quality, conventional nutritional components and muscle amino acids in Leizhou black ducks

Muscle quality is a key factor in determining consumer acceptance (Xiao et al., 2024). In this experiment, the YQ-6 supplementation group, particularly the M3 group, significantly reduced the b-value and shear force of the pectoral muscle. A lower b-value is typically associated with more desirable meat color, while the decrease in shear force directly reflects improved muscle tenderness (Zhu et al., 2023) (Namroud et al., 2008). These positive changes may be related to the rate of glycolysis, the pattern of pH decline, and the state of protein denaturation in post-slaughter muscle tissue. YQ-6 may indirectly influence these processes by regulating the body's metabolic or antioxidant status (Li et al., 2023b) (Byrne et al., 2000) (Jeleníková et al., 2008). Additionally, the M3 group exhibited a significant reduction in muscle crude fat content, which may facilitate the production of leaner meat products.

The nutritional value of muscle largely depends on its amino acid composition (Pereira and Vicente, 2013). Amino acids, as the fundamental building blocks of proteins, are absorbed by the human body and participate in tissue construction and metabolic processes (Ma et al., 2020). Consumers generally prefer meat products with natural flavors and textures (Ma et al., 2020) (Shin et al., 2023). This study found that the M3 group significantly increased the levels of aspartic acid, alanine, and phenylalanine in leg muscles, as well as the total amino acid (TFAA) and essential amino acid (EAA) content. Notably, umami amino acids such as glutamic acid and aspartic acid played a significant role in enhancing the meat's savory flavor (Hossain et al., 2024). The increased content of essential amino acids directly enhances the protein nutritional quality of muscle. The mechanism by which YQ-6 promotes optimization of the muscle amino acid profile remains incompletely understood. It may be related to its ability to enhance intestinal amino acid absorption, regulate host protein metabolism, or influence the production of related metabolites by gut microbiota (Macelline et al., 2021). Spearman correlation analysis also revealed associations between certain gut bacterial genera and flavor amino acid content, suggesting potential microbial-host interaction mechanisms. However, further validation through metabolomics and other approaches is required.

Effects of Marine-Derived enterococcus durans YQ-6 on the intestinal villi of Leizhou black ducks

The small intestine is the primary site for nutrient absorption in animals, and the morphology and structural integrity of its mucosa are crucial for its absorptive function (Liang et al., 2025). The villi and microvilli within the small intestine significantly increase surface area, enhancing the efficiency of nutrient absorption (Walton et al., 2016). In this study, the addition of YQ-6 significantly increased villus height (VH) and villus-to-crypt ratio (V/C) in the duodenum and ileum of Leizhou black ducks, while reducing crypt depth (CD). Higher VH and V/C ratios typically indicate a larger absorption surface area and more mature intestinal epithelial cells, signifying enhanced digestive and absorptive function (Cairo et al., 2018). This finding is consistent with reports by Yang He et al. (He et al., 2021) and Wageha et al. (Awad et al., 2008) regarding the improvement of intestinal morphology in poultry by enterococci. Probiotics may promote villus development and epithelial repair by supplying energy to intestinal epithelial cells through their metabolites (such as short-chain fatty acids) or by modulating local immune responses and reducing inflammatory damage (Zhu et al., 2023). The improved intestinal morphology provides a direct physiological explanation for the observed growth performance enhancement. However, the colonization status of YQ-6 in the gut and its interactions with intestinal epithelial cells require further investigation.

Marine-derived enterococcus durans YQ-6 improves the gut microbiota of leizhou black ducks

Within the intestinal ecosystem, the gut microbiota plays a crucial role. This study analyzed the effects of YQ-6 on the microbial community of the cecum through 16S rRNA gene sequencing. Analysis of alpha diversity (Shannon and Chao1 indices) revealed no significant differences between groups, indicating that the addition of YQ-6 did not disrupt the overall richness and evenness of the microbiota—characteristics typically considered ideal for probiotic applications (Farooq et al., 2023; Ghosh et al., 2021). However, Beta diversity analysis (PCoA) revealed significant intergroup differences, indicating that YQ-6 primarily altered the compositional structure of the microbiota rather than its overall diversity. Research indicates that the richness of gut microbiota and the balance of its distribution are positively correlated with the interdependent and regulatory mechanisms among its constituent members. This mechanism demonstrates that during periods of stress, a more diverse and balanced gut microbiota exhibits enhanced buffering and regulatory capacity. This effectively maintains microbial homeostasis and mitigates the adverse effects of stress on the body (Kittelmann et al., 2013).

In terms of microbial community composition, the addition of YQ-6 induced changes in the abundance of specific taxonomic groups. For example, at the phylum level, the relative abundance of the M3 group Deferribacterota was significantly reduced. This study first analyzed the bacterial community structure at different taxonomic levels to investigate changes in community composition and richness, thereby further exploring the effects of YQ-6 on the gut microbiota. The findings indicate that in the cecal environment, the intestines of newborn ducklings are nearly sterile during the initial stage. Over time, the Proteobacteria phylum dominated during the first three days after ducklings' birth; starting from the fourth day, the number of Bacillobacteria gradually increased and replaced the Proteobacteria as the dominant microbial community. During subsequent developmental stages, in addition to the Proteobacteria and Peptostreptococci phyla, a significant presence of the Bacteroidetes phylum was detected in the intestines of domestic ducks raised under greenhouse conditions (Best et al., 2016). Thick-walled bacteria possess the ability to degrade dietary fiber, producing metabolites such as acetic acid and butyric acid, thereby optimizing intestinal permeability. Bacteria belonging to the Bacteroidetes phylum are considered the primary decomposers of polysaccharides. It is noteworthy that the ratio of Firmicutes to Bacteroidetes is widely regarded as one of the key factors in assessing gut stability (Stojanov et al., 2020; Sun et al., 2023). In this study, the relative abundance of the phylum Firmicutes was elevated, suggesting that YQ-6 may influence lipid metabolism by regulating the ratio of Firmicutes to Bacteroidetes in the gut. Further research is needed to confirm this hypothesis.

Additionally, to investigate the specific effects of particular bacterial genera on meat duck production performance, this study conducted a detailed analysis of changes in the gut microbiota composition of Leizhou black ducks at the genus level. The results revealed that Bacteroides dominated the contents of the cecum, with increased abundance of this genus positively correlated with host body weight. While degrading intestinal carbohydrates, Bacteroides secretes multiple short-chain fatty acids including acetate, propionate, and butyrate. These metabolites actively participate in and influence energy metabolism processes within the host (Macfarlane and Macfarlane, 2003). Spearman correlation analysis between combined horizontal and vertical biomarkers and intestinal health, meat performance, meat quality, and flavor compounds in Leizhou Black Ducks revealed significant positive correlations. This further validates that the gut microbiota plays a positive role in enhancing growth performance and intestinal health in Leizhou Black Ducks.

Additionally, this study employed LEfSe analysis to identify gut microbiota biomarkers exhibiting significant differences between groups. The results indicate that different doses of marine-derived Enterococcus durans YQ-6 exerted differential effects on the gut microbiota of Leizhou black ducks, with corresponding alterations in the relative abundance of key gut microbiota across treatment groups. Combining the results of α-diversity and β-diversity analyses further confirms that YQ-6 exerts a significant regulatory effect on both the abundance and composition of the intestinal microbiota in Leizhou black ducks. However, the specific roles of these biomarkers identified through LEfSe analysis in the intestinal development of Leizhou black ducks, as well as whether they can induce differential expression of metabolites, require further in-depth research and investigation.

With the continuous advancement of high-throughput sequencing technology, the relationship between the gut microbiota and host health status as well as nutrient absorption has been explored and discussed in greater detail. Numerous studies have demonstrated that enhancing the biodiversity of the gut microbiota benefits host intestinal health, opening up new avenues for future research into gut microbial diversity (Clarke et al., 2014).

Conclusion

The addition of marine-derived Enterococcus durans YQ-6 to feed significantly improves the growth performance, slaughter yield, meat tenderness, and amino acid nutritional value of Leizhou black ducks. It also enhances intestinal morphology and structure while regulating gut microbiota composition. Within the dose gradient established in this trial (1 × 10⁷ to 1 × 10⁹ CFU/kg), the additive dose of 1 × 10⁹ CFU/kg yielded the optimal effect. These beneficial effects may be related to YQ-6′s ability to enhance nutrient absorption and optimize the intestinal microecology. This study provides valuable data on the application of YQ-6 as a potential antibiotic-free feed additive in meat duck production.

List of abbreviations

Acronym	Full English Name
a*	Green-Red Component
ADFI	Average Daily Feed Intake
ADG	Average Daily Gain
ASV	Amplicon Sequence Variant
b*	Blue-Yellow Component
BW	Body Weight
CD	Crypt depth
CFU	Colony-forming units
EAA	Essential Amino Acids
FCR	Feed Conversion Ratio
L*	Lightness
LA	Lactobacillus Acidophilus
LDA	Linear Discriminant Analysis effect size
LEfSe	Linear discriminant analysis Effect Size
M	Model group
NEAA	Non-Essential Amino Acids
PCR	Polymerase Chain Reaction
pH	Potential hydrogen
SV	Structural Variation
TFAA	Total Free Amino Acids
UAA	Umami Amino Acids
VH	Villus height

The funding source

This work was supported by program for scientific research start-up funds of Guangdong Ocean University (Grant No. R20061) and Guangdong Provincial Key Areas of Higher Education Project (Grant No. 2020ZDZX1043).

Ethics approval

The animal care, feeding and sampling procedures involved in the present study were approved by the Animal Ethics Committee of Guangdong Ocean University (IACUC No. GDOU‐NXYLAE‐2022‐DY004.1).

Data and model availability statement

The authors confirm that all the data used in the article supporting this study are available within the article.

CRediT authorship contribution statement

Yufeng Gou: Writing – original draft, Formal analysis, Data curation. Jiang Wu: Writing – original draft, Validation, Methodology. Qiucheng Yao: Software, Methodology. Namula Zhao: Software, Resources. Haiyan Wu: Validation, Supervision. Zhibao Chen: Writing – review & editing, Resources, Project administration, Funding acquisition, Conceptualization.

Disclosures

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.