Variations in gut microbiota composition and reproductive hormone levels between laying and broody Muscovy ducks.

During the laying period of Muscovy ducks, the ovarian cortex becomes filled with a large number of follicles. However, during the transition from laying eggs to nesting behavior, a significant number of follicles undergo various grades of atrophy, eventually leading to atresia. A comparison between the ovaries and follicles of Muscovy ducks during the laying stage and nesting stage reveals the following differences: During the laying stage, Muscovy ducks exhibit a larger ovarian volume, clear stages of follicle development, abundant dominant follicles, smooth and round follicle surfaces, thickened granular and membrane layers in follicles of all levels, thicker vascular walls, larger lumens, and lower vascular density. However, during the nesting stage, follicle development in the ovary ceases, and varying degrees of follicle atrophy occur. The boundary between the granular layer and membrane layer becomes blurred, ovarian atrophy intensifies, the number of atretic follicles increases, and the interstitial cells of atretic follicles multiply. Additionally, there is a higher density of blood vessels, thicker blood vessel walls, and smaller lumens observed during this stage ( Fig. 1 ). In addition, We used duck ovarian weight and ratio (ovarian weight/weight * 100%) and stromal weight to assess the condition of the ovaries ( Liu et al., 2018b ), and found that the broodiness group was significantly lower than the laying group ( P < 0.05). Fig. 1 The morphology of complete ovarian tissue in Muscovy duck. Laying, the structure of ovarian tissue in Laying Muscovy duck. Pre-laying, the structure of ovarian tissue in pre-Laying Muscovy duck. Broodiness, the structure of ovarian tissue in broody Muscovy duck. Fig. 1 The morphology of complete ovarian tissue in Muscovy duck. Laying, the structure of ovarian tissue in Laying Muscovy duck. Pre-laying, the structure of ovarian tissue in pre-Laying Muscovy duck. Broodiness, the structure of ovarian tissue in broody Muscovy duck. Compared to the laying group, the serum concentration of FSH was significantly decreased in Muscovy ducks in the broodiness group ( Fig. 2 A, P < 0.01). Conversely, the concentrations of PRL, FS and AMH were significantly higher in the broodiness group compared to the laying group ( Figs. 2 B- 2 D, P < 0.05). Fig. 2 Serum hormone levels of Muscovy ducks in broodiness group and laying group. Laying, the Laying Muscovy duck group. Broodiness, the broody Muscovy duck group. n = 9. “*” denotes a significant difference at the 0.01 < P < 0.05 level. “**” denotes a more pronounced significant difference as P < 0.01. Fig. 2 Serum hormone levels of Muscovy ducks in broodiness group and laying group. Laying, the Laying Muscovy duck group. Broodiness, the broody Muscovy duck group. n = 9. “*” denotes a significant difference at the 0.01 < P < 0.05 level. “**” denotes a more pronounced significant difference as P < 0.01. Based on the Venn diagram analysis, the following observations were made: In the ileum, the laying group had 92 unique OTUs, while the broodiness group had 226 unique OTUs. A total of 369 OTUs were found to be common between the 2 groups. In the cecum, the laying group exhibited 273 distinct OTUs, whereas the broodiness group displayed 840 unique OTUs. Moreover, there were 1020 OTUs that were shared between the Laying and Broodiness groups ( Figs. 3 A and 3 B). Fig. 3 Ileal and Cecal microbiota diversity in Muscovy ducks. (A–B) A Venn diagram depicting the common and unique flora species in the ileum and cecum between 2 groups. (C–D) The PCoA (Principal Coordinates Analysis) Score Plot of the ileum and cecum visualizes the dispersion of each sample in the 2 groups as indicated by the β diversity. Laying, the Laying Muscovy duck group. Broodiness, the broody Muscovy duck group. n = 9. Fig. 3 Ileal and Cecal microbiota diversity in Muscovy ducks. (A–B) A Venn diagram depicting the common and unique flora species in the ileum and cecum between 2 groups. (C–D) The PCoA (Principal Coordinates Analysis) Score Plot of the ileum and cecum visualizes the dispersion of each sample in the 2 groups as indicated by the β diversity. Laying, the Laying Muscovy duck group. Broodiness, the broody Muscovy duck group. n = 9. The results of the beta diversity indexes comparison analysis are presented in Figs. 3 C and 3 D. During the breeding cycle, the composition of the ileal and cecal microbiota was found to vary. Unconstrained principal coordinate analysis ( PCoA ) revealed the presence of 2 distinct clusters for both the ileal and cecal microbiota of nesting and laying. These clusters were separated along the first coordinate axis, indicating significant differences in the cecal microbiota between the nesting and laying phases. At the phylum level, the top 5 abundant bacteria in the ileum are Firmicutes, Proteobacteria, Actinobacteria, Bacteroidota, and Desulfobacterota. Compared with the laying group, the abundance of Actinobacteria decreased from 4.41 to 1.09% ( Fig. 4 A). In the cecum, the dominant bacteria consist of Firmicutes, Bacteroidetes, Actinobacteria, Desulfobacterota, and Spirochaetales. The abundance of Actinobacteria increased from 6.12 to 6.41%, while Desulfobacterota increased from 2.87 to 6.03% when compared to the laying group. On the other hand, Bacteroidetes decreased from 42.88 to 34.66%, and Spirochaetales decreased from 2.88 to 1.12% ( Fig. 4 B). Fig. 4 Ileal and Cecal microbiota composition in Muscovy ducks. Relative contribution of phylum (A and B) and genus (C and D) in the ileal and ceceal of Muscovy ducks in the Laying and Broodiness groups, n = 9. Fig. 4 Ileal and Cecal microbiota composition in Muscovy ducks. Relative contribution of phylum (A and B) and genus (C and D) in the ileal and ceceal of Muscovy ducks in the Laying and Broodiness groups, n = 9. At the genus level, the predominant genera identified in the ileal contents of ducks, as depicted in Fig. 4 C, included Staphylococcus, Escherichia-Shigella, Pseudomonas, Bacillus, Enterococcus, Macrococcus, Kurthia, Wohlfahrtiimonas, Rothia, Acinetobacter, Streptococcus, unclassified_f__Lachnospiraceae, Lactobacillus, Achromobacter and Stenotrophomonas. In contrast, the major genera in the cecal contents of ducks were Bacteroides, unclassified_o__Bacteroidales, Desulfovibrio, Rikenellaceae_RC9_gut_group, Butyricicoccus, Alistipes, Subdoligranulum, unclassified_f__Oscillospiraceae, unclassified_f__Ruminococcaceae and Christensenellaceae_R-7_group ( Fig. 4 D). Compared to the laying group, the broodiness group exhibits higher abundance levels of Rothia, Streptococcus , and Lactobacillus , whereas the abundance of Turicibacter, Aliicoccus , and Facklamia were lower in ileum ( P < 0.05, Fig. 5 A). In the cecum, the broodiness group demonstrates a significant decrease in the abundance of Butyricicoccus and unclassified_f_Rikenellaceae , whereas the abundance of Christensenellaceae_R-7_group, unclassified_f_Lachnospiraceae, Ruminococcus_torques_group, Parabacteroides, norank_f_Oscillospiraceae, Oscillospira, Cloacibacillus, Sellimonas, Shuttleworthia, norank_f_UCG-010, Synergistes, Family_XIII_AD3011_group , and Eubacterium _nodatum_group is notably increased compared to the laying group ( Fig. 5 B). Fig. 5 The relative abundances of different bacterial genera in the ileum and cecum of Muscovy ducks. Laying, the Laying Muscovy duck group. Broodiness, the broody Muscovy duck group. n = 9. “*” denotes a significant difference at the 0.01 < P < 0.05 level. “**” denotes a more pronounced significant difference as P < 0.01. Fig. 5 The relative abundances of different bacterial genera in the ileum and cecum of Muscovy ducks. Laying, the Laying Muscovy duck group. Broodiness, the broody Muscovy duck group. n = 9. “*” denotes a significant difference at the 0.01 < P < 0.05 level. “**” denotes a more pronounced significant difference as P < 0.01. In the ileum, the primary short-chain fatty acids ( SCFAs ) are acetate, propionate, and butyrate. In the cecum, the major SCFAs are acetate, propionate, butyrate, isobutyrate, isovalerate, and valerate. Compared to the laying group, the broodiness group exhibits a significant reduction in isobutyrate levels specifically in the cecum ( P < 0.05). However, there are no significant differences observed in the levels of other SCFAs between the 2 groups, both in the ileum and cecum ( Figs. 6 A and 6 B). Fig. 6 The short-chain fatty acids ( SCFAs ) in the ileum and cecum of Muscovy ducks. Laying, the Laying Muscovy duck group. Broodiness, the broody Muscovy duck group. n = 9. “*” denotes a significant difference at the 0.01 < P < 0.05 level. “**” denotes a more pronounced significant difference as P < 0.01. Fig. 6 The short-chain fatty acids ( SCFAs ) in the ileum and cecum of Muscovy ducks. Laying, the Laying Muscovy duck group. Broodiness, the broody Muscovy duck group. n = 9. “*” denotes a significant difference at the 0.01 < P < 0.05 level. “**” denotes a more pronounced significant difference as P < 0.01. A Spearman correlation analysis was performed to examine the relationship between the bacteria with relatively high abundance in the ileum and cecum and the serum hormone expression levels ( Fig. 7 ). In the ileum, the relative abundance of Lactobacillus showed a negative correlation with FS levels ( P < 0.05). Streptococcus abundance exhibited negative correlations with PRL, AMH, and FS ( P < 0.05). Similarly, Rothia abundance was negatively correlated with PRL and FS levels ( P < 0.05). The relative abundance of Enterococcus in the ileum was significantly positively correlated with the secretion level of FSH and negatively correlated with FS levels ( P < 0.05). In the cecum, The Ruminococcus_torques_group demonstrated negative correlations with FSH, but positive correlations with PRL and FS ( P < 0.05). The relative abundance of norank_f__norank_o__Clostridia_UCG-014, Olsenella and Christensenellaceae_R-7_group were significantly positively correlated with FS secretion levels ( P < 0.05). The unclassified_f__Lachnospiraceae were significantly positively correlated with FS and PRL, and negatively correlated with FSH ( P < 0.05). The relative abundances of Butyricicoccus was significantly positively correlated with FSH and negatively correlated with AMH secretion levels ( P < 0.05). norank_f__norank_o__ Clostridia_vadinBB60_group was significantly negatively correlated with isovalerate ( P < 0.05). Fig. 7 The correlation heatmap of bacterial genera and serum hormones in in the ileum and cecum of Muscovy ducks. A, The correlation heatmap of the 30 most abundant genera and serum hormones in the ileum. B, and the 15 most abundant genera and serum hormones in cecum. The level of serum hormones is represented on the X-axis, while the bacterial genera are represented on the Y-axis. Different colors on the right side of the legend indicate the correlation coefficients (R). Fig. 7 The correlation heatmap of bacterial genera and serum hormones in in the ileum and cecum of Muscovy ducks. A, The correlation heatmap of the 30 most abundant genera and serum hormones in the ileum. B, and the 15 most abundant genera and serum hormones in cecum. The level of serum hormones is represented on the X-axis, while the bacterial genera are represented on the Y-axis. Different colors on the right side of the legend indicate the correlation coefficients (R).

All management and experimental procedures followed the animal care protocols approved by the Fujian Agriculture and Forestry University Animal Care and Use Ethics Committee (Approval ID: PZCASFAFU24004). A total of 18 female Muscovy ducks, aged 300 ± 7 d, were randomly selected for the study. The group consisted of nine laying Muscovy ducks (laying group) and nine broody Muscovy ducks (broodiness group), sourced from Wens Food Group Co., Ltd. (Putian, Fujian, China). The rearing conditions for the ducks have been detailed in our previous study ( Huang et al., 2024 ). In brief, the duck house is equipped with a fully enclosed negative pressure ventilation system. It spans 61 meters in length, 10.5 meters in width, and 3.2 meters in height, and includes an evaporative cooling system with 3 wet curtains and 6 fans. Inside, there are 4 rows of 3-level metal cages, each measuring 57 meters in length, 1 meter in width, and 2.2 meters in height, subdivided into units (0.6 × 0.5 × 0.45 meters per unit). Each cage accommodates 3 Muscovy ducks, providing 1,000 cm² of space per duck. The ducks were granted unlimited access to food and water, and they were fed a commercial pellet diet devoid of antibiotics twice a day. The duck from broodiness group was selected based on the following phenotypic traits: after a period of egg laying, the ducks exhibited elevated body temperature, ruffled feathers, reduced appetite, and nesting behavior, which eventually led to the cessation of egg production. These ducks were then dissected to confirm ovarian regression and follicular atresia ( Liu et al., 2018a ; Wu et al., 2019 ; He et al., 2022 ). The ducks from the laying group were selected from the same cage or nearby cages as the brooding ducks whenever possible. All ducks were fasted for 12 hours after group assignment, and blood samples were collected the next morning at 8 am. The blood was left at room temperature for 2 h and then centrifuged at 3,000 × g , 4°C for 10 min. The serum was collected and stored at -20°C until further analysis for hormone levels. The ducks underwent laparotomy to expose the abdominal cavity. The mesentery was carefully dissected to fully uncoil the intestines, allowing access to the ileum and cecum. The entire ileum and cecum were then excised. To collect the chyme, the intestine was gently massaged from one end, propelling the contents towards the opposite end. The chyme was extracted from both the ileum and cecum and immediately transferred into cryopreservation tubes for subsequent analysis ( Zhang et al., 2020 ). These samples were immediately immersed in liquid nitrogen and stored at -80°C for later use. The frozen samples were then stored at -80°C for subsequent analysis, including 16S rDNA sequencing and short-chain fatty acid ( SCFA ) analysis. The hormones were quantified using ELISA kits following the manufacturer's instructions (Shanghai Enzyme-linked Biotechnology Co., Ltd, China): Duck prolactin (PRL, cat. no. YJ061209), Duck Mullerian Inhibiting Substance (AMH, cat. no. YJ421547), Duck follicle-stimulating hormone (FSH, cat. no. YJ061240), and Duck Follistatin (FS, cat. no. YJ105869). These kits utilize a double-antibody one-step sandwich ELISA method, where hormone inhibitors are pre-coated in wells with capture antibodies. Samples, standards, and HRP-labeled detection antibodies are sequentially added to the coated microplate, followed by incubation and thorough washing. The substrate TMB is then added for color development. Under the catalytic action of peroxidase, TMB turns blue and subsequently turns yellow upon acid addition. The intensity of the color is directly proportional to the hormone concentration in the sample. The absorbances (OD value) are measured at 450 nm using a microplate reader to calculate sample concentrations. The detection ranges of the AMH, FS, FSH, and PRL kits are as follows: 125 pg/mL – 4,000 pg/mL, 0.375 ng/mL - 12 ng/mL, 0.625 mIU/mL - 20 mIU/mL, and 62.5 μIU/mL - 2000 μIU/mL, respectively. The minimum detectable concentrations for these kits are less than 10 pg/mL, 0.1 ng/mL, 0.1 mIU/mL, and 10 μIU/mL, respectively. The intra-assay coefficient of variation is less than 10%, and the inter-assay coefficient of variation is less than 15%. The procedure of 16S rDNA sequencing was described in detail according to a previous study ( Liu et al., 2018b ). The simplified workflow was presented here. The bacterial DNA in digesta were extracted by using Stool DNA Kit (D4015-01, Omega Bio-tek, Norcross, GA), then amplified with the primers of V3-V4 region in bacterial 16S rDNA. The amplicons were purified and then sequenced via the Illumina MiSeq Platform. After quality control and assembly, the clean sequencing data were used for analysis on the online platform of Majorbio Cloud Platform ( www.majorbio.com ). Ileal and cecal PCoA on OTU level and Variation between groups at the phylum and genus levels were analyzed. Ileum and cecum microorganism among different group were visualized by Venn plot. All raw sequencing data have been deposited in the NCBI Sequence Read Archive under the BioProject PRJNA1134353 and PRJNA1134410. Concentrations of the main short-chain fatty acids ( SCFAs ), including acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate, in the digesta samples were analyzed using gas chromatography (7890A, Agilent, Santa Clara, CA), following the method described in our previous study ( Huang et al., 2020 ). Briefly, Thaw 0.7 g of digesta and suspend it in 1.5 mL of ultrapure water. Vortex the mixture and let it sit on ice for 30 min. Centrifuge each sample at 10,000 × g and 4°C for 15 min. Transfer 1 mL of the supernatant into a 1.5 mL centrifuge tube and mix it with 0.2 mL of crotonic acid-metaphosphate acid. Let it sit at 4°C for 30 min, followed by centrifugation at 10,000 × g and 4°C for 10 min. Collect 0.3 mL of the supernatant and add it to 0.9 mL of methanol. Vortex the mixture and centrifuge it at 8,000 × g and 4°C for 5 min. Finally, analyze the supernatant using gas chromatography with a flame ionization detector. The oven temperature should be set to increase from 100°C to 190°C, with nitrogen “N 2 ” used as the carrier gas at a flow rate of 1 mL/min. Data on hormone obtained from serum were analyzed using student t-test. Values are expressed as the means ± SEMs. Figures were created with Graphpad prism 6. Wilcoxon rank-sum test was used to compare variation of ileal and cecal digesta samples on Genus level, and the test level was 0.05. In figures, significances are annotated with the following markers: *, P < 0.05; **, P < 0.01.

The ovary serves not only as the reproductive organ responsible for egg production and release but also as an endocrine gland that synthesizes and secretes estrogen, directly influencing the egg-laying performance of poultry ( Kang et al., 2010 ; Conti et al., 2012 ). During the laying period, the ovarian cortex of female birds is filled with a large number of follicles ( Jiang et al., 2005 ). Various factors, such as genetics, nutrition, and environmental conditions, influence poultry during the transition from egg laying to nesting behavior, which includes follicular atrophy and subsequent atresia in multiple grades ( Cheng et al., 2017 ). Muscovy ducks exhibit a high broody rate, significantly reducing egg production, establishing them as a crucial model for studying broody mechanisms. Current research on broody mechanisms primarily focuses on the regulation of hormone-gene expression ( Zhao et al., 2023 ; Shen et al., 2024 ). However, the specific reasons remain unclear, limiting the identification and use of appropriate molecular markers for breeding Muscovy ducks to enhance egg production rates. Recent studies have shown that the gonadal axis interacts with intestinal microbiota, regulating reproductive physiology in animals ( Franasiak and Scott, 2015 ; Qi et al., 2021 ; Wang and Xie, 2022 ). Therefore, in this study, the composition of intestinal microbiota will be analyzed and key microbial communities identified to demonstrate the role of gut microbiota in the nesting behavior of Muscovy ducks, laying the foundation for future research to use methods such as fecal microbiota transplantation or nutritional modulation of gut microbiota to reduce the broody rate of Muscovy ducks, thereby enhancing the economic efficiency of Muscovy duck production. In poultry, the development of ovarian follicles and reproductive performance is primarily regulated by hypothalamic hormones such as FSH ( Ricke et al., 2022 ) and AMH ( Lemcke et al., 2018 ). As the main hormone of the reproductive axis, FSH is considered to be the primary survival factor for follicle growth in low-yield laying hens ( Elokil et al., 2020 ; Zhong et al., 2023 ) and for preventing ovarian aging in chickens ( Dong et al., 2022 ). The FSH reactivity of prehierarchical follicles is regulated by AMH ( Huang et al., 2021 ) and PRL ( Pan et al., 2022 ), which leads to the inhibition of prehierarchical follicle development. Additionally, Follistatin is a cysteine-rich monomeric glycoprotein that belongs to the transforming growth factor beta ( TGF-β ) family of signaling molecules and was originally isolated from ovarian follicular fluid ( de Kretser et al., 2002 ). In cooperation with FOXL2, follistatin is involved in intracellular FSHR transcription and granulosa cell proliferation through positive or negative autocrine regulatory mechanisms during follicular development in hens ( Qin et al., 2015 ). In this study, the levels of FSH, AMH, PRL, and FS were differentially expressed between the broodiness and laying groups, indicating that the broody behavior of Muscovy ducks is regulated by hormones of the reproductive axis, consistent with previous research ( Ye et al., 2019 ). These findings highlight the complex interplay of endocrine hormones in modulating reproductive behaviors and provide valuable insights into the hormonal mechanisms underlying broody behavior in Muscovy ducks. Cross-talk between gut microbiota and endocrine hormones can regulate the host's metabolism, immune function, and reproductive behavior ( Qi et al., 2021 ). Disruptions in gut microbiota can contribute to conditions such as human polycystic ovary syndrome and endometriosis, thereby impacting normal ovulation and overall ovarian function health ( Chen et al., 2022 ). The gut microbiome diversity differs significantly between chickens with high and low egg-laying performance ( Elokil et al., 2020 ). Transplanting fecal microbiota from high-yield to low-yield laying hens notably enhanced the recipients' egg production performance ( Wang et al., 2020 ). Studies have shown that supplementing nutrients in feed, such as essential oils ( Liu et al., 2020 ) and medium-chain triglycerides ( Feng et al., 2021 ), can promote gut microbiota abundance and increase serum levels of FSH, luteinizing hormone, and estradiol, thereby enhancing laying performance in hens. In this study, notable variations in the composition of intestinal microbiota were found between the broodiness and laying groups, suggesting a regulation by microbiota on poultry egg-laying performance. What is more, correlation analysis between microbiota and hormone levels showed correlations among the top 20 differentially abundant genera. Notably, among the genera significantly correlated with Follistatin— Enterococcus, Streptococcus, Lactobacillus, Ruminococcus torques group, norank_f__norank o Clostridia UCG-0014, unclassified_f__Lachnospiraceae, Christensenellaceae R-7 group , and Butyricicoccus —all belong to the phylum Firmicutes. This indicates that the interaction between hormones and gut microbiota can regulate follicle development in Muscovy ducks, with the interaction between Follistatin and Firmicutes being particularly significant. Future research will further explore the mechanisms of interaction between Follistatin and microbiota. Targeting these interactions, strategies such as nutritional regulation or fecal microbiota transplantation will be developed to lay the foundation for improving poultry reproductive performance.

In conclusion, combining the results of serum hormones and intestinal microbiota, it can be reasonably deduced that the broody behavior of Muscovy ducks can be regulated by the interaction between hormones and gut microbiota. Notably, the relationship between Follistatin and the composition of gut microbiota, specifically Firmicutes, is the most prominent.

China leads global waterfowl trade, with Muscovy ducks prized for their lean, delicious meat. However, the Muscovy ducks industry grapples with challenges, particularly the high nesting rate, dampening egg production and industry growth. Observations show 53.76% of female Muscovy ducks exhibit nesting behavior, impacting laying rates—31.75% for those with nesting ability, and over 80% for those with weak nesting tendencies ( Ye, 2020 ). Physically, broodiness in Muscovy ducks is identified by the degeneration and closure of ovarian follicles. Consequently, this condition leads to the cessation of ovulation ( Moley and Schreiber, 1995 ; Zhao et al., 2020 ). However, the specific reasons and precise underlying cause of this behavior have not been fully clarified or explained to date. Follicular atresia in broody ducks is a complex biological process regulated by endocrine hormones and various paracrine or autocrine factors ( He et al., 2022 ). The gut microbiota is indeed considered an endocrine organ capable of influencing distant organs and related biological pathways. Microbiota directly or indirectly interact with endocrine hormones such as follicle-stimulating hormone ( FSH ), anti-Mullerian hormone ( AMH ), prolactin ( PRL ), and follicular stimulating hormone ( FS ), thereby impacting ovarian function and development ( Qi et al., 2021 ). In humans, the gut microbiome plays a role in regulating the maturation of ovarian follicles and oocytes, as well as influencing fertilization and embryo migration. Alterations in the gut microbiome can specifically affect the reproductive endocrine system. For example, dysbiosis has been linked to the development of polycystic ovary syndrome ( PCOS ) ( Guo et al., 2016 ) and correcting abnormal microbiota may potentially enhance reproductive outcomes ( Franasiak and Scott, 2015 ). Additionally, improvement in the intestinal microbiome has shown a positive correlation with elevated serum FSH levels, promoting ovarian development ( Xu et al., 2023 ) and increased egg production in hens ( Qin et al., 2024 ). Our previous research indicates significant differences in gut microbiota between ammonia-induced low egg-laying ducks and normal ducks ( Huang et al., 2024 ), highlighting the need for further investigation into potential disparities in gut microbiota colonization between nesting and egg-laying ducks. Therefore, we hypothesize that follicular atresia induced by broodiness is associated with the composition of gut microbiota and reproductive hormone levels. In this study, broody Muscovy ducks were selected as subjects to examine changes in ovarian morphology, serum hormone levels, and structural variations of microorganisms in the ileum and cecum. The aim of this research is to demonstrate that gut microbiota play an important role in duck reproduction, laying a theoretical foundation for future studies focused on regulating gut microbiota to reduce the brooding rate of Muscovy ducks.

The authors declare no conflicts of interest.