Application of taurine in improving egg production, intestinal health and reproductive performance of muscovy ducks

Abstract

The application relates to the field of biotechnology, in particular to application of taurine in improving egg production performance, intestinal health and reproductive performance of Muscovy ducks. It is found that adding 1600 mg / kg of taurine in feed can improve the reproductive performance of Muscovy ducks by improving intestinal health, optimizing body metabolic homeostasis and regulating ovary gene expression. The application provides a theoretical basis for practical application of taurine as a functional feed additive in Muscovy duck breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of taurine in improving the egg production performance, intestinal health, and reproductive performance of Muscovy ducks. Background Technology

[0002] The Muscovy duck originated in Central and South America, centered around Mexico, and was later domesticated for farming. Muscovy ducks are relatively large, averaging about 4.3 kg in weight. Their bodies are narrower at the front and back and wider in the middle, giving them an olive-shaped appearance. They have a large head and a relatively short neck. They have a ring of red fleshy growths around the base of their beak and eye sockets, a row of long, longitudinal feathers on the top of their head, and their tail feathers extend horizontally, with wing feathers extending to the tail tip. Their plumage comes in a variety of colors, including brownish-black, pure white, speckled, light yellow, brownish-tan, and chestnut brown. Female Muscovy ducks typically weigh between 2 and 2.5 kg, and lay an average of 80-120 eggs per year. The incubation period is 35 days, with peak egg production occurring around the 40th week of age.

[0003] Muscovy ducks are an important specialty economic waterfowl in my country, characterized by rapid growth, good meat quality, and tolerance to roughage. They are widely raised in southern China, and the industry enjoys good economic benefits. However, their low reproductive performance and pronounced seasonality in egg production severely hinder the scale and economic efficiency of the industry. The reproductive physiology of Muscovy ducks, such as the speed and number of follicle development and ovulation cycle, differs from that of native egg-laying ducks, resulting in lower reproductive performance. The nutritional composition and ratio of their feed also significantly affect their reproductive performance, leading to increased farming costs. Therefore, in-depth research into the reproductive performance of Muscovy ducks and improving their ovarian development level is one of the key issues currently facing the development of the Muscovy duck industry. Summary of the Invention

[0004] The purpose of this invention is to provide the application of taurine in improving the egg production performance, intestinal health, and reproductive performance of Muscovy ducks, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the use of taurine in any of the following: (1) Prepare products to improve the egg production performance of Muscovy ducks; (2) To prepare products that improve the quality of Muscovy duck eggs; (3) Prepare products that improve the intestinal tract of Muscovy ducks; (4) Prepare products that improve follicle development; (5) Prepare products that improve the reproductive performance of Muscovy ducks; (6) Prepare products that improve the intestinal flora of Muscovy ducks; (7) Prepare products that improve ovarian development.

[0006] Optionally, improving Muscovy duck egg production performance includes increasing egg production rate and egg weight.

[0007] Optionally, improving the quality of Muscovy duck eggs includes increasing shell strength, yolk percentage, and Haugh unit, and improving yolk color.

[0008] Optionally, the improvement of Muscovy duck intestines includes increasing the villus height and villus-to-villus ratio in the Muscovy duck intestines, and increasing the content of acetic acid, propionic acid and butyric acid in the intestines.

[0009] Optionally, the improvement in follicle development includes increasing the ovarian index, grade follicle count, and oviduct index of Muscovy ducks.

[0010] Optionally, the improvement of gut microbiota includes increasing the relative abundance of Flavobacterium, Pseudomonas, Riken Bacteriaceae, and Bacillus spp., and decreasing the relative abundance of Halococcus and Enterococcus.

[0011] Optionally, the product includes formulations, feed additives, and feed.

[0012] More preferably, the product includes the following: (1) Preparation of a formulation to improve the egg production performance of Muscovy ducks; (2) Preparation of feed additives to improve the egg production performance of Muscovy ducks; (3) Prepare feed to improve the egg production performance of Muscovy ducks; (4) Preparation of formulations to improve the quality of Muscovy duck eggs; (5) Prepare feed additives to improve the quality of Muscovy duck eggs; (6) Prepare feed to improve the quality of Muscovy duck eggs; (7) Preparation of a formulation to improve the intestinal tract of Muscovy ducks; (8) Prepare feed additives to improve the intestinal tract of Muscovy ducks; (9) Prepare feed to improve the gut health of Muscovy ducks; (10) Prepare a formulation to improve follicle development; (11) Preparation of a formulation to improve the reproductive performance of Muscovy ducks; (12) Prepare feed additives to improve the reproductive performance of Muscovy ducks; (13) Prepare feed to improve the reproductive performance of Muscovy ducks; (14) Preparation of a formulation to improve the intestinal flora of Muscovy ducks; (15) Prepare feed additives to improve the intestinal flora of Muscovy ducks; (16) Prepare feed to improve the intestinal flora of Muscovy ducks; (17) Prepare a formulation to improve ovarian development.

[0013] The present invention provides a formulation in which the additives include taurine and excipients.

[0014] This invention provides a feed additive, which includes taurine and excipients.

[0015] The present invention provides a feed comprising the above-mentioned feed additives and a basal diet.

[0016] More preferably, the amount of taurine used in the feed is 1600 mg / kg.

[0017] The present invention discloses the following technical effects: 1. Adding taurine to the diet can significantly improve the egg production performance of Muscovy ducks, and also has a certain effect on improving egg quality. Among the gradient dosages set in the experiment, the medium dose level of 1600 mg / kg had the most significant effect on improving the production performance of Muscovy ducks.

[0018] 2. Regarding gut health, 1600 mg / kg taurine optimizes the gut microecological environment of Muscovy ducks, while regulating the body's absorption and metabolism of nutrients. Combined with 16S rRNA sequencing results, it can be concluded that taurine can enrich beneficial bacteria and inhibit potentially harmful bacteria such as Enterococcus, thus restoring the balance of the gut microecology.

[0019] 3. Adding 1600 mg / kg taurine to the diet has a significant promoting effect on the development of reproductive organs in Muscovy ducks, significantly increases the ovarian index and oviduct index, increases the number of graded follicles with developmental potential in the ovary, and also promotes the growth and development of the ovary and oviduct, effectively improving the overall tissue structure of the ovary, reducing follicular atresia and improving ovarian function.

[0020] 4. Coordinated regulation of the body's metabolism is an important pathway for taurine to exert its effects. LC-MS studies show that N-palmitoyl taurine, N-oleoyl taurine, and catechins are significantly upregulated, indicating that taurine provides systemic support for the reproductive process by enhancing antioxidant capacity, promoting energy supply, and maintaining amino acid metabolic homeostasis.

[0021] 5. This study performed transcriptome sequencing analysis on ovarian granulosa cells and found that taurine can regulate the expression levels of key genes such as COL1A2, ITGA4, LAMB1, and THBS2. These genes are enriched in signaling pathways related to extracellular matrix-receptor interactions and cell adhesion molecules. Therefore, it can be concluded that taurine may ensure the normal functioning of granulosa cells by making follicles more intact and promoting intercellular communication and signal transduction.

[0022] In conclusion, dietary supplementation with 1600 mg / kg taurine can improve the reproductive performance of Muscovy ducks by enhancing gut health, optimizing metabolic homeostasis, and regulating ovarian gene expression. This invention provides a theoretical basis for the practical application of taurine as a functional feed additive in Muscovy duck farming. Attached Figure Description

[0023] Figure 1 The effect of taurine on the morphology of Muscovy duck ovaries; where (a): ovarian sections of the CON group; (b): ovarian sections of the Tau group; PrF: primordial follicles; PF: primary follicles; SF: secondary follicles; POF: mature follicles; AF: atretic follicles; Figure 2 Flowchart for 16S rRNA bioinformatics analysis; Figure 3 Figures show the dilution curve (a) and the species abundance distribution curve (b); Figure 4 Alpha diversity analysis of Muscovy duck cecal flora; Figure 5 This is a principal component analysis (PCA) diagram based on the cecal flora of Muscovy ducks; Figure 6 A bar chart showing species organized by phylum, family, genus, and species; Figure 7 Heatmap of species composition at the genus level; Figure 8 This is a graph showing the effect size (LEfSe) analysis in linear discriminant analysis. Figure 9 This is an evolutionary clade diagram of different species; Figure 10 Scoring charts for Muscovy duck serum metabolites PCA (a), PLS-DA (b), and OPLS-DA (c); Figure 11 This is a volcano plot of differential metabolites; where the horizontal axis represents the log2 (Fold Change) value and the vertical axis represents -log. 10 (P-value), red dots represent upregulated metabolites, green dots represent downregulated metabolites, gray dots represent indifferent metabolites, and dashed lines represent the screening criteria for differential metabolites; Figure 12 A bar chart of differential metabolites; Figure 13 A graph showing the enrichment analysis of differentially metabolized metabolites in the KEGG pathway; Figure 14 For differential metabolite association heatmaps; Figure 15 This is a schematic diagram of the transcriptome sequencing experimental workflow; Figure 16 A flowchart of transcriptome bioinformatics analysis; Figure 17 Screening for differentially expressed genes; where (a): Volcano plot of differentially expressed genes; (b): Heatmap of differentially expressed gene clusters; the horizontal axis is log2 (Fold Change) value, and the vertical axis is -log. 10 (P-value), red dots represent upregulated genes, green dots represent downregulated genes, gray dots represent non-differentially expressed genes, and dashed lines represent the value lines of the differentially expressed gene screening criteria; Figure 18 Bubble chart for GO enrichment analysis of differentially expressed genes; Figure 19 Bubble diagram for KEGG pathway enrichment analysis of differentially expressed genes; Figure 20 To validate differentially expressed genes using RT-qPCR; Figure 21 This is a heatmap showing the correlation between the transcriptome and metabolome. Detailed Implementation

[0024] Example 1 1. Feeding and grouping of Muscovy ducks Two hundred and forty Muscovy ducks (192 females and 48 males, a male-to-female ratio of 1:4) of similar health and living conditions, aged 25 weeks, were included in the experimental study. They were housed indoors for 20 weeks. All ducks were kept under identical conditions, including consistent temperature, humidity, lighting, and ventilation. They had free access to feed and water throughout the study. The duck house was regularly cleaned and disinfected, and other management practices followed standard duck farm requirements. The experimental animals in this study were obtained from Anhui Yongqiang Agricultural Technology Co., Ltd.

[0025] Animals were randomly divided into 4 groups, with 12 replicates per group and 5 animals per replicate (4 females and 1 male). The control group was fed a basal diet without taurine. The low-dose taurine group was fed a basal diet supplemented with 800 mg / kg taurine (Tau-L). The medium-dose taurine group was fed a basal diet supplemented with 1600 mg / kg taurine (Tau-M). The high-dose taurine group was fed a basal diet supplemented with 2400 mg / kg taurine (Tau-H). Taurine (97% effective ingredient) was purchased from Shaanxi Rankang Biotechnology Co., Ltd. The basal diet of the control and experimental groups was the same, provided by Anhui Yongqiang Agricultural Technology Co., Ltd., and its specific composition and nutritional components are shown in Table 1.

[0026] Table 1. Composition and nutrient levels of the basal diet Note: Crude protein is the measured value, and the rest are calculated values.

[0027] 2. Effects of different doses of taurine added to the diet on the reproductive performance of Muscovy ducks 2.1 Sample Collection Six Muscovy ducks from each of the medium-dose taurine group and the control group were randomly selected. Blood was collected using non-anticoagulated vacuum blood collection tubes and needles. After collection, the blood collection tubes were placed at an angle in a foam box containing ice packs for temporary storage. Serum was then separated by centrifugation at 3000 rpm for 10 min and aliquoted into three cryovials, stored at -80°C for subsequent serum metabolomics analysis. The ducks were then slaughtered, and the ovaries and oviducts were completely removed. Follicles with a diameter greater than 8 mm were removed, and their fresh weight was immediately measured for ovarian and oviduct index calculations. A tissue block of no more than 1.2 × 1.2 × 0.6 cm from the same ovarian region was placed in a 10 mL centrifuge tube containing 4% paraformaldehyde fixative and stored at 4°C for hematoxylin-eosin (H&E) staining to observe follicular morphology and structure. Small yellow follicles were harvested, and the granulosa cell layer was extracted for transcriptomics analysis. After flash freezing in liquid nitrogen, the extracted cells were stored at -80°C. The jejunum and cecum were separated. Tissue samples from the mid-jejunum (2-3 cm) were fixed in 10% neutral formalin (for short-term preservation) or 70% ethanol (for long-term preservation) for sectioning and evaluation of villus height (VH), crypt depth (CD), and the VH / CD ratio. Approximately 1 g of cecal contents were aliquoted into cryovials and immediately stored at -80°C for volatile fatty acid detection and 16S rRNA gene sequencing. All intestinal tissue samples were rinsed with physiological saline before sampling.

[0028] 2.2 Test Methods 2.2.1 Egg production performance determination During the experiment, the initial and final weights of the Muscovy ducks were recorded, and the number of eggs laid, egg weight, and feed intake of each group of experimental Muscovy ducks were recorded daily. The egg production rate, average egg weight, average daily feed intake, and feed conversion ratio were calculated. The calculation formula is: Egg production rate (%) = Total number of eggs laid during the experiment / (Number of female Muscovy ducks during the experiment) (Number of days of trial) 100%; Average egg weight = total egg weight / total number of duck eggs; Average daily feed intake = total feed intake / total number of Muscovy ducks; Feed conversion ratio = total feed consumption during the trial period (kg) / total egg weight during the trial period (kg).

[0029] 2.2.2 Egg quality determination At the end of the laying period, six undamaged, qualified eggs were selected from each replicate for quality testing of Muscovy duck eggs. The testing method is as follows: Egg weight: Weighed using an electronic scale, accurate to 0.1 g; Egg shape index: Measure the longest longitudinal diameter and the widest transverse diameter of the egg using vernier calipers, accurate to 0.01 mm. Egg shape index = (longitudinal diameter / transverse diameter) 100%; Eggshell strength: Eggshell strength is measured using an eggshell strength tester; Egg white height: Break the duck egg and place it on a glass plate. Use calipers and toothpicks to measure the egg white height (accurate to 0.01 mm). The color of the egg yolk was determined using a Roche colorimetric fan. Eggshell thickness: The eggshell thickness at the blunt end, middle and tip of the egg is measured using an eggshell thickness gauge. The average value is calculated in millimeters, accurate to 0.01 mm. Haugh units: Based on egg weight and albumen height, calculated using the formula "Haugh units = 100". lg(H-1.7) The formula is calculated as W(0.37+7.57), where H is the albumen height and W is the egg weight. Egg yolk ratio: The weight of the egg yolk is measured to the nearest 0.01 g. Egg yolk ratio = egg yolk weight / total egg weight.

[0030] 2.2.3 Follicle development measurement At the end of the experiment, based on the results of previous observational traits (such as egg production performance and body weight), the experimental Muscovy ducks in the medium-dose taurine group were selectively slaughtered. Immediately after slaughter, intact oviduct and ovarian tissues were separated and collected. The total weight of the oviduct and ovary was accurately weighed using an electronic analytical balance (accurate to 0.01g), and the number of graded follicles was observed and recorded. The oviduct index and ovarian index were calculated separately using the following formulas: Oviduct index = Oviduct weight (g) / Muscovy duck body weight (g); Ovarian index = Ovarian weight (g) / Muscovy duck body weight (g).

[0031] 2.2.4. Determination of jejunal morphology and structure Immediately after slaughter, a 2-3 cm segment of the mid-jejunum from the Muscovy duck was harvested. The intestinal contents were gently rinsed with pre-cooled physiological saline, and then fixed in 4% paraformaldehyde phosphate buffer (PBS) for subsequent paraffin sectioning. After routine paraffin embedding, sectioning (5 μm thickness), and hematoxylin-eosin (H&E) staining, the morphology of the jejunal mucosa was observed under an optical microscope. Six clearly visible and structurally intact villi and crypts were randomly selected from each sample for morphological measurement through image analysis. The following key morphological parameters were measured and recorded: villus height (VH): the vertical distance from the villus tip to the crypt opening; crypt depth (CD): the vertical distance from the crypt opening to the base; and villus-to-crypt ratio (V / C): the ratio of villus height to crypt depth, an important parameter reflecting intestinal digestive and absorptive function.

[0032] 2.2.5 Determination of Volatile Fatty Acids Sample pretreatment: Immediately after slaughter at the end of the experiment, cecal chyme from Muscovy ducks was collected and placed in sterile centrifuge tubes, then stored at -80°C for analysis. Before analysis, the cecal chyme samples were thawed at room temperature. Approximately 1.0 g of chyme was accurately weighed and placed in a centrifuge tube, and a certain volume of pre-cooled ultrapure water (1:3, w / v) was added. After vortexing to mix, the mixture was centrifuged at 4°C and 12,000 rpm for 15 min. 1 mL of the supernatant was taken and 0.2 mL / mL of a crotonic acid metaphosphate solution was added. Store overnight at -20℃. After thawing, centrifuge at 12000 rpm for 10 min, collect the supernatant and store it. Before analysis, centrifuge again at 12000 rpm for 10 min, collect the supernatant, filter it through a 0.22 μm microporous membrane (or pass a small amount through a 0.22 μm needle filter), collect the filtrate for instrumental analysis, and directly inject the filtrate into the chromatograph using a 1.0 μL microsyringe, with an injection volume of 0.2-1.0 μL.

[0033] Chromatographic conditions: Gas chromatography (GC) was used for determination.

[0034] Chromatograph: GC-14B gas chromatograph (Shimadzu Corporation, Japan); Gas chromatograph parameters: The chromatographic column is a capillary column (30m×0.32mm×0.25 μm), the column temperature is 130℃, the vaporization temperature is 180℃, the hydrogen ion flame detector is used, the detection temperature is 180℃, the carrier gas is nitrogen with a pressure of 60 kPa, the hydrogen pressure is 50 kPa, the oxygen pressure is 50 kPa, the sensitivity (range) is 101, and the decay is 3.0.

[0035] Quantification was performed using the external standard method. A series of gradient concentration solutions of mixed volatile fatty acid standards (acetic acid, propionic acid, butyric acid, etc.) were prepared to establish a standard curve. Based on the peak area of ​​each component in the sample, its concentration in the actual chyme sample was calculated using the standard curve. The calculation formula is as follows: .

[0036] 2.2.6 Observation of ovarian morphology Immediately after slaughter, the ovaries were separated, and the surrounding connective tissue was carefully removed. Ovarian tissue pieces of approximately 1.0 cm × 1.0 cm × 0.5 cm were taken and quickly fixed in 4% paraformaldehyde solution for 24–48 h. The fixed tissue blocks were then dehydrated with a gradient of alcohols, cleared with xylene, and embedded in paraffin. Serial sections were prepared using a paraffin microtome at a thickness of 5 μm. After spreading and drying, the sections were stained with hematoxylin and eosin (H&E). The prepared ovarian tissue sections were placed under an optical microscope to observe the overall structure of the ovary, identify follicles at different developmental stages, and carefully observe the morphology and structure of primordial follicles, primary follicles, secondary follicles, mature follicles, and atretic follicles.

[0037] 2.2.7 Data Statistics and Analysis Data were organized using Excel, and statistical analysis was performed using one-way ANOVA in SPSS 26.0. Multiple comparisons were conducted using Duncan's method. Based on the ANOVA, orthogonal multinomial comparison analysis was performed to assess the linear and quadratic effects between each indicator and the amount of taurine added. For post-slaughter data, independent samples t-tests were used to analyze the significance of differences between two groups. Results are expressed as mean ± standard error of mean, with P < 0.05 indicating statistical significance. Graphs were generated using Origin 2024 software.

[0038] 2.3 Results and Analysis 2.3.1 Effects of different levels of taurine supplementation in diet on egg production performance of Muscovy ducks As shown in Table 2, compared with the control group, the egg production rate and average egg weight of Muscovy ducks were significantly increased after dietary supplementation with a medium dose of taurine (P<0.05). The feed conversion ratio of the medium-dose taurine group was significantly lower than that of the control group and the high-dose taurine group (P<0.05). Furthermore, there was no significant difference in average daily feed intake among the groups (P>0.05). Average egg weight and feed conversion ratio showed a quadratic correlation with the amount of taurine added. In conclusion, the medium-dose taurine group showed the best effect.

[0039] Table 2. Effects of different doses of taurine added to the diet on the egg production performance of Muscovy ducks. Note: Different letters on the shoulder label indicate significant differences (P<0.05), while the same letter or no letter indicates no significant differences (P>0.05). The same applies to the table below.

[0040] 2.3.2 Effects of different levels of taurine supplementation in feed on the quality of Muscovy duck eggs As shown in Table 3, compared with the control group, the low and medium dose taurine groups significantly improved the eggshell strength, eggshell percentage, and Haugh units of Muscovy ducks (p<0.05). The concentrated albumen percentage in the medium dose taurine group was significantly higher than that in the high dose taurine group (p<0.05), and the eggshell thickness was significantly higher than that in the control group (p<0.05). In the low dose taurine group, the yolk color and yolk percentage were significantly higher than those in the control group (p<0.05). Dietary taurine supplementation had no significant effect on egg shape index, albumen height, yolk height, and yolk diameter (p>0.05). Eggshell strength, concentrated albumen percentage, albumen height, yolk percentage, eggshell thickness, and Haugh units were all linearly correlated with the amount of taurine added, while eggshell strength, yolk color, and eggshell percentage showed a quadratic correlation with the amount of taurine added.

[0041] Table 3. Effects of different doses of taurine added to the diet on the quality of Muscovy duck eggs. 2.3.3 Effects of dietary supplementation with medium doses of taurine on intestinal morphology in Muscovy ducks As shown in Table 4, dietary supplementation with a medium dose of taurine significantly increased the villus height and V / C ratio in the Muscovy duck intestine (P<0.05). However, crypt depth had no significant effect (P>0.05).

[0042] Table 4. Effects of medium-dose taurine supplementation in diet on jejunal morphology in Muscovy ducks. 2.3.4 Effects of medium-dose taurine supplementation in diet on volatile fatty acids in Muscovy ducks As shown in Table 5, adding a medium dose of taurine to the diet significantly increased the content of acetic acid, propionic acid, and butyric acid in the intestines of Muscovy ducks (P<0.05). Furthermore, compared with the control group, the addition of taurine to the diet significantly increased the content of total volatile fatty acids (P<0.05). However, there were no significant differences in the content of isobutyric acid, isovaleric acid, and valeric acid between the two groups (P>0.05).

[0043] Table 5. Effects of dietary supplementation with medium doses of taurine on volatile fatty acids in the cecum of Muscovy ducks. 2.3.5 Effects of dietary supplementation with medium doses of taurine on follicle development in Muscovy ducks As shown in Table 6, the addition of a medium dose of taurine to the diet significantly increased the ovarian index and graded follicle count in Muscovy ducks compared to the control group (P<0.05). Furthermore, taurine significantly increased the oviduct index in Muscovy ducks (P<0.01).

[0044] Table 6. Effects of dietary supplementation with medium doses of taurine on follicle development in Muscovy ducks. 2.3.6 Effects of dietary supplementation with medium doses of taurine on ovarian morphology in Muscovy ducks Figure 1 The images show typical section morphology of the ovaries of the two groups of Muscovy ducks at the end of the experiment. Figure 1 (b) shows that the follicles in the ovaries of the taurine group are actively growing and have clear structural layers, indicating good reproductive performance; Figure 1 (a) shows that most follicles in the control group were atretic, and the granulosa cell layer was shrunken and indented, indicating that their development was significantly worse than that of the taurine group.

[0045] In summary, adding taurine to the diet can significantly improve the egg production performance of Muscovy ducks and improve egg quality to some extent. The medium dose of 1600 mg / kg showed the best effect. Adding 1600 mg / kg taurine can improve the intestinal morphology and structure of Muscovy ducks and increase the content of volatile fatty acids in the intestinal contents. These results indicate that taurine can promote intestinal homeostasis and improve intestinal health in Muscovy ducks. Adding 1600 mg / kg taurine significantly promoted the development of the ovary and oviduct in Muscovy ducks, specifically manifested as a significant increase in the number of graded follicles, a decrease in follicle atresia rate, and a significant improvement in ovarian tissue structure.

[0046] 3. 16S rRNA sequencing analysis of the effects of taurine on the gut microbiota structure of Muscovy ducks 3.1 Test Methods Samples of the cecal contents of Muscovy ducks were collected after slaughter, and 16S rRNA sequencing of the samples was performed by Sanshu Biotechnology Co., Ltd.

[0047] (1) DNA extraction: DNA was extracted using a kit. After the genomic DNA was extracted, the extracted genomic DNA was detected by 1% agarose gel electrophoresis.

[0048] (2) PCR amplification: Synthesize specific primers with barcodes or fusion primers with misaligned bases according to the specified sequencing region. To ensure the accuracy and reliability of subsequent data analysis, two conditions must be met: ① Use the lowest possible cycle number for amplification; ② Ensure that the cycle number for amplification is consistent for each sample. Randomly select representative samples for preliminary experiments to ensure that the vast majority of samples can amplify products of appropriate concentrations within the lowest possible cycle number. PCR was performed using TransGen AP221-02: TransStart Fastpfu DNA Polymerase; the PCR products were detected by 1% agarose gel electrophoresis to determine the size of the target band and purified using the Agencourt AMPure XP nucleic acid purification kit.

[0049] (3) Library construction on the second-generation high-throughput sequencing platform: ligating “Y”-shaped adapters; using magnetic beads to screen and remove adapter self-ligated fragments; enriching the library template using PCR amplification; denaturing with sodium hydroxide to produce single-stranded DNA fragments.

[0050] (4) Sequencing on a second-generation high-throughput sequencing platform: One end of the DNA fragment is complementary to the primer bases and fixed on the chip; the other end is randomly complementary to another nearby primer and is also fixed, forming a "bridge"; PCR amplification produces DNA clusters; DNA amplicon is linearized into single strands; a modified DNA polymerase and dNTPs with 4 fluorescent labels are added, and only one base is synthesized in each cycle; the surface of the reaction plate is scanned with a laser to read the types of nucleotides polymerized in the first round of reaction for each template sequence; the "fluorescent group" and "terminator group" are chemically cut to restore the 3' end stickiness and continue to polymerize the second nucleotide; the fluorescence signal results collected in each round are counted to obtain the sequence of the template DNA fragment.

[0051] 3.2 Data Processing and Analysis After the raw sequencing data were processed, data quality control was first performed. Optimized sequences were obtained through sequence assembly, filtering, and chimera removal. Then, OTU / ASV clustering and annotation were performed. Based on the clustering results, alpha and beta diversity analyses were conducted. Based on the annotation results, classification information at each level was obtained, enabling correlation analysis of sample composition and differences in community structure among samples. Data from the control group and the 1600 mg / kg taurine-supplemented group were analyzed using independent samples t-tests in SPSS 26.0 software. Experimental results are expressed as mean ± standard error of mean, with P < 0.05 indicating statistical significance. The entire analysis process followed the guidelines. Figure 2 .

[0052] 3.3 Results and Analysis 3.3.1 Analysis of Dilution Curves and Species Abundance Distribution Curves To ensure the scientific rigor and reliability of the Muscovy duck cecal microbiota analysis, this study used dilution curves and rank abundance curves to evaluate the quality and coverage of the sequencing data. Dilution curves ( Figure 3 As shown in (a) of the figure, the Observed_otus values ​​of both groups of samples first increased and then stabilized with increasing sequencing depth, reaching a plateau at 4000 sequences. This indicates that the current sequencing depth can effectively reflect the main structural characteristics of the target microbial community, laying a reliable foundation for subsequent analysis. Species abundance distribution curves ( Figure 3 (b) shows a typical characteristic of rapid decline followed by gradual decline, which is consistent with the natural distribution pattern of bacterial species. Among them, the taurine group has a wider flat section and a slower rate of decline, while the control group has a steeper decline in the dominant species area. This indicates that the addition of 1,600 mg / kg taurine to the diet can make the abundance of dominant species in the Muscovy duck cecal flora more uniform and the proportion of rare species higher, thus initially improving the balance of the bacterial community structure.

[0053] 3.3.2 Alpha diversity analysis of cecal contents microbiota There were no significant differences in Chao1, ACE, Simpson, and Shannon indices between the Tau and CON groups. P >0.05)( Figure 4 ).

[0054] 3.3.3 Analysis of Beta Diversity of Cecal Content Microbiota Principal component analysis (PCA) results are as follows Figure 5 As shown in the PCA score chart, it is clear that the samples in the taurine group and the control group have obvious clustering trends. The samples in the taurine group have a high degree of clustering, and the clustering between groups is significant.

[0055] 3.3.4 Differences in the phylum, family, genus, and species level of the bacterial community in the cecal contents of Muscovy ducks Bar chart of species composition at different taxonomic levels ( Figure 6 The data showed that the dominant phylum in the cecal microbiota of Muscovy ducks was Bacteroidetes (Bacteroidetes). Bacteroidota Firmicutes ( Firmicutes Proteobacteria ( Proteobacteria ) and Actinobacteria ( Actinobacteriota The dominant phyla composition of the two groups of samples was basically the same, but there were slight differences in relative abundance. Compared with the CON group, the Tau group had a higher abundance. Bacteroidota The abundance was significantly increased, especially in the Tau group. Firmicutes / Bacteroidota The (F / B) ratio is relatively low. Proteobacteria The abundance was also relatively low. Meanwhile, the Tau group of desulfobacteria ( Desulfobacterota ), deferrobacteria ( Deferribacterota The abundance of harmful bacteria phyla such as ) was significantly reduced. Actinobacteriota Verruciformis (Periphera) Verrucomicrobiota The abundance of beneficial bacteria such as phyla 1 and 2 showed an increasing trend. Regardless of the taxonomic level of analysis, the overall structure of the cecal flora in the Tau group was more balanced and stable.

[0056] 3.3.5. Genus-level differential clustering heatmap analysis Clustering heatmap ( Figure 7 The data clearly showed the expression patterns of differentially expressed bacterial genera in the two groups of samples. Samples within the same group clustered together, while the differences between groups were significant. The taurine group was enriched with beneficial functional genera such as Corynebacterium and Unclassified Trichophyceae, while the CON group was enriched with potentially harmful genera such as g_Desulfovibrio.

[0057] 3.3.6 LEfSe Analysis The LEfSe analysis method was used to screen for microbial groups with significant differences between the Tau and CON groups. The results are as follows: Figure 8 As shown, the differentially enriched species in the Tau Formation include f_Marinifilaceae Xanthomonas spp. g_ Pseudoflavonifractor ) and Rumenococcus ( g_Ruminococcus ); while the differentially enriched species in the CON group include RF39 (o_RF39) and Enterococci ( f_Enterococcaceae ), RF39 ( f_RF39 ), Enterococcus ( g_ Enterococcus ), g_Jeotgalicoccus and RF39 ( g_RF39 ). Figure 9 This is a cladogram of the differentially expressed species, visually illustrating the distribution characteristics of these taxa on the phylogenetic tree. The branching structure from phylum to genus level shows that they form clear partitions on the phylogenetic tree, further validating the results of the LDA analysis.

[0058] In summary, taurine can improve the intestinal microecology of Muscovy ducks, enhancing their intestinal health and barrier function. Taurine can increase intestinal flora diversity, improve flora composition, increase the content of beneficial bacteria, inhibit the growth and reproduction of harmful bacteria, and improve the intestinal health of Muscovy ducks, laying an important theoretical foundation for the application of functional feed additives in the healthy breeding of Muscovy ducks.

[0059] 4. LC-MS analysis of the effects of taurine on the serum metabolome of Muscovy ducks 4.1 Test Methods Muscovy duck serum samples were collected, and metabolome sequencing was performed on the samples by Sanshu Biotechnology Co., Ltd.

[0060] (1) Metabolite extraction: The sample was slowly thawed at 4℃. An appropriate volume of sample (0.5-1 mL, freeze-drying concentration can be performed if the sample volume is too large) was accurately measured into a centrifuge tube. Four times the volume of extraction solution (methanol / acetonitrile, 1:1, v / v) was added. The sample was vortexed for 60 s and extracted by low-temperature ultrasonication for 30 min. The protein was precipitated at -20℃ for 1 h. Then, the sample was centrifuged at 12,000 rpm for 10 min at 4℃. The supernatant was vacuum dried and reconstituted with 100 μL of 30% acetonitrile solution. After vortexing, the sample was centrifuged at 12,000 rpm for 10 min at 4℃. The supernatant was then used for instrumental analysis.

[0061] (2) On-machine testing: During the testing process, equal amounts of all test samples are mixed to prepare quality control samples (QC), which are then uniformly inserted into the analytical sequence and tested together with the test samples to ensure the stability and reliability of the analysis process. The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high resolution mass spectrometry (Q Exactive HFX, Thermo, USA).

[0062] Liquid chromatography parameters: Column: Waters HSS T3 (100 × 2.1 mm, 1.8 μm); Mobile phase: Phase A was 0.1% formic acid-water solution, Phase B was 0.1% formic acid-acetonitrile solution; Flow rate: 0.3 mL / min; Column temperature: 40℃; Injection volume: 2 μL; Elution gradient: 0 min Phase A / Phase B (100:0, v / v), 1 min Phase A / Phase B (100:0, v / v), 4 min Phase A / Phase B (40:60, v / v), 6.5 min Phase A / Phase B (5:95, v / v), 6.6 min Phase A / Phase B (100:0, v / v), 8.0 min Phase A / Phase B (100:0, v / v). Samples were placed in an autosampler at 4℃ throughout the analysis. To avoid the influence of instrument signal fluctuations, continuous analysis of samples was performed in a randomized order. QC samples are uniformly inserted into the sample analysis sequence to monitor and evaluate the stability of the system and the reliability of the test data.

[0063] Mass spectrometry conditions: Primary and secondary spectra were acquired using a Thermo Q Exactive HFX high-resolution mass spectrometry system (USA). The system was equipped with an electrospray ionization (ESI) source, 40 arb sheath gas, 10 arb auxiliary gas, an ion spray voltage of +3000 V / -2800 V, a temperature of 350℃, and an ion transmission tube temperature of 320℃. The scanning mode was Full-ms-ddMS2, and the scanning method was positive / negative ion. The primary mass spectrometry scan range was 70-1050 Da, with a primary resolution of 70,000 and a secondary resolution of 17,500.

[0064] 4.2 Data Processing and Analysis (1) Metabolomics data preprocessing: The raw data were first preprocessed using Progenesis QI (Waters Corporation, Milford, USA) software for baseline filtering, peak identification, peak matching, retention time correction, and peak alignment to obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity. Peaks containing secondary mass spectrometry data were identified using commercial databases and Sanshu Bio's self-built metabolite secondary mass spectrometry database and corresponding fragmentation patterns.

[0065] (2) Multidimensional Statistical Analysis: Multidimensional statistical analysis mainly includes three parts: unsupervised principal component analysis (PCA), supervised partial least squares discriminant analysis (PLS-DA), and supervised orthogonal partial least squares discriminant analysis (OPLS-DA). Import the data matrix from the "Data Matrix" file into R software. First, use PCA analysis to observe the overall distribution among samples and the degree of dispersion between groups. Then, use PLS-DA and OPLS-DA analyses to distinguish the overall differences in metabolic profiles among samples and identify differentially expressed metabolites between groups. Variable importance of projection (VIP) represents the contribution of each metabolite to the differences in the PLS-DA and OPLS-DA models. Generally, a VIP value greater than 1 is considered an important variable.

[0066] (3) Differential Metabolite Analysis: The screening of differential metabolites mainly refers to two parameters: variable importance of projection (VIP) and probability value (P value). The VIP value refers to the variable projection importance of the first principal component in the PLS-DA or OPLS-DA model. It represents the contribution of each metabolite in the PLS-DA or OPLS-DA model to the difference. Generally, a VIP value greater than 1 is considered an important variable. The P value is the statistical test value of the difference in metabolite expression. It is calculated by the t-test (Studen's test) and is used to measure the significance of the hypothesis test results. Generally, a P value < 0.05 is considered statistically significant. Using the t-test combined with multivariate analysis of OPLS-DA, metabolites with P value < 0.05, VIP > 1, FC > 1.5 or < 0.667 are considered differential metabolites for subsequent bioinformatics analysis.

[0067] (4) Advanced Analysis: This mainly involves a series of metabolic pathway analyses based on the KEGG database. The KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.kegg.jp / ) database is a commonly used resource for pathway research, providing insights into the advanced functions and benefits of biological systems such as cells, organisms, and ecosystems at the genomic and molecular levels. Metabolites can be annotated using the KEGG database to obtain metabolic pathway maps. By integrating information from the HMDB, KEGG compound, and LIPID MAPS databases, KEGG IDs of differentially expressed metabolites are obtained. Then, pathway annotation analysis is performed on these differentially expressed metabolites using the KEGG database to obtain their metabolic pathway maps.

[0068] 4.3 Results and Analysis 4.3.1 Multidimensional Statistical Analysis Figure 10 LC-MS analysis was performed on Muscovy duck serum samples from the CON group (pink dots) and the Tau group (blue dots). Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed sequentially. The results are as follows: (1) PCA analysis was performed on two principal components. The R2X value (explaining the variance of the sample variable) was 34.29%. The contribution rate of the first principal component (PC1) was 20%, and the contribution rate of the second principal component (PC2) was 14.29%. The contribution rate of the first principal component (20%) was greater than that of the second principal component (14.29%), indicating that the largest direction of variation in the data may be related to the treatment effect. The samples of the CON group and the Tau group showed partial overlap in the PCA score plot, but a clear grouping trend was already shown, and the samples within the group had good clustering.

[0069] (2) PLS-DA analysis revealed two principal components: R2X = 41.5%, R2Y = 0.997, and Q2 (model predictive ability) = 0.891. The contribution rate of the first principal component (Comp1) was 25.7%, and the contribution rate of the second principal component (Comp2) was 15.8%. The R2Y of 0.997 indicates that the model can distinguish between the CON group and the Tau group well. The two groups of samples are clearly grouped in the PLS-DA score graph, and the samples within the groups are clustered. The Q2 of 0.891 indicates that the model has good generalization ability.

[0070] (3) OPLS-DA analysis yielded one principal component and two orthogonal components, with R2X of 40.5%, R2Y of 0.997, and Q2 of 0.423. The contribution rate of the first principal component (Comp1) was 21.9%, and the contribution rate of the second principal component (Comp2) was 18.6%. The high R2Y value indicates a significant difference in the metabolic profiles of the Tau group and the CON group. The two groups were significantly separated in the OPLS-DA score map, further demonstrating the significant difference in metabolic profiles between the groups.

[0071] 4.3.2 Screening of Differential Metabolites The OPLS-DA method, combining t-test and multivariate analysis, was used to screen metabolites with P value < 0.05, VIP > 1, FC > 1.5 or < 0.67 as the screening threshold for differentially expressed metabolites. A total of 31 differentially expressed metabolites were screened in the CON group and the Tau group, of which 10 were significantly upregulated and 21 were significantly downregulated. Figure 11 The specific differences in metabolites between the Tau group and the CON group are shown in Tables 7 and 8. In the metabolomics analysis of the Tau-treated group and the CON group, 20 metabolites with the most significant fold changes were identified. The upregulated metabolites mainly involved antioxidant and lipid metabolism processes, while the downregulated metabolites were concentrated in glycolysis and purine metabolism pathways, suggesting that Tau may affect ovarian function by regulating energy metabolism and redox status. Figure 12 ).

[0072] Table 7. Differential metabolites in the serum of Muscovy ducks in the taurine group and the control group. Table 8. Differential metabolites in the serum of Muscovy ducks in the taurine group and the control group. 4.3.3. KEGG Pathway Enrichment Analysis of Differential Metabolites The metabolic pathways of differentially metabolized serum metabolites in the taurine group and the control group were analyzed, and the results are as follows: Figure 13 As shown, 10 significantly differentially affected metabolite pathways related to ovarian development were identified, including: tyrosine metabolism, cysteine ​​and methionine metabolism, pyrimidine metabolism, phenylalanine metabolism, ABC transporters, sulfur metabolism, pyruvate metabolism, nicotinamide and nicotinamide metabolism, purine metabolism, and the tricarboxylic acid cycle (TCA cycle).

[0073] 4.3.4 Correlation Heatmap Analysis of Differential Metabolites Correlation heatmap analysis was used to analyze the association patterns of differentially expressed metabolites in the ovary. Figure 14 The results showed that most metabolites were strongly correlated, indicating that their changing trends were very consistent; some negatively correlated clusters also existed, indicating that there was antagonistic regulation between different functional modules. These correlation patterns suggest that taurine, by regulating the core metabolic network and secondary metabolic processes, alters the overall metabolic profile of Muscovy duck ovaries, providing targeted metabolic support for follicle development.

[0074] 5. Effects of taurine on ovarian development in Muscovy ducks based on transcriptome analysis 5.1 Test Methods 5.1.1 Transcriptome Sequencing After slaughter, granulosa cell samples were collected from the ovaries of Muscovy ducks, and transcriptome sequencing was performed on these samples by Sanshu Biotechnology Co., Ltd. A schematic diagram of the transcriptome sequencing experimental procedure is shown below. Figure 15 As shown.

[0075] (1) Total RNA extraction and quality control: The basic principle of RNA extraction is to prevent RNA degradation during the extraction process and ensure that high-quality RNA with good integrity and purity is extracted from the target sample. Therefore, appropriate extraction reagents and extraction methods should be selected according to the species of the sample. After extracting total RNA from the tissue sample, the concentration and purity of the extracted RNA were detected using Nanodrop 2 000 (Thermo Fisher), and RNA integrity was detected using Agilent 2 100 / 5 400 (Agilent Technologies, CA, USA). Library construction can only be carried out after the sample quality control meets the standards.

[0076] (2) Sequencing library construction: mRNA was enriched from purified Total RNA using Oligo (dT) magnetic beads and polyA base pairing. The enriched mRNA was a complete RNA sequence with an average length of several kb. However, second-generation high-throughput sequencing platforms are designed for short sequence fragments. Therefore, fragmentation buffer was added to randomly break down the enriched mRNA. Using reverse transcriptase as a template, six-base random primers were added to synthesize the first strand of cDNA. Buffer, dNTPs, and DNA polymerase I were added to synthesize the second strand of cDNA. The double-stranded cDNA was then purified using AMPure XP beads to form a stable double-stranded structure. The double-stranded cDNA had sticky ends, which were padded with End Repair Mix to form blunt ends. An "A" base was then added to the 3' end to connect the Y-shaped sequencing adapter.

[0077] (3) Sequencing on the second-generation high-throughput sequencing platform: library enrichment, PCR amplification; 2.2% agarose gel recovery of the target band; Qubit for library detection; TBS380 (Picogreen) quantification, mixed according to the data ratio for sequencing; sequencing on the second-generation high-throughput sequencing platform (sequencing strategy: PE150).

[0078] 5.1.2 Validation of RNA-Seq results by qPCR This study used qPCR to verify the accuracy of RNA-Seq data. Ten genes were randomly selected, and their sequences were retrieved from the NCBI website. Primers were designed using Primer 5.0 software. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the primer sequences are shown in Table 9 below. Real-time quantitative PCR amplification was then performed, and the specific amplification reaction system is shown in Table 10 below.

[0079] Table 9 Primer Sequences Table 10 Specific reaction system for real-time quantitative PCR Reaction program: Each sample was replicated in 3 steps. The reaction program was: 95℃ for 30 sec; 95℃ for 15 s, 60℃ for 40 sec, with a cycle number of 40. Melting curve stage: 95℃ for 15 s; 60℃ for 1 min; 95℃ for 30 s; 60℃ for 15 s.

[0080] 5.2 Data Processing and Analysis (1) The FastP software was used to perform data quality control on all raw reads obtained from the second-generation high-throughput sequencing platform. After removing low-quality reads and adapter contamination, high-quality reads were obtained. All subsequent analyses were based on the clean reads.

[0081] (2) Use STAR software to map the Clean Data with the reference genome, obtain the location information of the Reads on the reference genome, and the characteristic information of the sequencing samples, and generate a bam file.

[0082] (3) The transcriptome data were assessed for overall quality using RSeQC software. Sequencing data that passed the quality assessment were then subjected to bioinformatics analysis. In-depth transcriptome analysis included differential expression analysis, alternative splicing analysis, new transcript prediction, and variation analysis. The specific analysis workflow is as follows: Figure 16 As shown.

[0083] 5.3 Results and Analysis 5.3.1 Reference sequence alignment analysis This experiment compared the effects of dietary taurine supplementation on gene expression changes in Muscovy duck ovaries and analyzed the gene expression differences between the CON group and the taurine group. After sequencing and filtering, each sample yielded 5.63–8.74 G of valid data, with a Q30 percentage greater than 98.26% and GC content consistently between 45.91% and 48.59%. Alignment of the high-quality sequences from 12 samples with the Muscovy duck reference genome yielded an overall alignment rate of 64.29%–90.24%, a unique position alignment rate of 61.77%–87.92%, and a multi-position alignment rate of 2.00%–4.59%. The results indicate that the sequencing data is sufficient in quantity and of excellent quality, with low interference from multi-position alignments, meeting the needs of subsequent gene expression quantification and differential gene screening analyses (Tables 11 and 12).

[0084] Table 11 Comparison of Sample and Reference Sequences Table 12 Comparison results of effective sequences and reference genome 5.3.2 Differential Gene Screening and Identification Based on |log2Fold Change|≥2 and P<0.05, a total of 8 differentially expressed genes (DEGs) were identified between the control group and the taurine group, of which 7 were significantly upregulated and 1 was significantly downregulated. Figure 17 (a) Compared with CON, the Tau group significantly increased the relative abundance of elastic microfibril interface protein 2 (EMILIN2), peptidylprolyl cis-trans isomerase (FKBP10), sugar-phosphate exchanger 2 (SLC37A2), G protein-coupled receptor family C5 member B (GPRC5B), protein homology domain 2-included protein (CBLN4), and Kruppel-like factor 4 (KLF4), while significantly decreasing the relative abundance of adenylate cyclase (ADCY3). Correlation analysis showed significant interactions among EMILIN2, FKBP10, SLC37A2, GPRC5B, CBLN4, and KLF4, indicating that these genes interact ( Figure 17 (3b in the middle).

[0085] 5.3.3 GO enrichment analysis GO functional classification and statistical analysis were performed on the differentially expressed genes in the two groups of samples. The results showed that the differentially expressed genes covered biological processes (BP), cellular components (CC), and molecular functions (MF). The top five differentially expressed genes with the highest enrichment were: glycerol-3-phosphate transmembrane transport, negative regulation of leukocyte adhesion to arterial endothelial cells, negative regulation of leukocyte adhesion to vascular endothelial cells, positive regulation of hemoglobin biosynthesis, and regulation of muscle hyperplasia. In the cellular component classification, the top five differentially expressed genes with the highest enrichment were: soluble guanylate cyclase complex, euchromatin, extracellular exosomes, extracellular vesicles, and extracellular organelles. In the molecular function classification, the top five differentially expressed genes with the highest enrichment were: glycerol-3-phosphate transmembrane transporter activity, RNA polymerase II-specific DNA-binding transcription factor activity, hexose-phosphate:inorganic phosphate antitransporter activity, organic phosphate:inorganic phosphate antitransporter activity, and hexose-phosphate transmembrane transporter activity. Figure 18 ).

[0086] 5.3.4 KEGG enrichment analysis KEGG enrichment analysis was used to screen for the main signaling pathways involved in differentially expressed genes between the CON and Tau groups. Figure 19As shown, among the top 30 enriched pathways, the top ten are, in order: Chemical carcinogenesis-receptor activation, Longevity regulating pathway-multiple species, Gap junction, Ovarian steroidogenesis, Regulation of lipolysis inadipocytes, Thyroid hormone synthesis, Cortisol synthesis and secretion, Circadian entrainment, GABAergic synapse, and Progesterone-mediated oocyte maturation. The enriched signaling pathways related to the ovary include Ovarian steroidogenesis and Progesterone-mediated oocyte maturation. In this sequencing study, genes related to these two signaling pathways, including GPRC5B, SLC37A2, and KLF4, were significantly upregulated in granulosa cells of Muscovy duck ovarian tissue after feeding them a diet supplemented with Tau. This indicates that Tau has a regulatory effect on granulosa cells and the ovary.

[0087] 5.3.5 RT-qPCR Validation Based on the above analysis, seven differentially expressed genes in ovarian tissue were selected for RT-qPCR detection. The relative expression levels of these differentially expressed genes in Muscovy duck ovarian granulosa cells are shown in the table below. Figure 20 Compared to the CON group, the Tau group GPRC5B, SLC37A2, KLF4 CBLN4, FKBP10 and EMILIN2 The expression level was significantly increased. ADCY3 The expression level of tau was significantly reduced. These results indicate that tau can promote the proliferation of granulosa cells in Muscovy duck ovaries, which is of great significance for improving ovarian development. The experimental results are consistent with the expression trend of RNA-seq data, demonstrating the reliability of this transcriptome sequencing and analysis.

[0088] 5.3.6 Correlation analysis between differentially expressed genes and metabolites To further analyze the regulatory relationship between differentially expressed metabolites and key genes, this study constructed a Pearson intergroup correlation heatmap, which shows the correlation between differentially expressed metabolites and differentially expressed genes. Figure 21 The results showed that N-Oleoyl Taurine and N-Palmitoyl Taurine were related to... GPRC5B, SLC37A2, KLF4, CBLN4, FKBP10, EMILIN2 Both showed a significant positive correlation; (±)-Catechin and GPRC5B, SLC37A2, KLF4, CBLN4, FKBP10, EMILIN2 It shows a significant positive correlation with ADCY3 It shows a significant negative correlation.

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of taurine in any of the following: (1) Prepare products to improve the egg production performance of Muscovy ducks; (2) To prepare products that improve the quality of Muscovy duck eggs; (3) Prepare products that improve the intestinal tract of Muscovy ducks; (4) Prepare products that improve follicle development; (5) Prepare products that improve the reproductive performance of Muscovy ducks; (6) Prepare products that improve the intestinal flora of Muscovy ducks; (7) Prepare products that improve ovarian development.

2. The application according to claim 1, characterized in that, The improvement of Muscovy duck egg production performance includes increasing egg production rate and egg weight.

3. The application according to claim 1, characterized in that, The improvement of Muscovy duck egg quality includes increasing eggshell strength, yolk percentage, and Haugh unit, as well as improving yolk color.

4. The application according to claim 1, characterized in that, The improvement of Muscovy duck intestines includes increasing the height of the intestinal villi and the villi-to-villi ratio, and increasing the content of acetic acid, propionic acid and butyric acid in the intestines.

5. The application according to claim 1, characterized in that, The improvement of follicle development includes increasing the ovarian index, grade follicle count, and oviduct index in Muscovy ducks.

6. The application according to claim 1, characterized in that, The improvement of gut microbiota includes increasing the relative abundance of Flavobacterium, Pseudomonas, Riken Bacteriaceae, and Bacillus spp., and decreasing the relative abundance of Halococcus and Enterococcus.

7. The application according to claim 1, characterized in that, The products include formulations, feed additives, and feed.

8. A formulation, characterized in that, The formulation includes taurine and excipients.

9. A feed additive, characterized in that, The feed additives include taurine and auxiliary ingredients.

10. A feed, characterized in that, The feed includes the feed additives and basal diet as described in claim 9.

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