Application of dihydromyricetin in promoting fish ovary development

Abstract

This invention relates to the application of dihydromyricetin in promoting ovarian development in fish, particularly its use in increasing the gonadal index and improving ovarian tissue structure and ultrastructure.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to the application of dihydromyricetin in promoting ovarian development in fish. Background Technology

[0002] Ovarian development in fish is a crucial physiological process affecting their reproductive efficiency and population propagation. Especially in aquaculture, improving gonadal development is essential for ensuring seedling supply and enhancing farming profitability. Currently, common methods for promoting ovarian development in fish include exogenous hormone induction (such as gonadotropin-releasing hormone analogs GnRHa and human chorionic gonadotropin HCG), fortified feeds (such as supplementing with highly unsaturated fatty acids, vitamin E, and lecithin), and environmental factor regulation (such as periodic regulation of light and water temperature). However, these methods still have certain limitations: exogenous hormones may cause endocrine disorders, residue risks, and germplasm degradation in fish; conventional fortified feeds have unstable effects and high costs; and environmental regulation methods are limited by facility conditions and cannot achieve precise and stable development promotion.

[0003] In recent years, natural plant extracts have gradually become a research hotspot in the aquaculture field due to their advantages such as being green, safe, and less likely to produce residues. Dihydromyricetin (DMY), as a natural flavonoid compound, has been reported to have various biological activities such as antioxidant, anti-inflammatory, and lipid-lowering effects; however, there are currently no publicly available reports on dihydromyricetin promoting ovarian development or improving oocyte quality in fish. Summary of the Invention

[0004] The present invention aims to provide the application of dihydromyricetin in promoting ovarian development in fish, particularly its use in increasing the gonadal index and improving the structure and ultrastructure of ovarian tissue.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: the application of dihydromyricetin in promoting ovarian development in fish.

[0006] Furthermore, dihydromyricetin is added to fish diets as a feed additive.

[0007] Furthermore, the amount of dihydromyricetin added is 0.1% to 0.5% of the diet mass.

[0008] Furthermore, the purity of the dihydromyricetin is ≥90%.

[0009] Furthermore, the promotion of ovarian development in fish includes increasing the gonadal index, inhibiting ovarian degenerative changes, maintaining the morphological and functional integrity of oocytes, protecting the ovarian microenvironment structure, and / or optimizing the ovarian ultrastructure.

[0010] Furthermore, the inhibition of ovarian degenerative changes refers to the inhibition of degenerative changes in oocytes, oogonia, primary oocytes, follicular membranes, corona radiata, and / or zona pellucida.

[0011] Furthermore, maintaining the morphological and functional integrity of oocytes refers to stabilizing the subcellular structure of oocytes and / or ensuring the normal progress of yolk synthesis and gene expression.

[0012] Furthermore, the protection of the ovarian microenvironment structure refers to maintaining the homeostasis of the ovarian stroma and ensuring the microenvironment for the synthesis of reproductive hormones and the exchange of substances.

[0013] Furthermore, the optimization of ovarian ultrastructure refers to coordinating yolk and lipid reserves, enhancing the stability of oocyte membrane and cortical structures, improving cellular transcription and energy metabolism efficiency, promoting protein synthesis and processing, and / or regulating autophagy and homeostasis.

[0014] The beneficial effects of this invention are: 1. This invention provides a green and safe new approach to promote development: The novel application of dihydromyricetin in promoting ovarian development in fish is discovered and disclosed for the first time. Compared to traditional exogenous hormone induction methods, the dihydromyricetin used in this invention is a natural plant extract, possessing advantages such as being green, safe, residue-free, and unlikely to cause endocrine disorders in fish, thus meeting the needs of the aquaculture industry's transformation towards environmentally friendly and sustainable development.

[0015] 2. Effectively Enhances Key Indicators of Ovarian Development: By using dihydromyricetin as a feed additive, the gonadal index of fish can be significantly increased, demonstrating its promoting effect on ovarian development at a macroscopic level. This provides a direct and effective technical means to improve the reproductive performance of broodstock and ensure an efficient supply of fry.

[0016] 3. Significantly improves ovarian tissue and cell structure: This invention can play a positive role at the histological and cellular levels. On the one hand, it can effectively inhibit ovarian degenerative changes and protect the integrity of key structures such as oocytes and follicular membranes; on the other hand, by maintaining the stability of the subcellular structure of oocytes and optimizing the ovarian microenvironment, it ensures the normal expression of yolk synthesis and reproduction-related genes, thereby comprehensively improving the quality and function of oocytes.

[0017] 4. Refined Regulation of Ovarian Ultrastructure to Improve Oocyte Quality: This invention delves into the ultrastructural level, optimizing oocyte development quality from multiple dimensions by coordinating yolk and lipid reserves, enhancing the stability of cell membrane and cortical structures, improving the energy metabolism efficiency of organelles such as mitochondria, promoting protein synthesis and processing in structures such as the endoplasmic reticulum, and regulating autophagy to maintain homeostasis. This refined regulation lays a solid material foundation for cultivating high-quality oocytes and improving fertilization and hatching rates.

[0018] 5. Controllable cost and convenient application: Dihydromyricetin can be used as a feed additive at a dosage of only 0.1% to 0.5% of the daily ration, with moderate purity requirements (≥90%), making it easy to integrate with existing feed production processes. This method does not require complex equipment modifications or environmental control, and the cost is relatively controllable, facilitating large-scale promotion and application in aquaculture practices. Attached Figure Description

[0019] Figure 1 This is a histological diagram of a specific part of the ovary in the control group of *Hymenopause chinensis*. Figure 2 This is a histological diagram of another part of the ovary in the control group, *Hymenopause chinensis*. Figure 3 Histological diagram of a specific part of the ovary in DMY group flower; Figure 4 Histological diagram of another part of the ovary in DMY group flower; Figure 5 Ultrastructure of the ovary in the control group (e.g., *Hymenopausal*); AF shows the ultrastructure of the ovary at different magnifications in different locations. Figure 6 The image shows the ultrastructure of the ovary in the DMY group of *Rhizopus chinensis*; AF shows the ultrastructure of the ovary at different magnifications in different locations.

[0020] Figures 1-4 In this diagram, Oo1: Phase I oocyte; Oo2: Phase II oocyte; Oo3: Phase III oocyte; Oo4: Phase IV oocyte; Oo5: Phase V oocyte; CA: Cortical vesicle; CR: Cortical vacuole; FT: Follicular layer; YG: Yolk granules; ZR: Radial bands; FA: Follicular atresia; LAO: Late atretic oocyte; LAF: Late atretic follicle; OD: Oocyte fragments; FC: Follicular cells; OC: Oocyte cytoplasm; YM: Yolk matrix.

[0021] Figures 5-6 In this context, N represents the nucleus; ZR represents the zona radiata; YP represents the yolk platelets; CA represents the cortical vesicles; Mt represents the mitochondria; FCP represents the follicular cell processes; MC represents the mitochondrial cloud; Rib represents the ribosomes; AV represents the autophagosomes; ER represents the endoplasmic reticulum; Golgi represents the Golgi apparatus; DB represents the compact body; Vesicle represents the vesicle; OCM represents the oocyte cytoplasm; LD represents the lipid droplets; and YG represents the yolk granules. Detailed Implementation

[0022] The following detailed description illustrates the specific implementation method: 1. Materials and Methods 1.1 Chemical reagents Dihydromyricetin (DMY, purity > 98.0%) was purchased from Shaanxi Angxu Biotechnology Co., Ltd.

[0023] 1.2 Fish farming Healthy and disease-free carp (body length: 20.0 ± 3.0 cm, weight 55.0 ± 5.2 g) were purchased from a breeding farm in Jiangkou County, Tongren City. They were temporarily raised in a circular pond with a diameter of 2 meters for one week, with 20 carp in each pond. The water temperature was kept constant at 18±1℃, and the light cycle was 16:8 h (light:dark). They were fed with Tongwei Fishery Carp Compound Feed 101 at 7 am and 7 pm each time, with 12 g of feed per pond each time. Half of the water was changed every day.

[0024] One week later, the healthy and vigorous tilapia were randomly divided into two groups of 60 each and placed in three circular ponds for rearing. The control group was fed a basal diet and the DMY group was fed a diet supplemented with 0.25% dihydromyricetin. The experiment lasted for 6 weeks.

[0025] 1.3 Sample Collection After the feeding experiment, the fish were weighed and euthanized by soaking in MS-222 (300 mg / L) until the gill covers stopped moving. The fish were dissected, the ovaries were collected, weighed, and the tissue was divided into two parts: (1) a 0.1 cm × 0.1 cm × 0.2 cm tissue block was placed in 2.5% glutaraldehyde fixative and fixed overnight at 4°C; (2) a 1 cm × 1 cm × 0.5 cm tissue block was fixed overnight at 4°C with 4% paraformaldehyde. Three replicate samples were collected from each pond in each group.

[0026] Gonadal Index (GI) = [Glandular weight / Body weight] × 100 1.4 Histological analysis After fixation with 4% paraformaldehyde, the ovaries were dehydrated by graded ethanol, embedded in paraffin, and cut into 4 μm thick serial sections, which were then stained with hematoxylin and eosin (H&E) (Fuzhou Feiji, China). The morphology and structure of the ovaries were observed under an Olympus CX31 microscope (Tokyo, Japan).

[0027] 1.5 Transmission Electron Microscopy Technique Ovaries were fixed in 2.5% glutaraldehyde solution for 24 hours, then post-fixed with 1% osmium tetroxide, dehydrated via an ethanol gradient, and then embedded in epoxy resin. Ultrathin sections of 70 nm thickness were cut, stained with uranium acetate and lead citrate, and observed under a Hitachi HT7700 transmission electron microscope.

[0028] 2. Results 2.1 Dihydromyricetin significantly increased the gonadal index of *Gynostemma pentaphyllum*. The results of calculating the gonadal index showed that the gonadal index of female crape myrtle in the diet supplemented with 0.25% dihydromyricetin was 5.73±1.22, which was significantly higher than that of the control group, indicating that dihydromyricetin can effectively promote the gonadal development of crape myrtle.

[0029] Table 1. Statistics of the Gonadal Index of the Flower Bud.

[0030] Note: represent p <0.05 2.2 Dihydromyricetin significantly improved the morphology and structure of the ovaries in *Gynostemma pentaphyllum*. The results are as follows Figures 1-4 As shown in the figure; the explanations of the relevant reference numerals in the figure are shown in Table 2 below.

[0031] Table 2 Figures 1-4 Explanation of the meaning of the symbols in the attached figures

[0032] 2.2.1 Inhibiting ovarian degenerative changes As attached Figure 1 , Figure 2 As shown, the control group had oocytes in all phases, but fewer oocytes were observed in phases IV (Oo4) and V (Oo5), and numerous follicular atresia (FA) were present. This was characterized by oocyte vacuolation leading to oocyte fragmentation (OD) (numerous large and irregular vacuoles appeared inside the oocyte, the yolk matrix (YM) showed cracks and dissolution, and the yolk material almost completely dissolved and disappeared, leaving only vacuolated structures), and follicular membrane dissociation (the follicular membrane, composed of granulosa cells and tunica vaginalis cells, completely separated from the oocyte, with only a few cells remaining at the edges; the entire structure was loose and collapsed), among other degenerative trends. (See attached image.) Figure 3 , Figure 4As shown, no obvious oocyte degeneration structures were observed in the ovarian sections of the DMY group. The developmental hierarchy of oogonia (Oo1), primary oocytes (Oo2-Oo4), and secondary oocytes (Oo5) was clear, and the structures such as the follicular layer (FT), cortical vesicles (CA), cortical vacuoles (CR), and zona pellucida (ZR) were intact. Furthermore, the proportion of oocytes in phase III and above (Oo3-Oo5) was increased. Although late atretic oocytes (LAO) were present (characterized by significant shrinkage, deformation, and reduced volume of the oocytes; cytoplasmic vacuolation, dissolution, and uneven staining); cells... The nucleus undergoes condensation, fragmentation, dissolution, or even complete disappearance; yolk granules disintegrate, fuse, and dissolve. Late atretic follicles (LAF) are characterized by severe oocyte dissolution, with almost no visible structure; follicular cells proliferate extensively, invade, and engulf oocyte remnants; the zona radiata or zona pellucida ruptures, shrinks, or remains; the entire follicle shrinks in volume, becoming a mass of residual structure; and is eventually completely absorbed by the ovarian tissue. However, the overall extent of degeneration is far lower than in the control group, suggesting that DMY can inhibit degenerative changes in ovarian tissue and maintain the normal developmental process of germ cells.

[0033] 2.2.2 Maintaining the morphological and functional integrity of oocytes As attached Figure 1 , Figure 2 As shown, although oocytes were visible at all time stages in the control group, some follicles exhibited atresia (FA), and phenomena such as uneven cytoplasmic staining and abnormal nucleocytoplasmic ratio were observed. (See attached image.) Figure 3 , Figure 4 As shown, the nuclei and nucleoli of oocytes in the DMY group were clearly visible, and the yolk granules (YG) in the cytoplasm were evenly distributed, suggesting that DMY can stabilize the subcellular structure of oocytes, ensure the normal functioning of physiological functions such as yolk synthesis and gene expression, and reduce the risk of germ cell apoptosis.

[0034] 2.2.3 Protecting the ovarian microenvironment structure As attached Figure 1 , Figure 2 As shown, in the control group, the cellular arrangement of the ovarian stroma was disordered, the connection stability between the follicular layer (FT) and the oocyte was slightly weaker, and follicular atresia (FA) was observed, with obvious separation and gaps between the follicular layer (FT) and the oocyte, and a significant decrease in connection stability. In Oo4, the connection between the follicular cells (FC) in the oocyte cytoplasm (OC) and the oocyte became loose, and the integrity of the follicular layer (FT) was disrupted. In Oo5, the connection between the follicular cells (FC) in the oocyte cytoplasm (OC) and the oocyte was disrupted, and the follicular layer (FT) showed breakage and curling. (See attached image) Figure 3 , Figure 4As shown, in the ovarian stroma of the DMY group, the follicular cells (FC) were arranged regularly, and the connective tissue was not loose or inflammatory, indicating that DMY can maintain the homeostasis of the ovarian stroma, ensure the microenvironment for the synthesis of reproductive hormones and the exchange of substances, and provide support for oocyte development.

[0035] 2.3 Dihydromyricetin significantly improved the ultrastructure of the ovary in *Gynostemma pentaphyllum*. The results are attached. Figures 5-6 As shown; Figures 5-6 The relevant reference numerals in the accompanying drawings are explained in Table 3 below.

[0036] Table 3 Figures 5-6 Explanation of the meaning of the symbols in the attached figures

[0037] 2.3.1 Optimization of Reproduction-Related Structures 2.3.1.1 More coordinated egg yolk and lipid reserves like Figure 5 As shown, in the control group, the yolk precursor material (YP) in oocytes was in a basal developmental state, with only a few scattered lipid-related structures in the cytoplasm. The spatial distribution and synergy between yolk development and lipid reserves were weak. Figure 6 As shown, the yolk granules (YG) in the DMY group were more plump, and the yolk platelets (YP) and lipid droplets (LD) showed a typical cooperative distribution pattern—the lipid droplets were arranged in an orderly manner around the yolk structure, and the two were highly matched in spatial distribution. This feature indicates that DMY can effectively promote the coordinated development of yolk synthesis and lipid reserves, and provide more sufficient and balanced nutritional support for the maturation process of oocytes.

[0038] 2.3.1.2 The oocyte membrane and cortex structure are more stable. As attached Figure 5 As shown, although the cytoplasmic matrix (OCM) of oocytes in the control group was intact, the distribution of cortical vesicles was relatively random, and structures such as mitochondria (Mt) and ribosomes (Rib) were loosely arranged. The connection between follicular cell processes (FCPs) and the cytoplasm was also relatively loose. (See attached image.) Figure 6 As shown, the oocytes in the DMY group exhibited more regular and orderly arrangement of vesicles on the surface of the cytoplasmic matrix (OCM), denser structural connections between follicular cell processes (FCPs) and the cytoplasm, and more regular distribution of membrane-associated organelles such as mitochondria (Mt) and endoplasmic reticulum (ER). This suggests that DMY can enhance the efficiency of material transport in the oocyte membrane, optimize the structural stability of the cortex, and lay a better structural foundation for the subsequent fertilization process.

[0039] 2.3.2 Optimization of organelle function 2.3.2.1 Improved transcriptional and energy metabolism efficiency As attached Figure 5 As shown, although the mitochondria (Mt) and mitochondrial clouds (MC) in the oocytes of the control group were structurally intact, their distribution density was at a basal level, and their spatial association with the endoplasmic reticulum (ER) was weak. Meanwhile, the nucleus (N) had a regular morphology, an intact nuclear membrane, chromatin predominantly euchromatin, sparsely distributed nuclear pores, and transcriptional activity at a basal level. (See attached image.) Figure 6 As shown, the DMY group had a slightly higher mitochondrial (Mt) density, a tighter fit between mitochondria and endoplasmic reticulum (ER), and a clearer mitochondrial cristae structure. Correspondingly, the nucleus (N) volume was slightly larger, the nucleolus was more prominent, and the number of nuclear pore complexes was abundant, suggesting that nucleocytoplasmic communication was more active and overall transcriptional and metabolic capacity was enhanced.

[0040] 2.3.2.2 Protein synthesis and processing are more active. As attached Figure 5 As shown, in the control group, ribosomes were evenly distributed in a basal state, Golgi vesicle secretion was at a normal level, and the binding of the endoplasmic reticulum (ER) to ribosomes was relatively loose; as shown in the attached figure. Figure 6 As shown, the DMY group exhibits a denser distribution of ribosomes (Rib), more active Golgi vesicle budding, and tighter membrane connections between the endoplasmic reticulum (ER) and the Golgi apparatus (Golgi), suggesting more active protein synthesis and processing.

[0041] 2.3.2.3 Fine Regulation of Autophagy and Homeostasis As attached Figure 5 As shown, the mitochondrial (Mt) structure in the control group remained intact, while autophagosomes (AV) and dense bodies (DB) were at basal levels; as shown in the attached figure. Figure 6 As shown, the mitochondria (Mt) in the DMY group had a more regular morphology and no obvious structural disorder was observed, but the number of dense bodies (DB) increased, indicating that DMY can maintain cell homeostasis and self-renewal, and perform organelle quality control more efficiently.

[0042] 2.3.3 Enhanced intercellular communication As attached Figure 5 As shown, in the control group, the follicular cell process (FCP) interface with the oocyte was smooth, with fewer processes, loose connections, and intercellular communication at a basal level. (See attached image.) Figure 6 As shown, the DMY group showed an increase in follicular cell protrusions (FCPs) and a tighter connection with the oocyte, indicating that DMY can enhance intercellular communication between the oocyte and follicular cells.

[0043] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. Application of dihydromyricetin in promoting ovarian development in fish.

2. The application of dihydromyricetin according to claim 1 in promoting ovarian development in fish, characterized in that: Dihydromyricetin is added to fish diets as a feed additive.

3. The application of dihydromyricetin according to claim 2 in promoting ovarian development in fish, characterized in that: The amount of dihydromyricetin added is 0.1% to 0.5% of the diet weight.

4. The application of dihydromyricetin according to claim 3 in promoting ovarian development in fish, characterized in that: The purity of the dihydromyricetin is ≥90%.

5. The application of dihydromyricetin according to any one of claims 1 to 4 in promoting ovarian development in fish, characterized in that: The promotion of ovarian development in fish includes increasing the gonadal index, inhibiting ovarian degenerative changes, maintaining the morphological and functional integrity of oocytes, protecting the ovarian microenvironment structure, and / or optimizing the ovarian ultrastructure.

6. The application of dihydromyricetin according to claim 5 in promoting ovarian development in fish, characterized in that: The inhibition of ovarian degenerative changes refers to the inhibition of degenerative changes in oocytes, oogonia, primary oocytes, follicular membranes, corona radiata, and / or zona pellucida.

7. The application of dihydromyricetin according to claim 5 in promoting ovarian development in fish, characterized in that: Maintaining the morphological and functional integrity of oocytes refers to stabilizing the subcellular structure of oocytes and / or ensuring the normal synthesis of yolk and gene expression.

8. The application of dihydromyricetin according to claim 5 in promoting ovarian development in fish, characterized in that: The protection of the ovarian microenvironment structure refers to maintaining the homeostasis of the ovarian stroma and ensuring the microenvironment for the synthesis of reproductive hormones and the exchange of substances.

9. The application of dihydromyricetin according to claim 5 in promoting ovarian development in fish, characterized in that: The optimization of ovarian ultrastructure refers to coordinating yolk and lipid reserves, enhancing the stability of oocyte membrane and cortical structures, improving cellular transcription and energy metabolism efficiency, promoting protein synthesis and processing, and / or regulating autophagy and homeostasis.

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