A Chinese herbal medicine compound feed additive for promoting the growth and ovary development of rice field eel

Abstract

The application discloses a Chinese herbal medicine compound feed additive for promoting the growth and ovary development of ricefield eels, which comprises 2.5-3.5 parts of semen cuscutae, 2.5-3.5 parts of herba epimedii, 1.0-2.0 parts of radix astragali, 1.0-2.0 parts of angelica sinensis and 0.5-1.5 parts of ligustrum lucidum. The five kinds of dried Chinese herbal medicines are ground into medicinal powder respectively and uniformly mixed to obtain the Chinese herbal medicine compound feed additive. The Chinese herbal medicine compound feed additive is weighed according to the wet weight of the surimi before feeding, directly added into the surimi, uniformly dispersed in the surimi through repeated kneading or mechanical stirring, and the surimi feed containing the Chinese herbal medicine compound is prepared. The application realizes the reproductive regulation through the feed adding mode, does not depend on the injection of exogenous hormones, and is convenient for large-scale application in the intensive cultivation of ricefield eel parents.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture feed additive technology, specifically relating to a compound feed additive of traditional Chinese medicine for aquaculture, and particularly to a compound feed additive of traditional Chinese medicine that targets and promotes the growth of swamp eels and regulates the development of female swamp eel ovaries, which is applied to the breeding of superior swamp eel parent stock and the technology of improving the quality and efficiency of commercial swamp eel farming. Background Technology

[0002] The swamp eel is an important and distinctive freshwater aquaculture fish in my country, exhibiting a unique reproductive physiology characterized by sex reversal. Individuals reach sexual maturity primarily as females, but after spawning once, their ovaries gradually degenerate, leading to male transformation. This physiological characteristic not only gives the swamp eel significant value in basic research on gonadal development and sex differentiation regulation in fish, but also significantly increases the technical difficulty of artificial broodstock breeding and large-scale seedling propagation. In actual production, female swamp eel broodstock generally suffer from asynchronous gonadal development, low egg load, and inconsistent reproductive performance, resulting in prominent problems such as poor fertilization rate of fertilized eggs and unstable hatching rate of larvae. The physical condition of the broodstock and the level of ovarian development directly determine the production of high-quality fertilized eggs and the efficiency of large-scale seedling propagation. The industry urgently needs a technical means to simultaneously promote the growth performance of swamp eels, optimize the physiological state of female broodstock, and directionally promote the orderly development of ovaries under conventional aquaculture models.

[0003] Currently, there are two main technical approaches to improve the reproductive performance of swamp eels in production, both of which have significant limitations. One is the exogenous hormone-induced spawning program, which involves injecting exogenous oxytocin into the parent eels to induce terminal maturation of the gonads and ovulation in the short term. While this has the advantage of rapid effectiveness, it only acts on the spawning stage at the end of the reproductive period and cannot systematically improve the growth, feed utilization, and ovarian development synchronicity of the parent eels during the long-term intensive breeding phase. At the same time, the injection procedure is prone to causing stress damage to the parent eels, the hormone dosage is difficult to control, and long-term use poses a risk of drug residues. This does not meet the requirements of green aquaculture and cannot be routinely mixed into feed as a feed additive. It is also difficult to adapt to large-scale daily breeding management and cannot fundamentally improve issues such as gonadotropin secretion and oocyte development at the endogenous level. Secondly, there are traditional Chinese medicine feed additives for fish. While existing research has confirmed that single or compound traditional Chinese medicines can improve fish feeding, growth, digestion, and immunity through their multi-component, multi-target, and low-residue advantages, and some medicinal and edible plants also possess sex hormone-like activities that can regulate animal reproductive endocrine function and promote gonadal development, a small number of studies have also verified the potential of traditional Chinese medicine additives to improve the reproductive performance of swamp eels. However, current technologies only provide broad descriptions of functional effects and have not designed specific compound formulations for the physiological characteristics of sex reversal in swamp eels, nor have they specified the specific ratio of medicinal materials or the dosage of feed additives. Most studies only verify the effects of promoting growth or gonadal development, lacking experimental evidence of the synergistic effect of the two. They have not verified the effects from multiple dimensions such as serum reproductive hormones, ovarian tissue sections, and gonad-related gene expression, nor have they screened and determined a safe and efficient dosage range. This can easily lead to problems such as insufficient effects or excessive dosage, resulting in increased costs and physiological disorders.

[0004] In summary, existing methods of inducing spawning with exogenous hormones are stressful, lack sustainability, and do not align with green aquaculture principles. Conventional herbal additives suffer from numerous shortcomings, including unclear compound formulations, ambiguous dosages, unproven synergistic effects on growth promotion and ovarian development, and weak targeting. Furthermore, a fixed-ratio herbal compound formula with Cuscuta chinensis and Epimedium as core gonadotropic components, Astragalus membranaceus and Angelica sinensis as supplementary qi-tonifying and blood-nourishing herbs, and Ligustrum lucidum for balancing regulation has not yet been publicly disclosed. Moreover, the optimal addition ratio of this compound formula in eel feed is not clearly defined, making it impossible to achieve the dual goals of simultaneously promoting eel weight gain and growth, and ensuring high-quality ovarian development in female parents under routine feeding conditions. Based on the deficiencies of the existing technologies, this invention proposes a targeted herbal compound feed additive solution. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a traditional Chinese medicine compound suitable for feed addition and parent enhancement of swamp eels, and its method of use, so that it can improve the growth performance and feed utilization efficiency of swamp eels without relying on exogenous hormone injections, and promote the transformation of oocytes from the primary growth phase to the cortical vesicle phase, thereby promoting the ovarian development process.

[0006] Specifically, the following technical issues are included:

[0007] This addresses the issues of insufficient growth efficiency and low feed utilization in the breeding of swamp eels.

[0008] This addresses the issues of asynchronous ovarian development and slow oocyte development in female parent eels before artificial breeding.

[0009] This provides a green feed additive solution with clearly defined ingredients, proportions, and dosages that can be directly added to wet fish paste feed.

[0010] By regulating reproductive endocrine and molecular response indicators such as Fsh, Lh, T, Vtg, Amh, and fshr, the responsiveness of the ovarian tissue of the yellow eel to endogenous gonadotropin signals was improved.

[0011] Compound composition

[0012] This invention provides a compound feed additive made from traditional Chinese medicine, which is composed of Cuscuta chinensis, Epimedium brevicornu, Astragalus membranaceus, Angelica sinensis, and Ligustrum lucidum. The preferred ratio verified by experiments is: Cuscuta chinensis: Epimedium brevicornu: Astragalus membranaceus: Angelica sinensis: Ligustrum lucidum = 3:3:1.5:1.5:1 (mass ratio).

[0013] Components Quality Percentage of mass Functional positioning in compound preparations dodder 3.0 copies 30% Main drug; involved in ovarian function repair, endocrine regulation and follicle development support. Epimedium 3.0 copies 30% Main drug; possesses estrogen-like activity and serves as a core component for gonadotropin development. Astragalus 1.5 copies 15% Adjuvant drugs; improve metabolism, anti-stress and physical condition, and provide basic support for growth and gonadal development. Angelica sinensis 1.5 copies 15% Adjuvant medication; improves nutrient supply and the local microenvironment of the ovary, and assists in follicle development. Privet 1.0 copy 10% Adjuvant drugs; participate in endocrine balance and synergistic effects, and reduce the risk of unilateral strong stimulation.

[0014] Additive preparation method

[0015] The preparation and use of the herbal compound of this invention involves the direct addition of dried herbal powder, without any additional processing such as decoction, alcohol extraction, concentration, fermentation, granulation, extract preparation, or purification of active ingredients. The implementation method verified by experiments in this paper is as follows:

[0016] 1. Take dried dodder seed, epimedium, astragalus, angelica, and privet fruit separately, remove mud, impurities, moldy or insect-infested parts, and set aside.

[0017] 2. Grind the above five dried Chinese herbs into powder; the fineness of the powder should be such that it can be fully mixed with the fish paste, and the eels can eat it normally without obvious separation. Preferably, it can be passed through an 80-mesh sieve.

[0018] 3. Weigh out the powdered herbs according to the mass ratio of Cuscuta chinensis: Epimedium: Astragalus membranaceus: Angelica sinensis: Ligustrum lucidum = 3:3:1.5:1.5:1, mix them evenly, and obtain the compound powder of traditional Chinese medicine.

[0019] 4. Before feeding, weigh the compound powder according to the wet weight of the fish paste, add it directly to the fish paste, and mix it repeatedly or mechanically to make the powder evenly dispersed in the fish paste, thus obtaining fish paste feed containing Chinese herbal medicine compound.

[0020] 5. Fish paste containing medication should be prepared and used immediately; for short-term storage, it should be sealed, protected from light and moisture, and stored at low temperature to avoid moisture absorption, clumping, mold growth, or decreased appetite due to prolonged storage.

[0021] The key to the above method is to mix dried Chinese herbal medicine powder in a certain proportion and then add it directly to the fish paste.

[0022] Feed addition amount and feeding method

[0023] The feed carrier verified by experiments in this invention is wet fish paste. The recommended addition amount, based on the wet weight of the fish paste, is 0.2%–0.6%, or 2–6 g / kg; the preferred addition amount is 0.4%, or 4 g / kg.

[0024] The preferred implementation method, as verified by experiments in the paper, is as follows:

[0025] Select healthy eels, temporarily raise them for 1 week and train them to eat fish paste for 2 weeks, and then start feeding them. Add the compound powder of traditional Chinese medicine directly to the wet fish paste at a ratio of 0.4%, mix it thoroughly or stir it evenly, and feed them once a day in the evening. The daily feeding amount is about 5% of the total weight of the eels in the net cage, and continue feeding for 4 weeks.

[0026] If used for large-scale broodstock enhancement, the daily feeding amount and cycle can be adjusted according to water temperature, feeding intensity, broodstock size, and gonadal development stage.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. Application in green feed: This invention achieves reproductive regulation through feed addition, without relying on exogenous hormone injections, making it easy to apply on a large scale in the intensive breeding of swamp eels.

[0029] 2. Clear growth-promoting effect: In the 4-week feeding trial, the body weight of each supplemented group was higher than that of the control group at 4 weeks, and the body weight difference in the 0.4% group reached a highly significant level. The weight gain rate and specific growth rate also showed better performance.

[0030] 3. Improved feed utilization efficiency: After 4 weeks, the feed conversion ratio of each herbal medicine addition group was lower than that of the control group, indicating that the compound can improve feed utilization.

[0031] 4. Promotes ovarian development: In the 0.4% compound group, the number of PGOs decreased and the number or proportion of CAOs increased, indicating that the compound can promote the transformation of oocytes from the primary growth phase to the cortical vesicle phase.

[0032] 5. The endocrine regulation pathway is relatively clear: the 0.4% group can increase indicators such as Fsh, Lh, and Vtg and downregulate Amh, suggesting that it has a positive regulatory effect on early follicle recruitment, yolk formation and oocyte differentiation in swamp eels.

[0033] 6. Molecular evidence supports the findings: in the 0.4% group, fshr was significantly upregulated in ovarian tissue, female development-related genes were upregulated, and male differentiation-related genes were downregulated overall, indicating that the compound not only has phenotypic effects but also has an explainable molecular response. Attached Figure Description

[0034] Figure 1 Flowchart for the preparation and application of the compound in the feeding of yellow eels;

[0035] Figure 2 Charts showing growth indicators for different breeding cycles;

[0036] Figure 3 A graph showing the changes in the number of oocytes during different culture cycles;

[0037] Figure 4 A graph showing changes in serum hormone levels over different breeding cycles;

[0038] Figure 5 Images of ovarian sections from the 0-week, 2-week, and 4-week experiments; where: A, B, C, D, E, F: ovarian sections from the 0-week experiment; G, H, I, J, K, L: ovarian sections from the 2-week 0.4% supplementation group; M, N, O, P, Q: ovarian sections from the 4-week 0.4% supplementation group.

[0039] Figure 6 Heatmap of differentially expressed genes among ovarian groups;

[0040] Figure 7 Volcano plot showing differences between ovarian groups;

[0041] Figure 8 A graph showing the intergroup GO function enrichment analysis;

[0042] Figure 9 A graph showing KEGG pathway enrichment between groups;

[0043] Figure 10 Significance plot of candidate genes for female development. Note: ns P > 0.05, * P < 0.05;

[0044] Figure 11 A salience plot of candidate genes for male development;

[0045] Figure 12 This is a saliency plot of candidate genes for hormone receptors. Detailed Implementation

[0046] To illustrate the technical content, structural features, objectives, and effects of the present invention in detail, the following description is provided in conjunction with the embodiments.

[0047] the term definition PGO Primary growth phase oocyte. CAO Cortical vesicle stage oocytes. Fsh Follicle-stimulating hormone (FSH) is used to evaluate reproductive endocrine regulation. Lh Luteinizing hormone (LH) is used to evaluate endocrine activities related to further oocyte development. T Testosterone, a steroid hormone in fish, is also an important precursor to estrogen synthesis. Vtg Vitellogenin is involved in yolk formation and oocyte development. Amh Anti-Müllerian hormone is associated with inhibition / regulation of gonadal development. WGR / SGR / FCR / CF These are weight gain rate, specific growth rate, feed conversion ratio, and condition factor.

[0048] Example 1

[0049] A compound feed additive made from traditional Chinese medicine to promote the growth and ovarian development of eels, comprising:

[0050] 2 parts Cuscuta chinensis, 3 parts Epimedium brevicornu, 1.5 parts Astragalus membranaceus, 1.5 parts Angelica sinensis, and 1 part Ligustrum lucidum.

[0051] (1) Take dried dodder seed, epimedium, astragalus, angelica and privet fruit, remove mud, impurities, moldy or insect-eaten parts, and set aside;

[0052] (2) Grind the above five dried Chinese herbal medicines into 80-mesh powder for later use;

[0053] (3) Take the powders of Cuscuta chinensis, Epimedium, Astragalus membranaceus, Angelica sinensis and Ligustrum lucidum according to the mass ratio, mix them evenly, and you will get the compound feed additive of Chinese herbal medicine.

[0054] Example 2

[0055] A compound feed additive made from traditional Chinese medicine to promote the growth and ovarian development of eels, comprising:

[0056] Cuscuta chinensis 2.5 parts, Epimedium brevicornu 2.5 parts, Astragalus membranaceus 1.0 part, Angelica sinensis 1.0 part, Ligustrum lucidum 0.5 parts.

[0057] The preparation method is the same as in Example 1.

[0058] Example 3

[0059] A compound feed additive made from traditional Chinese medicine to promote the growth and ovarian development of eels, comprising:

[0060] Cuscuta chinensis 3.5 parts, Epimedium 3.5 parts, Astragalus membranaceus 2.0 parts, Angelica sinensis 2.0 parts, Ligustrum lucidum 1.5 parts.

[0061] The preparation method is the same as in Example 1.

[0062] Application Example 1

[0063] Before feeding, weigh the herbal compound feed additive according to the wet weight of the fish paste, and add it directly to the fish paste at a rate of 0.4%. Disperse the powder evenly in the fish paste through repeated kneading or mechanical stirring to obtain fish paste feed containing the herbal compound. Feeding time is 4 weeks.

[0064] Application Example 2

[0065] Before feeding, weigh the herbal compound feed additive according to the wet weight of the fish paste, and add it directly to the fish paste at a concentration of 0.2%. Disperse the powder evenly in the fish paste through repeated kneading or mechanical stirring to obtain fish paste feed containing the herbal compound. Feeding time is 6 weeks.

[0066] Application Example 3

[0067] Before feeding, weigh out the herbal compound feed additive according to the wet weight of the fish paste, and add it directly to the fish paste at a rate of 0.6%. Disperse the powder evenly in the fish paste through repeated kneading or mechanical stirring to obtain fish paste feed containing the herbal compound. Feeding time is 2 weeks.

[0068] Test case

[0069] 1. Experimental animals, grouping, and housing conditions

[0070] The experimental eels were purchased from a group caught in wild traps in Deyang, Sichuan. After being transported back, they were temporarily kept for one week without being fed. Then, they were trained to eat fish paste and earthworms for two weeks, with the proportion of earthworms gradually reduced and the proportion of fish paste increased. Once the eels were able to stably feed on fish paste, the formal experiment was carried out.

[0071] Eighty-four hundred healthy yellow eels with an initial weight of approximately (20±2.6) g were selected and randomly assigned to four compound supplementation levels: 0%, 0.2%, 0.4%, and 0.6%, with three replicates per level and seventy eels per replicate. The addition ratios were calculated based on the wet weight of the fish paste, corresponding to 0, 2, 4, and 6 g / kg.

[0072] 0% control group 3 70 20.1±2.6 0 0.2% group 3 70 19.9±2.5 2 0.4% group 3 70 20.2±2.7 4 0.6% group 3 70 19.6±2.8 6

[0073] Outdoor aquaculture cages measuring 100 cm × 100 cm × 100 cm with a 40-mesh mesh were used, each with a water area of ​​1 m². The cages were covered with a suitable amount of water hyacinth. Feeding was conducted once daily in the evening at a rate of 5% of the total mass of the eels in each cage. The formal experimental period was 4 weeks, with samples taken at weeks 0, 2, and 4. During the experiment, the water temperature ranged from 19.9 to 33.4℃, with an average of (27.5 ± 0.5)℃; pH ranged from 6.04 to 7.69, with an average of (6.86 ± 0.23); ammonia nitrogen ranged from 0.1 to 0.2 mg / L; conductivity ranged from 275 to 351 μS / cm, with an average of (308.2 ± 20.6) μS / cm; dissolved oxygen was maintained above 5.0 mg / L; and nitrite nitrogen was below 0.1 mg / L.

[0074] 2. Sampling Steps

[0075] Sampling was performed at weeks 0, 2, and 4. Ten fish were randomly selected from each net cage at each time point, for a total of 120 fish from 12 net cages. Fish were fasted for 24 hours prior to sampling and anesthetized with 150 mg / L MS-222. Body length and weight were measured after anesthesia, followed by tail vein blood collection. Blood was placed in enzyme-free centrifuge tubes containing anticoagulant, incubated at 4°C for 16 hours, and then centrifuged at 3500×g for 10 minutes at 4°C. The supernatant serum was collected and stored at -80°C. After blood collection, the ovaries were dissected and isolated. Approximately 0.5 cm of ovarian tissue was fixed in Born's fixative, while the remaining tissue was temporarily stored in liquid nitrogen and then transferred to -80°C.

[0076] 3. Detection Method

[0077] Ovarian tissue was fixed in Born fixative for 24 h, trimmed, dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and sectioned before H&E staining. NDP.view2 software was used for observation, and CAO and PGO were counted separately in 2 mm² statistical regions. Two non-overlapping fields were selected for each section, and six replicates were set for each group. The average diameter of CAO was calculated as (major axis + minor axis) / 2.

[0078] Growth performance is calculated using the following formulas: WGR (%) = 100 × (Final Average Weight - Initial Average Weight) / Initial Average Weight; FCR = Total Feeding Amount / (Final Average Weight - Initial Average Weight); SGR (% / d) = 100 × (ln Final Average Weight - ln Initial Average Weight) / Number of Feeding Days; CF (%) = 100 × Fish Body Weight / Fish Body Length 3 .

[0079] Serum T, E2, Prl, Fsh, Vtg, Lh, and Amh were measured using ELISA. Each sample was tested in duplicate, and the OD value was read at 450 nm and the concentration was calculated from the standard curve.

[0080] Transcriptome analysis was performed using ovarian tissues from the 0.4% group (fed for 4 weeks) and the 0% control group (fed for 4 weeks). Six samples were initially collected from each group, for a total of 12 samples. After quality control, nine samples were used for subsequent analysis: four from the control group and five from the 0.4% group. Paired-end 150 bp sequencing was performed using the Illumina NovaSeq 6000 platform.

[0081] Experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 26.0 and graphing was conducted using GraphPad Prism 10.0. Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Duncan's method was used for multiple comparisons when significant. P < 0.05 indicated a significant difference, and P < 0.01 and P < 0.001 indicated a higher level of significance.

[0082] 4. Experimental Results and Corresponding Conclusions

[0083] 4.1 Growth performance

[0084] Survival rate No deaths were observed in any of the cages within 4 weeks, with a survival rate of 100%. The compound did not cause mortality in the farmed animals at the added levels in this experiment. Body weight(BW) At 2 weeks, the levels in each of the supplemented groups were higher than the control, but the differences were not significant; at 4 weeks, the P values ​​were <0.01 in the 0.2% group, <0.001 in the 0.4% group, and <0.05 in the 0.6% group. The compound has a weight-gain-promoting effect, with the 0.4% group showing the most outstanding performance. Body length (BL) There was no significant difference at 2 weeks; at 4 weeks, the 0.4% group was higher than the control (P<0.05). Adding 0.4% can promote body length growth. SGR The highest level was observed in the 0.6% group at 2 weeks (P<0.05 between groups); the highest level was observed in the 0.4% group at 4 weeks (P<0.05). Appropriate dosage can improve specific growth rates, and the 0.4% group showed stable effects in the later stages. FCR At 2 weeks, the intergroup P value was greater than 0.05; at 4 weeks, all added groups were lower than the control group. The compound preparation showed a trend of improving feed utilization in the later stages of the experiment. CF Both the 2-week and 4-week groups showed P>0.05. The compound did not cause any abnormal changes in body fat percentage. WGR At 2 weeks, the intergroup P > 0.05; at 4 weeks, all added groups were higher than the control, and the 0.4% group P < 0.01. The 0.4% group showed a significant increase in weight gain over 4 weeks.

[0085] 4.2 Serum reproductive hormones

[0086] T At 2 weeks, the 0.4% and 0.6% groups were higher than the control (P<0.01), with the 0.6% group being the highest; at 4 weeks, there were no significant differences between the additive groups and the control. The T response mainly appears in the early stage of the experiment. E2 There were no significant differences among the groups throughout the entire experimental period (P>0.05). This does not constitute grounds for claiming that the compound significantly increases E2. Prl There were no significant differences between the groups at 2 weeks and 4 weeks (P>0.05). This does not constitute grounds for claiming that the compound significantly regulates Prl. Fsh At 2 weeks, all groups with added antibodies had higher rates than the control, with the highest rate at 0.4%; at 4 weeks, the 0.4% group had higher rates than the control (P<0.05). The 0.4% group showed a clear upward response to Fsh. Vtg At 2 weeks, the levels were slightly higher than the control, but P>0.05; at 4 weeks, all added groups were higher than the control, with P<0.05 in the 0.4% group. The 0.4% group showed an increase in vitamin G after 4 weeks. Lh At 2 weeks, all groups with added antibodies showed higher levels than the control, with the 0.2% group showing a significant improvement; at 4 weeks, the 0.4% group showed higher levels than the control (P<0.05). The compound can affect Lh secretion, but the optimal response dose varies at different time points. Amh Between the 0-2 week groups, P>0.05; at 4 week, 0.4% of the group had a lower incidence than the control (P<0.01), while the difference was not significant in other groups. The 0.4% group showed a clear Amh downregulation response at 4 weeks.

[0087] 4.3 Ovarian histological data

[0088] control group 0 6.33±1.21 19.08±5.60 control group 2 6.50±1.22 17.08±4.04 control group 4 5.83±1.63 22.67±9.61 0.4% group 0 6.33±1.21 19.08±5.60 0.4% group 2 6.83±0.75 14.41±4.64 0.4% group 4 7.50±0.84 10.90±3.84

[0089] In the 0.4% group, the number of CAO cells increased with prolonged feeding time, but the difference between weeks 0, 2, and 4 was not statistically significant (P>0.05); the number of PGO cells gradually decreased. Hematologic and epithelial (H&E) observations showed that the overall structure of the ovarian tissue remained intact, and the distribution range of CAO cells expanded at week 4; the diameter of CAO cells did not change significantly between weeks 0, 2, and 4 (P>0.05). Therefore, the direct histological evidence for ovarian development presented in this invention is mainly manifested in changes in the composition of the oocyte stage, rather than a significant increase in the diameter of CAO cells.

[0090] 4.4 Transcriptome and Candidate Gene Data

[0091] Sample quantity Six ovarian samples were initially collected from the 0.4% group and the control group, for a total of 12 samples; after quality control, nine samples were used for subsequent analysis (four from the control group and five from the 0.4% group). sequencing quality A minimum of 42,476,288 raw reads and 39,846,142 clean reads were obtained per sample; both Q20 and Q30 were >95%; and the base error rate was 0.01%. Differential genes Using P < 0.05 and |log2FoldChange| > 0 as criteria, a total of 1635 differentially expressed genes were identified, of which 800 were upregulated, 835 were downregulated, and 26427 genes did not meet the screening criteria. KEGG enrichment These pathways include oxidative phosphorylation, ribosomes, ECM-receptor interaction, folic acid biosynthesis, focal adhesion, and RNA degradation.

[0092] Female development candidate genes foxl2, smad2, star, wnt4, lhr, p53, eral1, and sgk1 were all significantly upregulated (P<0.05). There were no significant differences in cyp19a1, nr5a2, gdf9, erb, mdm2, and bmp15 (P>0.05). Candidate genes for male development IgF1 was significantly downregulated (P<0.05); sox9b was significantly upregulated (P<0.05). Although dmrt1, dmrt2, dmrt3, dmc1, igf2, spef1, and spef2 generally showed a decreasing trend, the differences between groups were not significant (P>0.05). Hormone receptor candidate genes fshr was significantly upregulated (P<0.05). There were no significant differences in esr1, vldlr, amhr2, and prlr (P>0.05).

[0093] The above descriptions are examples of the preferred embodiments of the present invention, and the parts not described in detail are common knowledge to those skilled in the art. Those skilled in the art should recognize that the specific embodiments described above are merely exemplary and are intended to enable them to better understand the content of the present invention, and should not be construed as limiting the scope of protection of the present invention. Under the guidance of the technical solutions of the present invention, those skilled in the art can achieve the purpose of the present invention in other ways without creative effort, and any improvements made based on the technical solutions of the present invention fall within the scope of protection of the present invention.

Claims

1. A compound feed additive made from traditional Chinese medicine to promote the growth and ovarian development of eels, comprising: Cuscuta chinensis 2.5-3.5 parts, Epimedium brevicornu 2.5-3.5 parts, Astragalus membranaceus 1.0-2.0 parts, Angelica sinensis 1.0-2.0 parts, Ligustrum lucidum 0.5-1.5 parts.

2. The herbal compound feed additive according to claim 1, comprising: Cuscuta chinensis 3 parts, Epimedium brevicornu 3 parts, Astragalus membranaceus 1.5 parts, Angelica sinensis 1.5 parts, Ligustrum lucidum 1 part.

3. A method for preparing a traditional Chinese medicine compound feed additive for promoting the growth and ovarian development of eels according to claim 1 or 2, comprising: (1) Take dried dodder seed, epimedium, astragalus, angelica and privet fruit, remove mud, impurities, moldy or insect-eaten parts, and set aside; (2) Grind the above five dried Chinese herbal medicines into powder for later use; (3) Take the powders of Cuscuta chinensis, Epimedium, Astragalus membranaceus, Angelica sinensis and Ligustrum lucidum according to the mass ratio, mix them evenly, and you will get the compound feed additive of Chinese herbal medicine.

4. The preparation method according to claim 3, wherein: The medicinal powder in step (2) is passed through an 80-mesh sieve.

5. A method of using a traditional Chinese medicine compound feed additive for promoting the growth and ovarian development of eels according to claim 1 or 2, comprising: Before feeding, weigh the herbal compound feed additive according to the wet weight of the fish paste, add it directly to the fish paste, and then repeatedly knead or mechanically stir to evenly disperse the powder in the fish paste to obtain fish paste feed containing herbal compound.

6. The method of use according to claim 5, wherein: The amount of the herbal compound feed additive added is 0.2% to 0.6% based on the wet weight of the fish paste.

7. The method of use according to claim 6, wherein: The amount of the herbal compound feed additive added is 0.4% based on the wet weight of the fish paste.

8. The method of use according to claim 5, wherein: The feeding period is 2 to 6 weeks.

9. The method of use according to claim 8, wherein: The feeding period is 4 weeks.

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