Application of human chorionic gonadotropin in the preparation of drugs for repairing ovarian damage in tilapia

Abstract

The present invention provides the use of human chorionic gonadotropin (HCG) in the preparation of a drug for repairing ovarian damage in tilapia, belonging to the field of aquaculture technology. The present invention provides the use of human chorionic gonadotropin in the preparation of a drug for repairing ovarian damage in tilapia. Human chorionic gonadotropin can increase the content of hormones and vitellogenin in serum and increase the expression of sex hormone-related genes in the ovary. At the same time, one or more of estradiol, human chorionic gonadotropin, luteinizing hormone-releasing hormone and coumestrol can increase the body weight of tilapia exposed to copper and cadmium contaminated water phase, providing a new idea for the preparation of new growth-promoting drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and particularly relates to application of human chorionic gonadotropin (HCG) in preparing a medicine for repairing ovarian damage in tilapia. Background Art

[0002] With the rapid development of modern industrialization, heavy metal pollution is becoming increasingly serious. Heavy metal residues from industrial waste enter the bodies of animals and plants through water and soil, and then pass through the food chain system and gradually enter the human body, posing a threat to human health. [1] As an important part of the aquatic ecosystem, fish are at the top of the food chain and are very sensitive to heavy metal pollution in water bodies. [2] Current research on heavy metal pollution mainly focuses on the accumulation of heavy metal pollutants in fish and their impact on physiological activities. [3 -6], the specific effects of exposure to complex heavy metals in the environment on the physiological toxicity and population reproduction of farmed fish are still unclear, especially the damage and repair of the ovaries of fish when exposed to complex aqueous phases of multiple heavy metals are rare.

[0003] Copper and cadmium are the two main heavy metal pollutants found in aquaculture waters in most of the main tilapia production areas in Guangxi Zhuang Autonomous Region. [7] Copper (Cu) is an essential micronutrient for all living organisms and plays a key role in many biological processes, including antioxidant and cell proliferation. [8-9] However, excessive copper can have an adverse effect on the normal growth and development of fish. [10-12] In contrast, Cd is a completely non-essential element for fish and is one of the most toxic heavy metals in the environment.

[13] Excessive accumulation of Cd in fish can cause a series of adverse effects, including toxicity to the metabolism and immune system of fish.

[14] In addition, cadmium can seriously affect the reproductive axis (HPG) regulation of fish

[15] , suppressing hormone levels in serum

[16] Studies have shown that copper and cadmium combined exposure can interact with each other in fish, causing greater than additive damage to fish reproductive function.

[17] Excessive intake of copper and cadmium in fish can reduce the reproductive capacity of the ovary by increasing oocyte atresia, and significantly affect the synthesis of estrogen and the transcription of receptors in fish.

[18] Therefore, it is feasible to use hormone injections to make up for the hormone gap in fish during copper and cadmium combined aqueous exposure and repair the ovarian damage of fish exposed to copper and cadmium combined.

[0004] Estrogen drugs are mainly divided into two types according to their sources: synthetic and natural. Synthetic hormone drugs mainly include estradiol (E2), human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone (LHRH-α), etc. 17β-E2 is estrogen. Injection of 17β-E2 can regulate the expression of genes related to the sex steroid hormone synthesis pathway in the hypothalamus of goldfish through feedback effects.

[19] , and can directly induce the production of vitellogenin VTG in tilapia

[20] , which has the effect of promoting ovarian development and oocyte maturation. Similarly, human chorionic gonadotropin and luteinizing hormone-releasing hormone are analogs of luteinizing hormone-releasing hormone (LRH) and gonadotropin-releasing hormone GnRH in fish, respectively. LRH is a type of gonadotropin GTH. GTH and GnRH regulate the production and development of gonadal steroids by interacting with their receptors in the gonads, leading to the final maturation of the gonads.

[21] In vivo injection of HCG and LHRH-α can induce the development of fish ovaries and the maturation of oocytes. [22-23] Hormones from natural sources are also called phytoestrogens. Phytoestrogens are similar in structure to estradiol and can bind to estrogen receptors (ER) to exert estrogen-like effects.

[24] Coumestrol is a phytoestrogen with the most obvious effect. Intraperitoneal injection of a certain dose of coumestrol in Siberian sturgeon can effectively induce the production of yolk protein in its body.

[25] It is confirmed that phytoestrogens also have the function of promoting oocyte maturation similar to synthetic hormones in fish.

[0005] In response to the prevalence of copper and cadmium pollution in the main tilapia aquaculture areas in Guangxi, the research team previously studied the accumulation of copper and cadmium in various tissues and the damage to the ovaries of tilapia after exposure to these substances in aqueous solutions. The team found that copper and cadmium can accumulate in tilapia and cause some damage to their ovaries. However, there are few reports on the repair of ovarian damage caused by combined exposure to copper and cadmium in aqueous solutions.

[0006] References:

[0007] [1] Ma Tingjun, Lin Bingrong, Jia Changxi. Analysis of heavy metal residues in fish cultured in recycled water and their consumption risk[J]. Chinese Agricultural Science Bulletin, 2010, 26(5): 332-336.

[0008] [2] Kong Xiangzhen, He Wei, Qin Ning, et al. Species sensitivity distribution assessment of heavy metals to freshwater organisms ecological risks [J]. China Environmental Science, 2011, 31(9): 1555-1562.

[0009] [3]Wang X,Sato T,Xing B,et al.Health risks ofheavy metals to thegeneral public in Tianjin,China via consumption ofvegetables and fish[J].Science ofthe Total Environment,2005,350(1):28-37.[4]Cheung K C,Leung H M,Wong M H.Metal concentrations ofcommon freshwater and marine fish from thePearl River Delta,South China[J].Archives of environmental contaminationandtoxicology,2008,54(4):705-715.

[0010] [5]Omar W A,Saleh Y S,MarieM-A S.Integrating multiple fish biomarkersand risk assessment as indicators of metal pollution along the Red Sea coastof Hodeida,Yemen Republic[J].Ecotoxicology and environmental safety,2014,110:221-231.

[0011] [6]Pandey S,Parvez S,Ansari R A,et al.Effects of exposure to multipletrace metals on biochemical,histological and ultrastructural features ofgills of a freshwater fish,Channa punctate Bloch[J].Chemico-biologicalinteractions,2008,174(3):183-192.

[0012] [7] Ali MB, Tripathi RD, Rai UN, et al. Physico-chemical characteristics and pollution level of lake Nainital (UP, India): Role of macrophytes and phytoplankton in biomonitoring and phytoremediation of toxicmetal ions [J]. Chemosphere, 1999, 39 (12): 2171-2182.

[0013] [8] Wang Zhifang. Detection and risk assessment of heavy metals and antibiotics in aquaculture ponds in the main tilapia production areas in Guangxi [D]. Guangxi University, 2018.

[0014] [9]Gaetke LM, Chow-Johnson HS, Chow C K. Copper: toxicologicalrelevance and mechanisms[J]. Archives of toxicology, 2014, 88(11):1929-1938.

[0015]

[10] Clara, Balsano, Cristiana, et al. Is copper a new target to counteract the progression of chronic diseases? [J].Metallomics,2018,10(12):1712-1722.

[0016]

[11] Shaw BJ, Handy R D. Dietary copper exposure and recovery in Niletilapia,Oreochromis niloticus[J]. Aquatic Toxicology, 2006,76(2):111-121.

[0017]

[12] AM Shokr E.Effect of Copper on Hematological,Biochemical Changesand Reproductive Hormones ofthe Nile tilapia Oreochromis niloticus[J].Egyptian Journal ofAquatic Biology andFisheries,2020,24(2):1-18.

[0018]

[13] Cao J,Wang G,Wang T,et al.Copper caused reproductive endocrinedisruption in zebrafish(Danio rerio)[J].Aquatic Toxicology,2019,211:124-136.

[0019]

[14] Luo Y,Shan D,Zhong H,et al.Subchronic effects ofcadmium on thegonads,expressions of steroid hormones and sex-related genes in tilapiaOreochromis niloticus[J].Ecotoxicology,2015,24(10):2213-2223.

[0020]

[15] Zheng J L,Yuan S S,Wu C W,et al.Acute exposure to waterbornecadmium induced oxidative stress and immunotoxicity in the brain,ovary andliver ofzebrafish(Danio rerio)[J].Aquatic toxicology,2016,180:36-44.

[0021]

[16] Tilton SC, Foran CM, Benson W H. Effects of cadmium on therapeutic axis of Japanese medaka (Oryzias latipes) [J]. Comparative Biochemistry and Physiology Part C: Toxicology&Pharmacology, 2003, 136 (3): 265-276.

[0022]

[17] Masufumi T,Shin'Ichi Y.New aspects of cadmium as endocrinedisruptor[J].Environmental sciences:an internationaljournal ofenvironmentalphysiology and toxicology 2006,13(2):107-116.

[0023]

[18] Driessnack MK, Matthews AL, Raine JC, et al. Interactive effects of chronic waterborne copper and cadmium exposure on tissue-specific metalaccumulation and reproduction in fathead minnow (Pimephales promelas) [J]. Comparative Biochemistry and Physiology Part C: Toxicology&Pharmacology, 2016, 179: 165-173.

[0024]

[19] Shi Hongjuan, Ru Xiaoying, Liu Yuqi, et al. Transcriptome analysis of the hypothalamus of female golden croaker injected with 17β-estradiol[J]. Journal of Guangdong Ocean University, 2021, 41(02): 76-85.

[0025]

[20] Takemura A, Kim B H. Effects of estradiol-17βtreatment on in vitro and in vivo synthesis of two distinct vitellogenins in tilapia[J]. ComparativeBiochemistry and Physiology Part A: Molecular&Integrative Physiology, 2001, 129(2-3):641-651.

[0026]

[21] Xiao J, Zhou Y, Luo Y, et al.Suppression effect of LHRH-A and hCG onPiwi expression in testis ofNile tilapia Oreochromis niloticus[J].General and Comparative Endocrinology, 2013,189:43-50.

[0027]

[22] Mollah MFA, Tan ES P.HCG-induced spawning of the catfish,Clarias macrocephalus(Gunther)[J].Aquaculture,1983,35:239-247.

[0028]

[23] Further study on the effect of synthetic hypothalamic luteinizing hormone-releasing hormone (LRH) analogues on induced spawning in domestic fish [J]. Fisheries Science and Technology Information, 1976(Z2):8-14. DOI:10.16446 / j.cnki.1001-1994.1976.z2.003.

[0029]

[24] Kaldas RS, Hughes Jr C L. Reproductive and general metabolic effects of phytoestrogens in mammals [J]. Reproductive Toxicology, 1989, 3 (2): 81-89.

[0030]

[25] Pelissero C,Bennetau B,Babin P,et al.The estrogenic activity ofcertainphytoestrogens in the Siberian sturgeon Acipenserbaeri[J].The Journal of SteroidBiochemistry and MolecularBiology,1991,38(3):293-299. Summary of the Invention

[0031] In view of this, the purpose of the present invention is to provide an application of human chorionic gonadotropin in the preparation of a drug for repairing ovarian damage in tilapia, which can promote the recovery of ovarian damage in tilapia induced by heavy metal stress.

[0032] The present invention provides application of human chorionic gonadotropin in preparing a medicine for repairing ovarian damage in tilapia.

[0033] Preferably, the ovarian damage of tilapia is caused by heavy metal pollution.

[0034] Preferably, the heavy metals include copper and cadmium.

[0035] Preferably, the tilapia ovarian damage repair includes increasing the hormone and vitellogenin content in serum and improving the expression of sex hormone-related genes in the ovary.

[0036] Preferably, the hormones in the serum include GnRH, GTH and E2.

[0037] Preferably, the ovarian sex hormone-related genes include CYP11A1, CYP17, CYP19, 17β-HSD and 3β-HSD.

[0038] The present invention provides application of estrogen substances in increasing the body weight of tilapia.

[0039] Preferably, the estrogen substances include one or more of estradiol, human chorionic gonadotropin, luteinizing hormone-releasing hormone and coumestrol.

[0040] The invention provides a medicine for promoting the growth of tilapia, comprising an estrogen substance and a pharmaceutically acceptable excipient. The estrogen substance comprises one or more of estradiol, human chorionic gonadotropin, luteinizing hormone-releasing hormone and coumestrol.

[0041] The present invention provides the use of HCG in the preparation of a drug for repairing ovarian damage in tilapia. In the present invention, a fish model for drug repair after exposure to a copper and cadmium complex aqueous phase was constructed using female Gift tilapia as experimental animals. The repair effects of four hormone drugs on the ovaries of tilapia damaged by copper and cadmium were evaluated. Estradiol, human chorionic gonadotropin, luteinizing hormone-releasing hormone, and coumestrol all promoted the recovery of the ovaries of tilapia stressed by heavy metals. Ovarian heavy metal content detection, hormone level detection, and QRT-PCR analysis showed that human chorionic gonadotropin had a greater effect on promoting the recovery of the ovaries of tilapia stressed by heavy metals.

[0042] The present invention provides a drug for promoting the growth of tilapia, comprising an estrogen-like substance and a pharmaceutically acceptable excipient. The estrogen-like substance includes one or more of estradiol, human chorionic gonadotropin, luteinizing hormone-releasing hormone, and coumestrol. Experiments have shown that injection of the estrogen-like substance can promote weight gain in tilapia exposed to copper and cadmium contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The changes of tilapia weight and GSI index, where A: change of tilapia weight; B: change of tilapia GSI index; *: significant difference compared with the blank control group in the same time period (p < 0.05); #: significant difference compared with the negative control group in the same time period (p < 0.05);

[0044] Figure 2 Figure 2 Changes in heavy metal content in tilapia ovaries. A: Cu content in tilapia ovaries; B: Cd content in tilapia ovaries; *: Significant difference compared with the blank control group in the same time period (p < 0.05); #: Significant difference compared with the negative control group in the same time period (p < 0.05);

[0045] Figure 3 The changes of serum hormone levels in tilapia, including A: changes in serum GnRH content in tilapia; B: changes in serum GTH content in tilapia; C: changes in serum E2 content in tilapia; D: changes in serum VTG content in tilapia; *: significant difference compared with the blank control group in the same time period (p<0.05); #: significant difference compared with the negative control group in the same time period (p<0.05);

[0046] Figure 4The changes in the mRNA expression levels of sex hormone-related genes in the ovary of tilapia, including: A: changes in the mRNA expression levels of CYP11a1 in the ovary of tilapia; B: changes in the mRNA expression levels of CYP17 in the ovary of tilapia; C: changes in the mRNA expression levels of CYP19 in the ovary of tilapia; D: changes in the mRNA expression levels of 17β-HSD in the ovary of tilapia; E: changes in the mRNA expression levels of 3β-HSD in the ovary of tilapia *: significant difference compared with the blank control group in the same time period (p<0.05); #: significant difference compared with the negative control group in the same time period (p<0.05). DETAILED DESCRIPTION

[0047] The present invention provides application of human chorionic gonadotropin in preparing a medicine for repairing ovarian damage in tilapia.

[0048] In the present invention, the ovarian damage of tilapia is preferably caused by heavy metal pollution, and the heavy metals preferably include copper and cadmium.

[0049] In the present invention, the tilapia ovarian damage repair method preferably includes restoring copper and cadmium levels in ovarian tissue, increasing serum hormone and vitellogenin levels, and enhancing ovarian sex hormone-related gene expression. The serum hormones preferably include GnRH, GTH, and E2. The ovarian sex hormone-related genes preferably include CYP11A1, CYP17, CYP19, 17β-HSD, and 3β-HSD.

[0050] The present invention provides application of estrogen substances in increasing the body weight of tilapia.

[0051] In the present invention, the estrogen-like substances preferably include one or more of E2, HCG, LHRH-α and coumestrol. The method of increasing the weight of tilapia is to expose it to a water phase contaminated with copper and cadmium. 2+ The concentration of Cd was 300 μg / L. 2+ The concentration is 100 μg / L. The examples of the present invention demonstrate that, compared with tilapia injected with PBS solution contaminated with copper and cadmium, E2, HCG, LHRH-α and coumestrol can increase the weight of tilapia to varying degrees, with significant differences, and HCG has a better effect.

[0052] The invention provides a medicine for promoting the growth of tilapia, comprising estrogen substances and pharmaceutically acceptable excipients. The estrogen substances include one or more of E2, HCG, LHRH-α and coumestrol.

[0053] In the present invention, the drug is preferably an injection. The excipient is preferably physiological saline. When containing only one active ingredient, the concentration of E2 in the drug is preferably 2.5-10 μg / g body weight, more preferably 8 μg / g body weight; the concentration of HCG is preferably 0.6-1.5 IU / g body weight, more preferably 1.0 IU / g body weight; the concentration of LHRH-α is preferably 0.05-0.20 μg / g body weight, more preferably 0.15 μg / g body weight; and the concentration of coumestrol is preferably 0.025-0.10 μg / g body weight, more preferably 0.05 μg / g body weight.

[0054] The present invention does not particularly limit the preparation method of the drug; methods for preparing injectable drugs known in the art can be used. The drug is used as follows: when the drug contains only one active ingredient, the injection dose of E2 is 10 μg / g, the injection dose of HCG is 1.5 IU / g, the injection dose of LHRH-α is 0.20 lU / g, and the injection dose of coumestrol is 0.10 mg / g.

[0055] The application of human chorionic gonadotropin provided by the present invention in the preparation of a drug for repairing ovarian damage in tilapia is described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] Effects of estrogen-like substances on damage to tilapia induced by copper and cadmium pollution

[0058] 1 Materials and Methods

[0059] 1.1 Test materials

[0060] The experimental fish were 5-month-old female GIFF tilapia, sourced from the National Tilapia Breeding Farm of the Guangxi Academy of Fisheries Sciences (Nanning, Guangxi), with an average body length of 15.38 ± 2.13 cm and an average weight of 110 ± 13.84 g. The feed used was purchased from Baiyang Industrial Investment Group Co., Ltd., a complete extruded tilapia pellet, at a daily feed rate of 1–1.5% of the fish's body weight. The experimental water temperature was natural (25–32°C).

[0061] CuSO4·5H2O (analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd., and CdCl2·2.5H2O (analytical grade) was purchased from Tianjin Damao Chemical Reagent Factory.

[0062] 1.2 Experimental methods

[0063] Selected female tilapia were acclimated in aerated clean water for 14 days. During the acclimation period, they were fed twice a day, with each feeding amount being 1% to 1.5% of the fish body weight.

[0064] The entire experimental period was divided into two phases: the copper and cadmium aqueous joint exposure phase (30 days) and the repair phase (7 days), totaling 37 days.

[0065] Water phase combined exposure stage: The experimental fish were divided into two groups, a blank control group of 50 fish and a water phase exposure group of 300 fish. The water used in the blank control group was aerated tap water. The Cu 2+ The concentration was 300 μg / L, Cd 2+ The concentration was 100 μg / L. One-third of the heavy metal solution was replaced daily and the siphon method was used to remove the metabolites and residual feed of the tilapia.

[0066] Repair stage: Tilapia treated with aqueous copper and cadmium combined stress for 30 days were divided into six groups and injected with PBS (negative control group), 10 μg / g tilapia body weight of dimethyl sulfoxide DMSO (solvent group), 10 μg / g tilapia body weight of estradiol (E2, E2 group), 1.5 IU / g tilapia body weight of human chorionic gonadotropin (HCG group), 0.20 IU / g tilapia body weight of luteinizing hormone releasing hormone group (LHRH group) and 0.10 mg / g tilapia body weight of coumestrol (couestrol group). After the injection, the fish were placed in clean water and raised for 7 days.

[0067] Sampling was performed 30 days into the combined exposure experiment and 7 days after post-exposure recovery. Tilapia were anesthetized with 0.01% MS-222 anesthetic, and all procedures were performed on ice. Body length and weight of each group of tilapia were measured. Blood was collected from the tail vein and allowed to stand at room temperature for 1 hour before being placed in a 4°C refrigerator overnight at 8000 rpm / min for 5 minutes. The supernatant was collected and stored in a -80°C freezer until ready for use. Ovarian tissue was dissected and weighed, and the gonadal index (GSI) was calculated. The ovarian tissue was then divided into two portions, frozen in liquid nitrogen, and stored in a -80°C freezer for subsequent experiments.

[0068] 1.3 Detection of copper and cadmium content

[0069] Following the method reported by Luo Yongju et al. (Luo Yongju. Study on the Toxicity and Fertility of Cadmium on Gift Tilapia [D]. Nanjing Agricultural University, 2015), inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the copper and cadmium ion contents in ovarian tissue. The specific steps were as follows: the ovarian sample to be tested was weighed (in mg) (accuracy 0.0001 g), placed in a 10 mL digestion tube containing 1 mL of nitric acid, and the tube was placed in a graphite digester for complete digestion at 120°C. After the tube cooled, ultrapure water was added to the volume to 10 mL, and the solution was allowed to stand for 30 minutes. The copper or cadmium concentration (μg / mL) in the solution was determined using inductively coupled plasma mass spectrometry, and the copper and cadmium ion contents in the ovarian tissue were calculated.

[0070] 1.4 Determination of hormone levels in serum

[0071] The levels of gonadotropin (GTH), gonadotropin-releasing hormone (GnRH), estradiol (E2) and vitellogenin (VTG) in serum were determined according to the operating instructions of the corresponding ELISA kits provided by Shanghai Qiaodu Biotechnology Co., Ltd.

[0072] 1.5 Detection of related gene expression in ovarian tissue based on RT-qPCR

[0073] 1.5.1 Extraction of total mRNA

[0074] The ovarian tissues in each group were removed and ground under frozen conditions. The ovarian tissues were lysed according to the instructions of TaKaRa RNAiso Plus reagent, and the ovarian tissues were broken with a homogenizer to extract the total mRNA from the ovarian tissues.

[0075] 1.5.2 Reverse transcription

[0076] The extracted RNA sample was diluted to 500 ng and analyzed according to TaKaRa PrimeScript TM Reverse transcription was performed according to the RT MasterMix (Perfect Real Time) kit instructions. The reaction system is shown in Table 1. Reaction conditions: 15 min extension at 37°C, 5 s inactivation of the reverse transcriptase at 85°C, and storage at 4°C. The resulting cDNA was stored at -20°C.

[0077] Table 1 Reverse transcription system

[0078]

[0079] 1.5.3 Amplification

[0080] mRNA was reverse transcribed into cDNA according to the instructions of TB Green Premix Ex Taq kit, and real-time quantitative PCR was performed using SYBR green fluorescent dye reagent ( The premix was prepared using a real-time fluorescence quantitative PCR instrument (ABI System Sequence Detector 7500) and the amplification conditions were as follows: 95°C for 30s pre-denaturation, 95°C for 5s PCR reaction, 60°C for 30s (40 cycles), and dissociation. The reaction system is shown in Table 2. The experimental results used β-actin as an internal reference and 2 -ΔΔCt Methods The expression levels of all genes were calculated. The oligonucleotide sequences of the primers used for marker mRNA expression detection are shown in Table 3.

[0081] Table 2 qRT-PCR reaction system

[0082]

[0083] Table 3 Primer sequences

[0084]

[0085]

[0086] 1.6 Statistical analysis

[0087] SPSS 22 was used to analyze all indicators. One-way analysis of variance (ANOVA) and Dunnett's multiple comparison test were used to observe whether the differences between the groups were significant. Graphpad Prism 5 software was used to draw graphs based on the experimental results.

[0088] 2 Results

[0089] 2.1 Tilapia weight and GSI index

[0090] Depend on Figure 1Compared with the blank control group, 30-day exposure to the copper-cadmium combined aqueous solution significantly reduced tilapia weight and GSI (by 34.46% and 60.00%, respectively) (p < 0.05). Compared with the negative control group, 7 days after drug injection and recovery, the weight of tilapia exposed to the copper-cadmium combined aqueous solution increased to varying degrees, with the coumestrol group showing a significant 61.18% increase (p < 0.05). However, the GSI index showed the opposite effect, decreasing in all groups compared with the negative control group. Therefore, exposure to the copper-cadmium combined aqueous solution significantly reduced tilapia weight and GSI. While the four drugs administered after exposure increased weight in the short term, the improvement in GSI was not significant.

[0091] 2.2 Copper and cadmium contents in tilapia ovaries

[0092] like Figure 2 As shown in the results, compared with the blank control group, the Cu content in the ovarian tissue of tilapia exposed to the copper and cadmium composite aqueous solution for 30 days decreased by 68.48%, while the Cd content increased by 319.25%, and the differences were significant (p < 0.05). After exposure to the copper and cadmium composite aqueous solution, the four drugs were injected and the recovery period lasted for 7 days. The Cu content in each experimental group was significantly lower than that in the blank control group, while the Cd content was significantly higher than that in the blank control group (p < 0.05). Compared with the negative control group, there were no significant differences in the Cu and Cd contents among the drug groups (p > 0.05).

[0093] 2.3 Hormone and vitellogenin levels in serum

[0094] like Figure 3As shown, 30 days of exposure to a copper-cadmium complex aqueous solution reduced hormone levels and vitellogenin content in tilapia serum. After 30 days of exposure, GnRH, GTH, and VTG levels decreased by 9.17%, 13.01%, and 2.20%, respectively, compared to the blank control group (P>0.05). E2 content decreased significantly by 17.55% (P<0.05). Seven days after drug injection and recovery, serum hormone levels in tilapia in all drug groups remained lower than in the blank control group, while VTG levels were significantly elevated. Compared with the negative control group, GnRH levels in the HCG, LHRH, E2, and Cou groups increased by 11.05%, 17.41%, 10.03%, and 12.41%, respectively, and GTH levels increased by 14.92%, 18.71%, 19.22%, and 11.68%, respectively. E2 levels in the HCG, LHRH, and E2 groups increased by 49.31%, 42.39%, and 45.91%, respectively, with significant differences (P < 0.05). Concomitantly, VTG levels in the HCG group increased by 39.56%, with significant differences (P < 0.05). E2 levels in the Cou group and in all drug groups except the HCG group did not change significantly (P > 0.05). These results suggest that HCG and LHRH-α injections can help increase serum GnRH, GTH, E2 levels, and VTG levels, particularly E2 levels and VTG levels.

[0095] 2.5 Content of sex hormone-related genes in ovaries

[0096] like Figure 4As shown in the results, exposure to copper and cadmium combined aqueous solutions for 30 days significantly reduced the expression levels of sex hormone-related genes in the ovaries of tilapia. Compared with the blank control group, the expression levels of CYP11A1, CYP17, CYP19, 17β-HSD, and 3β-HSD in the ovaries of tilapia exposed to copper and cadmium combined aqueous solutions decreased by 86.74%, 77.54%, 73.13%, 91.04%, and 76.60%, respectively (p < 0.05). After drug injection and 7 days of recovery, the expression levels of sex hormone-related genes in each drug group were significantly lower than those in the blank control group, but significantly increased compared with the negative control group: the mRNA expression levels of CYP11A1 in the ovaries of tilapia in the HCG group and LHRH group increased by 282.26% and 255.08%, respectively, and the differences were significant (P < 0.05); the mRNA expression levels of CYP11A1 in the ovaries of tilapia in the E2 group and Cou group increased by 176.00% and 161.23%, respectively, and the differences were not significant (p > 0.05); the mRNA expression levels of CYP17 in the HCG group, LHRH-α group, E2 group and Cou group increased by 124.76%, 124.25%, 75.51% and 102.30%, respectively, and the mRNA expression levels of CYP19 in the HCG group, LHRH-α group, E2 group and Cou group increased by 124.76%, 124.25%, 75.51% and 102.30%, respectively. The mRNA expression levels increased by 62.18%, 59.09%, 35.58% and 26.84% in the HCG group, LHRH-α group and 17β-HSD group, respectively, and the differences were not significant (p>0.05); the mRNA expression levels of 17β-HSD in the Cou group and E2 group increased by 57.71% and 62.27% in the Cou group and E2 group, respectively, and the differences were not significant (p>0.05); the mRNA expression levels of 3β-HSD in the HCG group, LHRH-α group and E2 group increased by 100.64%, 113.16% and 81.53%, respectively, and the differences were significant (P<0.05). Meanwhile, the mRNA expression level of 3β-HSD in the Cou group increased by 69.66%, with no significant differences (p>0.05). These results indicate that injection of all four drugs can increase the expression levels of sex hormone-related genes in tilapia ovaries, especially HCG and LHRH-α, which can significantly increase the gene expression levels of CYP11A1, 17β-HSD, and 3β-HSD.

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of human chorionic gonadotropin in preparing a drug for repairing ovarian damage in tilapia, wherein the ovarian damage in tilapia is caused by heavy metal pollution, and the heavy metals are copper and cadmium.

2. The application according to claim 1, characterized in that The tilapia ovarian damage repair comprises increasing the hormone and vitellogenin content in serum and increasing the expression of sex hormone-related genes in the ovary; The hormones in the serum include GnRH, GTH and E2; The ovarian sex hormone-related genes include CYP11A1, CYP17, CYP19, 17β-HSD and 3β-HSD.

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