Gonadotropin releasing hormone analogue of megalobrama amblycephala, composite oxytocic and application

Abstract

The invention relates to the field of fishery medicines, and discloses a megalobrama amblycephala gonadotropin releasing hormone analogue, a composite oxytocic and application. The hormone analogue MaGnRH3a provided by the invention has a remarkable effect of promoting megalobrama amblycephala egg cell maturation. The effect time of the female parent fish to be parturified by brood on the MaGnRH3a oxytocic is short, and the parturition induction efficiency is high. The modified and designed MaGnRH3a is an efficient active peptide specifically aiming at artificial spawning induction of megalobrama amblycephala. The ovulation time can be shortened to 10-13 hours and is increased by 41% compared with that of a traditional method, and the death rate of parent fishes is reduced to 0% from 16%; and through specific design, the endocrine interference risk of non-target organisms is remarkably reduced. When the active peptide and domperidone are compounded into an oxytocic, the compound oxytocic has obvious economic and ecological benefits when being applied to megalobrama amblycephala artificial breeding production.

Description

Technical Field

[0001] The present invention relates to the field of fishery drugs, and more particularly to a gonadotropin-releasing hormone analogue for blunt snout bream, a compound oxytocic agent and their applications. Background Art

[0002] Blunt snout bream (Megalobrama amblycephala) belongs to Osteichthyes, Cypriniformes, Cyprinidae, and Megalobrama. For large-scale farming, a large number of fry are required. Currently, the cultured blunt snout bream are all from artificially propagated fry. Fry are the foundation of aquaculture and the sustainable development of fish resources; artificial fry breeding is the lifeline of aquaculture and an eternal theme of aquaculture. Artificially cultured fish usually cannot lay eggs by themselves and must be artificially induced to spawn. Artificial spawning induction is an important means of artificial fish reproduction; the purpose of artificial spawning induction is to promote the further development and maturation of the gonads of broodstock through physiological means, achieving the processes of ovulation, spawning, sperm ejaculation, and fertilization. The rational use of oxytocic agents is particularly important.

[0003] Reproductive activities are mainly regulated by the nervous and endocrine systems and are controlled by the HPG axis composed of the brain, pituitary gland, and gonads. After the nerve center of fish in the breeding season is stimulated accordingly, it sends instructions to the endocrine organ hypothalamus, prompting the hypothalamus to secrete gonadotropin-releasing hormone (GnRH) and gonadotropin-releasing inhibitory hormone or factor (GRIH or GRIF) (Luo Xiaonian et al., 2020). GnRH acts on the pituitary gland to secrete gonadotropin (GtH); while the role of dopamine (DA) in teleost fish is equivalent to GRIH, and DA can inhibit GtH secretion (Xie Jiahua, 2002). GtH is the most critical endocrine factor for promoting gonad development and maturation, acting on the gonads to regulate sex hormone secretion, germ cell maturation, and gonadal ovulation and sperm ejaculation (Chen Shijuan, 2010).

[0004] Currently, the commonly used spawning agents for fish on the market mainly include pituitary gland (PG), chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone analogue (LRH-A), domperidone (DOM), etc. The pituitary gland is generally a self-made carp pituitary gland. The principle is to use the follicle-stimulating hormone and luteinizing hormone contained in the pituitary gland of sexually mature carp to promote the further development and maturation of sperm and egg cells and induce female fish to come into heat and lay eggs. Due to limited sources of pituitary gland, high preparation cost, unstable spawning effect, and cumbersome configuration process, etc., it far cannot meet the production needs and is not green, economical, and efficient for spawning. In addition, injecting pituitary gland suspension is likely to introduce exogenous pathogens and cause the death of broodstock due to infection. Chorionic gonadotropin is a glycoprotein hormone extracted from the urine of pregnant women aged 2 - 4 months, with a relative molecular mass of about 360,000. Its main function is to promote the ovulation of broodstock and also has a certain effect on gonadal development, but it is more sensitive to temperature and is prone to drug resistance. Studies have found that the spawning effect of the second injection of spawning agent for broodstock is poor, far lower than that of the first spawning. Domperidone is a dopamine antagonist that can block the inhibition of dopamine (DA) on the release of gonadotropin (GtH), thus achieving the effect of promoting the release of GtH. When used in combination with gonadotropin-releasing hormone analogue (GnRH-A), it can promote the maturation of fish gonads and sperm ejaculation and egg laying faster, and it is a highly active fish spawning agent.

[0005] The current traditional spawning method for female bream and blunt snout bream in the market is: the injection amount of luteinizing hormone-releasing hormone analogue is 8 - 10 micrograms / kg, the injection amount of human chorionic gonadotropin is 400 - 800 IU / kg, and the injection dose of domperidone maleate is 1 mg / kg ("Good Breeds, Good Cultivation, and Good Marketing of Freshwater Fish", Liu Shaojun, 2024). The traditional spawning method often results in less than 100% successful spawning, and at the same time, the broodstock is prone to death after spawning, thus causing economic losses. In addition, because the traditional spawning agent does not have species specificity, the unmetabolized hormone enters the water body with excrement, which may interfere with the endocrine system of wild fish, affect the ecological balance, and cause ecological risks (Lan Guozhu et al., 2023).

[0006] The traditional spawning method has high costs, is not easy to preserve, and has unstable supply when using HCG. At the same time, it will increase the drug resistance of broodstock to the spawning agent. To sum up, the current commercial spawning agents have poor effects on inducing the ovulation of bream and blunt snout bream broodstock, and the broodstock is prone to infection and death after spawning. The production cost of fry is extremely high. The unmetabolized hormone enters the water body with excrement, which may interfere with the endocrine system of wild fish, affect the ecological balance, and cause ecological risks (Lan Guozhu et al., 2023). Summary of the Invention

[0007] The object of the present invention is to provide a gonadotropin-releasing hormone analogue for bream or blunt snout bream, and the analogue is Pyro-Glu-His-Trp-Ser-Tyr-[D-Trp]-Trp-Leu-Pro-Net (proline-glutamic acid-histidine-tryptophan-serine-tyrosine-D-tryptophan-tryptophan-leucine-proline-ethylamine).

[0008] Another object of the present invention is to provide a composite ovulation-inducing agent for bream or blunt snout bream, and the composite ovulation-inducing agent includes a gonadotropin-releasing hormone analogue for bream or blunt snout bream and domperidone.

[0009] Another object of the present invention is to provide the application of the gonadotropin-releasing hormone analogue for bream or blunt snout bream in the preparation of a composite ovulation-inducing agent for bream or blunt snout bream.

[0010] Another object of the present invention is to provide an artificial ovulation-inducing method for bream or blunt snout bream using the above composite ovulation-inducing agent, and the method is simple and has a high ovulation-inducing efficiency.

[0011] The last object of the present invention is to provide the application of the above analogue, composite ovulation-inducing agent or ovulation-inducing method in the breeding or artificial cultivation of bream or blunt snout bream.

[0012] In order to achieve the above object, the present invention takes the following technical measures:

[0013] A gonadotropin-releasing hormone analogue for blunt snout bream, and the analogue is Pyro-Glu-His-Trp-Ser-Tyr-[D-Trp]-Trp-Leu-Pro-Net (proline-glutamic acid-histidine-tryptophan-serine-tyrosine-D-tryptophan-tryptophan-leucine-proline-ethylamine).

[0014] A composite ovulation-inducing agent for bream or blunt snout bream, including a gonadotropin-releasing hormone analogue for blunt snout bream and domperidone. The ratio of the gonadotropin-releasing hormone analogue for blunt snout bream to domperidone is 1:(16.67 - 50);

[0015] For the above-mentioned method, preferably, when the composite ovulation-inducing agent is injected, the injection amount of the analogue in the composite ovulation-inducing agent is 40 - 60 micrograms per kilogram of fish; the injection dose of domperidone is 1 - 2 milligrams per kilogram of fish.

[0016] The protection scope of the present invention also includes the application of the above hormone analogue, composite ovulation-inducing agent or ovulation-inducing method in the breeding or artificial cultivation of bream or blunt snout bream.

[0017] Compared with the prior art, the present invention has the following advantages and effects:

[0018] Existing commercial oxytocics have poor oxytocic effects on blunt snout bream or black bream. After oxytocin injection, the parent fish are prone to death, and the ecological risk is high. The modified and designed Megalobrama amblycephala gonadotropin-releasing hormone analogue MaGnRH3a provided by the present invention has a significant effect on promoting the maturation and ovulation of oocytes of blunt snout bream or black bream, reduces the mortality rate of parent fish after oxytocin injection, and has obvious economic benefits. Moreover, the specifically designed MaGnRH3a oxytocic for blunt snout bream or black bream has no effect on other fish, can significantly reduce the ecological risk, and has a significant ecological effect. Description of the Drawings

[0019] Figure 1 It is the effect time diagram of Megalobrama amblycephala after injecting three different oxytocics. Detailed Embodiments

[0020] For the technical solutions described in the present invention, unless otherwise specified, they are all conventional solutions in the art; for the reagents or materials, unless otherwise specified, they are all from commercial channels. In nature, since the genetic relationship between blunt snout bream and black bream is extremely close, in this application, the applicant takes Megalobrama amblycephala as an example to illustrate the oxytocic effect of the analogue of the present invention. For other blunt snout bream or black bream, good effects can also be obtained by using the hormone analogue of the present invention or the oxytocic method of the present invention.

[0021] Example 1:

[0022] Obtaining of gonadotropin-releasing hormone analogue of blunt snout bream or black bream:

[0023] A. Analyze the structures of all endogenous gonadotropin-releasing hormone encoding genes of Megalobrama amblycephala to obtain two bioactive gonadotropin-releasing hormone core decapeptides, and their amino acid sequences are respectively: gonadotropin-releasing hormone 2: Gln-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly; gonadotropin-releasing hormone 3: Gln-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly.

[0024] B. Modify and design specific sites of the two core decapeptides: convert Gln at the first position to Pyro, convert Gly at the sixth position to D-Trp, and convert Gly at the tenth position to Net. Two gonadotropin-releasing hormone analogues are obtained.

[0025] C. Artificially synthesize the modified and designed MaGnRH2a gonadotropin-releasing hormone 2 analogue, and its structural formula is: Pyro-Glu-His-Trp-Ser-His-[D-Trp]-Trp-Tyr-Pro-Net;

[0026] D. Artificially synthesized modified MaGnRH3a gonadotropin-releasing hormone 3 analog: Pyro-Glu-His-Trp-Ser-Tyr-[D-Trp]-Trp-Leu-Pro-Net, i.e., proline-glutamic acid-histidine-tryptophan-serine-tyrosine-D-tryptophan-tryptophan-leucine-proline-ethylamine.

[0027] Example 2:

[0028] Obtaining a compound oxytocic for blunt snout bream or triangular bream:

[0029] Since follicle-stimulating hormone and luteinizing hormone in fish pituitary glands are inhibited by dopamine, when using gonadotropin-releasing hormone analogs for induced spawning, they are usually injected simultaneously with DOM to reduce the inhibitory effect. Therefore, the present invention also adopts a combined use method.

[0030] The compound oxytocic for blunt snout bream or triangular bream of the present invention is obtained by mixing the hormone analog MaGnRH2a or MaGnRH3a with domperidone in a mass ratio of 1:(16.67 - 50).

[0031] In this example, the weight ratio of MaGnRH2a or MaGnRH3a to domperidone is 1:30.

[0032] Example 3:

[0033] Artificial induced spawning of different oxytocics for blunt snout bream:

[0034] The experimental process is as follows:

[0035] Taking the commercially available oxytocic HCG + LHR-A2 as a control, the differences in the effects of the modified MaGnRH2a, MaGnRH3a and HCG + LHR-A2 in promoting the maturation and ovulation of female blunt snout bream oocytes were compared and analyzed.

[0036] During the breeding season of blunt snout bream, 75 female blunt snout bream parents were selected, with a weight of about 500 g per tail. They were randomly divided into 3 groups, with 25 blunt snout breams in each group placed in an indoor breeding pond (3.0 m × 3.0 m), with a water depth of 1.2 m and a water temperature of 20°C.

[0037] Control group: The oxytocic for the control group was 400 IU / kg HCG + 8 μg / kg LHR-A2 + 1.2 mg / kg domperidone maleate dissolved in 0.8 ml of normal saline, and was injected intrathoracically into female blunt snout bream. The injected blunt snout breams were placed in an indoor parent pond, and the temperature was maintained at 20°C.

[0038] Experimental Group 1: The oxytocic agent for the experimental group was 40 μg / kg MaGnRH2a (synthesized by Nanjing Genscript Biotech Co., Ltd.) + 1.2 mg / kg of dioctyl maleate dissolved in 0.8 ml of normal saline, and it was injected into the chest of female bream. The injected bream was placed in the same environment as the control group to await spawning.

[0039] Experimental Group 2: The oxytocic agent for the experimental group was 40 μg / kg MaGnRH3a (synthesized by Nanjing Genscript Biotech Co., Ltd.) + 1.2 mg / kg of dioctyl maleate dissolved in 0.8 ml of normal saline, and it was injected into the chest of female bream. The injected bream was placed in the same environment as the control group to await spawning.

[0040] Results:

[0041] Control Group: It was only detected that 6 female fish ovulated 12 h after injecting the oxytocic agent of the control group, 15 female fish ovulated at 13 h, 19 female fish ovulated at 14 h, and only 21 female fish successfully spawned after 20 h.

[0042] Experimental Group 1: After injecting the oxytocic agent containing MaGnRH2a, it was detected that 4 female fish matured and ovulated at 11 h, 6 female fish could ovulate at 12 h, 16 female fish ovulated at 13 h, 18 female fish ovulated at 14 h, and after 20 h, 20 female fish successfully ovulated.

[0043] Experimental Group 2: The results showed that after injecting the oxytocic agent containing MaGnRH3a, it was detected that the oocytes of 5 female bream matured and ovulated at 10 h, 16 female fish ovulated 11 h after injection, 20 female fish ovulated after 12 h, and 24 female fish successfully ovulated after 13 h.

[0044] In summary, the ovulation effect time of bream injected with the oxytocic agent containing MaGnRH3a was shorter than that of the control group, and the ovulation efficiency was also higher than that of the control group. The oxytocic efficiency of MaGnRH3a was significantly improved.

[0045] 48 h after oxytocin injection, no death of female parent fish occurred in the experimental group; 4 female parent fish died in the control group, and no death occurred in the female parent fish of the experimental groups injected with MaGnRH2a and MaGnRH3a in the experimental group. This indicates that the bream injected with the oxytocic agent containing MaGnRH2a or MaGnRH3a has less damage to the parents, and the economic benefits are significantly improved.

[0046] In summary, the MaGnRH3a + DOM compound oxytocic agent of the present invention can shorten the ovulation time to 10 - 13 h, speed up by 41% compared with the traditional method, reduce the mortality rate of parent fish from 16% to 0%, and significantly reduce the risk of endocrine disruption to non-target organisms through specific design.

Claims

1. A method for artificially inducing spawning of bream or bream based on a gonadotropin-releasing hormone 3 analogue of Megalobrama amblycephala, comprising the steps of injecting a composite oxytocic agent into the Megalobrama amblycephala; the composite oxytocic agent is composed of a gonadotropin-releasing hormone 3 analogue of Megalobrama amblycephala and dioconazole; the gonadotropin-releasing hormone 3 analogue of Megalobrama amblycephala is: proline-glutamic acid-histidine-tryptophan-serine-tyrosine-dextrorotatory tryptophan-tryptophan-leucine-proline-ethylamine.

2. According to claim 1, the mass ratio of the gonadotropin-releasing hormone 3 analogue of Megalobrama amblycephala and dioconazole is 1: (16.67-50), wherein the injection dose of each component of the composite oxytocin is 40-60 micrograms / kg of fish for the gonadotropin-releasing hormone 3 analogue of Megalobrama amblycephala MaGnRH3a; and the injection dose of dioconazole DOM is 1-2 mg / kg of fish.

3. Use of the amblycephalic gonadotropin analogue described in claim 1 and the composite oxytocin described in claim 2 in artificial breeding of bream and live bream.

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