A sea urchin bile oxytocin and its preparation method and application

Abstract

The invention discloses a sea urchin seed gall oxytocin agent and a preparation method and application thereof, and belongs to the technical field of sea treasure seedling cultivation in the marine aquaculture industry. The preparation method of the sea urchin seed gall oxytocin agent is: garlic is mixed with disinfected seawater, crushed, filtered, and the filtrate is taken to obtain the sea urchin seed gall oxytocin agent. The sea urchin seed gall oxytocin agent uses natural product garlic as its raw material, is non-toxic to the seed gall after induced ...

Description

Technical Field

[0001] The invention relates to the technical field of sea treasure seedling cultivation in the marine aquaculture industry, and in particular to a sea urchin seed gall bladder oxytocin agent and a preparation method and application thereof. Background Art

[0002] Sea urchins are widely distributed in coastal waters around the world. There are more than 100 species along the coast of my country, but only a few have economic value. Among them, the strongylocentrotus intermedius, also known as the intermediate ball sea urchin, is one of the main species of sea urchins cultivated in my country. It belongs to the phylum Echinodermata, class Echinoides, order Orthomorpha, family Echinoidea. The strongylocentrotus intermedius was introduced from Japan by the Aquaculture Department of Dalian Fisheries College in 1989. At present, the technology of indoor artificial breeding and offshore cultivation of commercial urchins has been mastered.

[0003] In the indoor artificial breeding of sea urchins, the gallbladder induction technology mainly adopts physical methods and chemical methods. The physical method mainly adopts shade drying, running water, and temperature stimulation. Although the physical method can meet the needs of production, it has the defects of low induction efficiency and instability. The chemical method mainly uses KCl as an induction agent to induce the urchins to spawn. Although it has high induction efficiency and strong operability, it has the problems of osmotic pressure disorder in the body after the induction of gallbladder, high toxicity, and high mortality rate, which brings difficulties to the preservation of gallbladder after spawning.

[0004] CN104208655B is a safe, efficient and non-toxic new endogenous oxytocic agent for sea urchins. It uses pentapeptide amide NGFFFamid as an oxytocic agent. When it is used to induce spawning of sea urchins, the average egg discharge of female sea urchins in the test group is 1.1 million / individual, and no dead individuals occur. CN101987196A is an oxytocic agent for inducing spawning of sea cucumbers in vivo. The oxytocic agent is NGLWY amide and KCl as oxytocic agents for spawning of sea cucumbers. When a single NGLWY amide is used, the survival rate of sea cucumbers is low. When KCl is used as an oxytocic agent in induced spawning of sea urchins, the mortality rate of seed gall after induced spawning is as high as more than 70%. It can be seen that although the use of KCl as an oxytocic agent can improve the induced spawning efficiency of seed gall, the mortality rate of seed gall after induced spawning is too high, which is not conducive to the preservation of seed gall after induced spawning. Therefore, developing an induced labor agent for sea urchin breeding galls, reducing the use of chemical drugs, and improving the survival rate of breeding galls while ensuring the efficiency of induced labor are still the key to indoor artificial breeding of sea urchins. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a preparation method and application of a sea urchin gallbladder oxytocin agent. The sea urchin gallbladder oxytocin agent is made of garlic juice, a natural product, has no toxic side effects on the gallbladder, has an anti-inflammatory effect, ensures the survival rate of the gallbladder, and has good oxytocin efficiency.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a method for preparing a sea urchin bile oxytocin agent, comprising the following steps:

[0008] The garlic and sterilized seawater are mixed at a solid-liquid ratio of (40-60) g: (80-120) mL, and then the mixture is crushed and filtered to obtain a filtrate, which is a sea urchin seed gall oxytocin agent.

[0009] Preferably, the preparation process of the disinfected seawater is as follows: filtering the seawater through a 450-500 mesh silk sieve, boiling it for 5 minutes, and cooling it for later use.

[0010] Preferably, 240-280 mesh sieve is used for filtering, which can effectively filter out the garlic residue.

[0011] The second aspect of the present invention provides a sea urchin bile oxytocin agent.

[0012] The third aspect of the present invention provides the use of a sea urchin seed bile oxytocin agent in inducing sea urchin spawning.

[0013] Preferably, the method for using the sea urchin seed bile oxytocin in inducing labor in sea urchins is: inducing labor by injecting the sea urchin seed bile oxytocin into the peristomal membrane of the sea urchin seed bile.

[0014] More preferably, the injection volume of each sea urchin seed is 2-4 mL.

[0015] More preferably, the concentration of the sea urchin bile oxytocin is 0.03 g / mL-0.05 g / mL.

[0016] Beneficial effects of the present invention:

[0017] The sea urchin gallbladder induced labor agent prepared by the invention uses garlic as the main raw material. Garlic, as a natural product, has no toxic side effects on the gallbladder after sperm or egg discharge, and garlic has an anti-inflammatory effect, which can ensure the survival rate of the gallbladder after induced labor. At the same time, garlic is cheaper than chemical drugs, saving costs.

[0018] The sea urchin seed gall oxytocin prepared by the present invention is injected into the seed gall from the seed gall perioral membrane to induce spawning, and the average number of eggs laid by the female seed gall can reach 2.6 million / piece, the average fertilization rate can reach 95%, and after the sea urchin seed gall oxytocin prepared by the present invention is used to induce spawning, the survival rate of the seed gall can reach 100%. It can be seen that the sea urchin seed gall oxytocin prepared by the present invention is used for sea urchin induced spawning, while ensuring its induced spawning efficiency, achieving the effect of reducing the use of chemical drugs and improving the survival rate of the seed gall after induced spawning, ensuring the subsequent storage and use of the seed gall, and solving the problem of low survival rate of the seed gall when KCl is used as the sea urchin seed gall oxytocin in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Microscope image of fertilized eggs corresponding to the sea urchin bile oxytocin prepared in Example 1;

[0020] Figure 2 : Microscopic image of fertilized egg division corresponding to the sea urchin bile oxytocin prepared in Example 1;

[0021] Figure 3 : Schematic diagram of the injection method of sea urchin catalyst;

[0022] Figure 4 :Diagram of fertilized egg hatching. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0024] As described in the background art, there are two methods for inducing ovulation of sea urchins: physical and chemical. The physical method has a low efficiency, and the chemical method has a high efficiency but is highly toxic. In the prior art, although amide is used as an oxytocin to induce ovulation of sea urchins, the effect is still poor. After inducing ovulation of female sea urchins, the amount of eggs released is 1.1 million per egg.

[0025] Based on this, the present invention provides a method for preparing a sea urchin bile oxytocin and its application. The method for preparing the sea urchin bile oxytocin comprises the following steps:

[0026] Mix 40-60g of garlic with 80-120mL of sterilized seawater, and crush them to obtain a mixed solution. Filter the mixed solution through a 240-280 mesh sieve to obtain the filtrate, which is the sea urchin seed gall oxytocin.

[0027] The preparation process of disinfected seawater is as follows: filter the seawater through a 450-500 mesh silk sieve, boil it for 5 minutes, and let it cool for later use.

[0028] The use of sterilized seawater can effectively prevent the introduction of bacteria into sea urchin gallbladder during the preparation and use of sea urchin gallbladder oxytocin. If too many bacteria are introduced, the garlic juice cannot completely sterilize them, which will cause the bacteria to enter the gallbladder after oxytocin induction, and then cause the gallbladder to die.

[0029] The induced labor is carried out by injecting 0.03-0.05 g / mL of sea urchin seed bile oxytocin into the perioral membrane of the sea urchin seed bile, wherein the injection amount of the sea urchin seed bile oxytocin is 2-4 mL.

[0030] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0031] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0032] Embodiment 1:

[0033] Take 50g of peeled garlic and 100mL of sterilized seawater, mix them, add them into a wall-breaking machine, and grind them for 2 minutes to obtain a mixed solution; wherein the preparation method of the sterilized seawater is: filter the seawater through a 500-mesh silk sieve, boil it for 5 minutes, and let it cool for later use;

[0034] The mixed solution was filtered through a 280-mesh silk sieve, and the filtrate was diluted with sterilized seawater to obtain a sea urchin seed gall oxytocin with a concentration of 0.05 g / mL, which was used for the catalysis of sea urchin seed gall.

[0035] Embodiment 2:

[0036] Take 40g of peeled garlic and mix it with 80mL of sterilized seawater, add it into a wall-breaking machine, and grind it for 2 minutes to obtain a mixed solution; wherein, the preparation method of the sterilized seawater is: filter the seawater through a 450-mesh silk sieve, boil it for 5 minutes, and let it cool for later use.

[0037] The mixed liquid was filtered through a 240-mesh silk sieve, and the filtrate was diluted with sterilized seawater to obtain a sea urchin seed gall oxytocin with a concentration of 0.04 g / mL, which was used for the catalysis of sea urchin seed gall.

[0038] Embodiment 3:

[0039] 60 g of peeled garlic and 150 mL of sterilized seawater were mixed, added into a wall-breaking machine, and crushed for 2 minutes to obtain a mixed solution; wherein the preparation method of the sterilized seawater is as follows: the seawater was filtered through a 450-mesh silk sieve, boiled for 5 minutes, and cooled for later use;

[0040] The mixed solution was filtered through a 240-mesh silk sieve, and the filtrate was diluted with sterilized seawater to obtain a sea urchin seed gall oxytocin with a concentration of 0.03 g / mL, which was used for the catalysis of sea urchin seed gall.

[0041] Comparative Example 1:

[0042] Take 50g of peeled garlic and 100mL of sterilized seawater, mix them, add them into a wall-breaking machine, and grind them for 2 minutes to obtain a mixed solution; wherein the preparation method of the sterilized seawater is: filter the seawater through a 500-mesh silk sieve, boil it for 5 minutes, and let it cool for later use;

[0043] The mixed solution was filtered through a 280-mesh silk sieve, and the filtrate was diluted with sterilized seawater to obtain a sea urchin seed gall oxytocin with a concentration of 0.2 g / mL, which was used for the catalysis of sea urchin seed gall.

[0044] Comparative Example 2:

[0045] Take 50g of peeled garlic and 100mL of sterilized seawater, mix them, add them into a wall-breaking machine, and grind them for 2 minutes to obtain a mixed solution; wherein the preparation method of the sterilized seawater is: filter the seawater through a 500-mesh silk sieve, boil it for 5 minutes, and let it cool for later use;

[0046] The mixed solution was filtered through a 280-mesh silk sieve, and the filtrate was diluted with sterilized seawater to obtain a sea urchin seed gall oxytocin with a concentration of 0.15 g / mL, which was used for the catalysis of sea urchin seed gall.

[0047] Comparative Example 3:

[0048] Take 50g of peeled garlic and 100mL of sterilized seawater, mix them, add them into a wall-breaking machine, and grind them for 2 minutes to obtain a mixed solution; wherein the preparation method of the sterilized seawater is: filter the seawater through a 500-mesh silk sieve, boil it for 5 minutes, and let it cool for later use;

[0049] The mixed solution was filtered through a 280-mesh silk sieve, and the filtrate was diluted with sterilized seawater to obtain a sea urchin seed gall oxytocin with a concentration of 0.1 g / mL, which was used for the catalysis of sea urchin seed gall.

[0050] Test Example 1:

[0051] From the sea urchin gallbladders of Yezoma dung preserved by Dalian Haibao Fishery Co., Ltd., sea urchin gallbladders with mature gonads verified by dissection were selected, and 36 gallbladders weighing 60g±2g were selected from them and divided into 6 groups on average. The sea urchin gallbladders in each group were injected with the sea urchin gallbladder oxytocin prepared in Examples 1-3 and Comparative Examples 1-3 from the peristomal membrane, and the injection amount of the oxytocin was 3mL. After the injection, the sea urchin gallbladders were placed on a sperm and egg collector, and were wetted with filtered fresh seawater every 5 minutes to keep the gallbladder moist and allow the gallbladder to discharge normally. Sperm and eggs were collected respectively after 1 hour. The egg production in each group of gallbladders is shown in Table 1.

[0052] After the collection is completed, the collected sperm is artificially inseminated. After the fertilization is completed, it is placed in a 25L incubation tank filled with water for incubation. The fertilized eggs corresponding to Examples 1-3 and Comparative Examples 1-3 are stirred evenly in water, and each group of fertilized eggs is quantified with a 1mL quantitative tube, and the fertilized eggs are examined under a microscope, and the fertilization status is recorded to calculate the fertilization rate. The results are shown in Table 1.

[0053] The induced breeding galls were raised separately and observed for 96 hours, and the mortality rate of the breeding galls was counted. The results are shown in Table 1.

[0054] Table 1 Egg laying and fertilization rate of gallbladder

[0055]

[0056] It can be seen from Table 1 that when the 0.03-0.05 g / mL seed gall bladder oxytocin prepared by the present invention is used for sea urchin induced spawning, the average egg-laying amount of female sea urchins can reach 2.6 million eggs / piece, and the fertilization rate of sperm obtained by induced spawning is as high as 95%. No matter what concentration of the seed gall bladder oxytocin prepared by garlic juice, there is no death of the seed gall bladder, which shows that garlic, as a natural nutrient, has no toxic side effects on the seed gall bladder, is safe, effective and can save costs.

[0057] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a sea urchin bile oxytocin agent, characterized in that: The following steps are involved: The seawater is filtered through a 450-500 mesh silk sieve and boiled for 5 minutes to obtain sterilized seawater. The garlic and sterilized seawater are mixed at a solid-liquid ratio of (40-60) g: (80-120) mL, and the mixture is crushed and filtered to obtain a filtrate, which is the sea urchin seed gall oxytocin agent. When the sea urchin oxytocin is used to prepare a sea urchin oxytocin drug, the concentration of the sea urchin oxytocin is 0.03 g / mL-0.05 g / mL.

2. The sea urchin oxytocin prepared by the preparation method according to claim 1.

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