Low-temperature preservation and ultralow-temperature preservation method for sperms of siniperca chuatsi subjected to feed domestication

Abstract

The invention provides a feed domesticated siniperca chuatsi sperm low-temperature preservation and ultralow-temperature preservation method, which comprises the following steps: firstly, providing a siniperca chuatsi sperm low-temperature preservation diluent which comprises Hanks liquid, sodium chloride, soya bean lecithin, vitamin E, curcumin, melatonin and SOD (superoxide dismutase); and adding an anti-freezing protective agent into the low-temperature preservation diluent to obtain the ultralow-temperature preservation anti-freezing liquid for the siniperca chuatsi sperms. The siniperca chuatsi semen and the low-temperature preservation diluent are uniformly mixed, so that low-temperature preservation can be realized; the siniperca chuatsi semen is uniformly mixed with the cryopreservation anti-freezing liquid, so that cryopreservation can be realized. The siniperca chuatsi sperm low-temperature preservation diluent is preferably selected, low-temperature preservation of the siniperca chuatsi sperms can be achieved, ultralow-temperature preservation can be conducted after an anti-freezing protective agent is added, the persistence of the preservation time of the siniperca chuatsi sperms can be guaranteed, the protection effect on the sperms is good, and the activation rate and activity of the unfrozen sperms are high.

Description

Technical Field

[0001] The invention relates to a technical solution for preserving fish genetic resources, and in particular to a method for low-temperature preservation and ultra-low-temperature preservation of sperm of Siniperca chuatsi after feed domestication. Background Art

[0002] Mandarin fish is a rare species unique to my country. At present, the annual production and sales volume is 300,000 to 350,000 tons, and the annual output value of finished fish is 24.5 billion yuan. It undertakes upstream and downstream and drives an industry worth over 100 billion yuan. The cost of traditional Mandarin fish farming is 28 to 40 yuan per catty, while the cost of feed Mandarin fish farming is 16 to 24 yuan per catty. Therefore, the next 3 to 5 years will be a rapid development stage for feed Mandarin fish. However, the research and production of feed-domesticated Mandarin fish is basically in the initial stage, and the technology is not mature enough. In particular, the reproduction of feed-domesticated Mandarin fish needs further research, and compared with the undomesticated Mandarin fish that eats live bait, the reproductive capacity is different due to the inconsistency of nutrition, environment and other conditions. Therefore, it is necessary to conduct relevant research on its sperm to improve artificial breeding technology and preserve excellent germplasm resources. We also found that the sperm of feed-domesticated Mandarin fish will be activated in conventional diluents, which also increases the difficulty of artificial breeding. It is necessary to study the cryopreservation and ultra-low temperature preservation methods of the sperm of feed-domesticated Mandarin fish.

[0003] The above-mentioned record of background technical knowledge is intended to help ordinary technicians in this field understand the existing technologies that are relatively close to the present invention, and at the same time facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clear that in the absence of clear evidence that the above-mentioned content has been disclosed before the filing date of this patent application, the above-mentioned background technology should not be used to evaluate the novelty of the technical solution of this application. Summary of the invention

[0004] Technical issues In order to solve the above problems, the purpose of the present invention is to provide a method for low-temperature preservation and ultra-low-temperature preservation of sperm of Siniperca chuatsi after feed acclimation, preferably a low-temperature preservation diluent for Siniperca chuatsi sperm, which can realize the low-temperature preservation of Siniperca chuatsi sperm, and ultra-low-temperature preservation can be performed after adding an antifreeze protective agent, which can ensure the durability of the storage time of Siniperca chuatsi sperm, and has a good protective effect on semen. After thawing, the activation rate of the sperm after storage is high and the vitality is good.

[0005] That is, the present invention is: (1) Siniperca chuatsi sperm cryopreservation diluent, comprising: Hanks solution, sodium chloride 0.01-1 g / L, soy lecithin 0.01-1 g / L, vitamin E 0.01-1 g / L, curcumin 0.01-1 g / L, melatonin 0.0001-0.1 g / L, SOD 0.0001-0.1 g / L.

[0006] Preferably, the low temperature is 1-6°C, more preferably 2-5°C, most preferably 4°C.

[0007] Preferably, the Siniperca chuatsi sperm cryopreservation diluent comprises: Hanks solution, 0.05-0.5 g / L sodium chloride, 0.05-0.5 g / L soy lecithin, 0.05-0.5 g / L vitamin E, 0.05-0.5 g / L curcumin, 0.001-0.05 g / L melatonin, and 0.001-0.05 g / L SOD.

[0008] More preferably, the Siniperca chuatsi sperm cryopreservation diluent comprises: Hanks solution, 0.5 g / L sodium chloride, 0.3 g / L soy lecithin, 0.5 g / L vitamin E, 0.1 g / L curcumin, 0.01 g / L melatonin, and 0.01 g / L SOD.

[0009] Preferably, the activation rate of Siniperca chuatsi sperm after cryopreservation for 192 hours with the Siniperca chuatsi sperm cryopreservation diluent is not less than 20%.

[0010] Soy lecithin is mainly composed of low-density lipoprotein (LDL) and lecithin. LDL can gather around the sperm cell membrane to form a protective barrier, which protects frozen sperm and makes it less likely for sperm to suffer cold shock. Lecithin is an antioxidant that plays a major role in antifreeze protection in egg yolk. Lecithin can regulate the cholesterol content in the sperm plasma membrane, making unsaturated fatty acids less susceptible to oxidative damage and ensuring that sperm has a higher plasma membrane integrity rate. Curcumin has a significant protective effect on sperm caused by reactive oxygen species, and its mechanism of action is related to reducing the content and resisting the production of lipid peroxides. Vitamin E also has a certain antioxidant effect and can neutralize harmful free radicals. Soy lecithin, curcumin and vitamin E used together have a significant additive effect, which can repair damaged cell membranes, increase the unsaturation of cell membrane fatty acids, and improve cell membrane function. In addition, melatonin is a class of powerful antioxidants and free radical scavengers that can protect the integrity and permeability of sperm membranes, reduce lipid peroxidation of unsaturated fatty acids in membranes, and maintain sperm structure. In addition, exogenous melatonin can cross the membrane and enter the sperm mitochondria, directly scavenging free radicals and reducing oxidative stress caused by excess ROS. In the process of scavenging free radicals, melatonin will produce AMK (N1-acetyl-5-methoxykynurenine) and AFMK (N1-acetyl-N2-formyl-5-methoxykynurenine), which are also powerful antioxidants. Melatonin can also exert its antioxidant effect by upregulating antioxidant enzymes (such as superoxide dismutase) and downregulating pro-oxidant enzymes (such as nitric oxide synthase). Therefore, adding melatonin to antifreeze is beneficial to maintaining sperm motility under ultra-low temperature conditions. In particular, the combined use of melatonin and SOD has a more obvious effect.

[0011] (2) Use of the aforementioned Siniperca chuatsi sperm cryopreservation diluent in cryopreservation of Siniperca chuatsi sperm.

[0012] Preferably, the low temperature is 1-6°C, more preferably 2-5°C, most preferably 4°C.

[0013] (3) An antifreeze solution for ultra-low temperature storage of sperm of Siniperca chuatsi, which comprises an antifreeze protective agent in addition to the aforementioned Siniperca chuatsi sperm low temperature storage diluent.

[0014] Preferably, the antifreeze protectant comprises dimethyl sulfoxide.

[0015] Preferably, the content of the antifreeze protectant is 1-15%, more preferably 5-10%, most preferably 10%.

[0016] More preferably, the antifreeze protectant further comprises γ-aminobutyric acid.

[0017] More preferably, the γ-aminobutyric acid accounts for 0.2-1.0% of the cryoprotectant. Studies have found that adding a specific amount of γ-aminobutyric acid to the cryoprotectant can inhibit ice formation and reduce osmotic pressure, provide extracellular and intracellular protection for sperm, further enhance the antifreeze effect of dimethyl sulfoxide, and improve the activation rate of sperm after ultra-low temperature storage.

[0018] (4) Use of the aforementioned antifreeze solution for ultra-low temperature preservation of Siniperca chuatsi sperm in ultra-low temperature preservation of Siniperca chuatsi sperm.

[0019] Preferably, the ultra-low temperature is not higher than -196°C.

[0020] (5) A method for cryopreservation of sperm of feed-acclimated Siniperca chuatsi, comprising: selecting male Siniperca chuatsi that have been acclimated to feed and have good gonad development, good health, no injuries or diseases on the body surface, and are sexually mature; collecting semen and mixing it with a Siniperca chuatsi sperm cryopreservation diluent at a volume ratio of 1:2 to 10; and storing it at a low temperature of 1 to 6°C.

[0021] (6) A method for ultra-low temperature preservation of sperm of feed-acclimated Siniperca chuatsi, comprising: selecting feed-acclimated male Siniperca chuatsi with well-developed gonads, good health, no injuries on the body surface, no diseases, and sexually mature, collecting semen and mixing it with Siniperca chuatsi sperm ultra-low temperature preservation antifreeze solution at a volume ratio of 1:2 to 10, equilibrating it at 4°C for 5 to 10 minutes, packaging it into containers with good thermal conductivity, placing it flat at 3 to 4 cm above the liquid nitrogen surface for 3 to 5 minutes, and then quickly transferring it to liquid nitrogen for storage.

[0022] Preferably, the method for ultra-low temperature preservation of sperm of Siniperca chuatsi after feed acclimation also includes a thawing step, wherein the container is quickly taken out from liquid nitrogen, immediately placed in a 35-45°C water bath and shaken to melt, and activated with pure water after melting.

[0023] Based on the common knowledge in this field, the above-mentioned preferred conditions can be combined with each other to obtain a specific implementation method.

[0024] The raw materials and reagents involved in the present invention are all common commercially available products, and the operations involved are all routine operations in the art unless otherwise specified.

[0025] Beneficial Effects According to the present invention, a preferred low-temperature preservation dilution solution for mandarin fish sperm is obtained by compounding Hanks solution, sodium chloride, soy lecithin, vitamin E, curcumin, melatonin and SOD, and the mandarin fish semen still has an activation rate of 21.67±1.53%, a movement time of 12.67±0.58s and a lifespan of 24.33±1.15s after being stored at 4°C for 192h, thereby facilitating the improvement of the artificial breeding technology of mandarin fish and the preservation of excellent germplasm resources; after adding an antifreeze protective agent to the low-temperature preservation dilution solution for mandarin fish sperm, ultra-low temperature cryopreservation of mandarin fish sperm at -196°C can be achieved, and after ultra-low temperature preservation, the activation rate of the sperm is still 55.67±4.04%, the movement time is 46.33±1.53s, and the lifespan is 74.67±1.53s. The scheme of the present invention can ensure the durability of the storage time of the sperm of Siniperca chuatsi, and has a good protection effect on semen. After the storage, the activation rate of the sperm is high and the vitality is good.

[0026] The present invention adopts the above technical solution to achieve the above purpose, which makes up for the shortcomings of the prior art and has reasonable design and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the above and / or other purposes, features, advantages and examples of the present invention more obvious and easy to understand, the drawings required for use in the specific implementation of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Schematic diagram showing the activation rate of sperm of Siniperca chuatsi after cryopreservation; Figure 2 Schematic diagram showing the movement time of sperm of Siniperca chuatsi after cryopreservation; Figure 3 Schematic diagram showing the life span of sperm of Siniperca chuatsi after cryopreservation; Figure 4 Schematic diagram showing the activation rate of sperm of Siniperca chuatsi after cryopreservation; Figure 5 Schematic diagram showing the movement time of sperm of Siniperca chuatsi after cryopreservation; Figure 6 Schematic diagram showing the lifespan of sperm of Siniperca chuatsi after cryopreservation. DETAILED DESCRIPTION

[0029] Those skilled in the art can refer to the content of this article and appropriately replace and / or modify the process parameters to achieve the same. However, it should be particularly noted that all similar replacements and / or modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products and preparation methods described in the present invention have been described through preferred examples. It is obvious that relevant personnel can modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0030] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The present invention uses the methods and materials described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail.

[0031] Unless otherwise specified, all percentages, parts, ratios, etc. are by weight; otherwise specified, including but not limited to, "%", "wt%", "mass%" means weight percentage, "mol%" means molar percentage, and "vol%" means volume percentage.

[0032] When a quantity, concentration or other numerical value or parameter is given as a range, a preferred range or a series of upper preferred values ​​and lower preferred values, it should be understood that it specifically discloses all ranges formed by any pair of numerical values ​​of any larger range limit or preferred value and any smaller range limit or preferred value, regardless of whether the range is disclosed separately. For example, when describing a range of "1 to 5 (1-5)", the described range should be understood to include a range of "1 to 4 (1-4)", "1 to 3 (1-3)", "1 to 2 (1-2)", "1 to 2 (1-2) and 4 to 5 (4-5)", "1 to 3 (1-3) and 5", etc. Unless otherwise stated, where a numerical range is described herein, the range includes the range end values ​​and all integers and fractions within the range.

[0033] Unless specifically stated, the materials, methods, and examples described herein are illustrative only and not limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are still described herein.

[0034] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other without conflict.

[0035] The present invention is described in detail below Embodiment 1: This embodiment provides a method for low-temperature storage and ultra-low-temperature storage of sperm of Siniperca chuatsi after feed acclimation, which specifically includes the following steps.

[0036] 1. Materials: Male Siniperca chuatsi parents that had been acclimated to feed were taken from the Deqing base of Zhejiang Freshwater Fisheries Research Institute. The fish with well-developed gonads, good health, no injuries or diseases on the body surface and sexual maturity were selected as experimental materials, and semen was collected for later use.

[0037] 2. Prepare the low-temperature preservation diluent for sperm of Siniperca chuatsi, which is the preservation solution: Hank's solution (D-Hank, s solution, does not contain calcium and magnesium ions, does not contain phenol red, Tianjin Haoyang Biological Products Technology Co., Ltd.) + Hanks solution + sodium chloride 0.5g / L + soy lecithin 0.3g / L + vitamin E 0.5g / L + curcumin 0.1g / L + melatonin 0.01g / L + SOD 0.01g / L and store in the refrigerator for use.

[0038] 3. Prepare the antifreeze solution for ultra-low temperature preservation of sperm of Siniperca chuatsi, which is also called antifreeze solution: it is prepared by preparing the low temperature preservation diluent of sperm of Siniperca chuatsi + antifreeze protective agent. The antifreeze protective agent is dimethyl sulfoxide, which is mixed with the preservation solution to form the antifreeze solution, wherein the content of the antifreeze protective agent is 10%. The above antifreeze solution is stored in a refrigerator at 4°C for use.

[0039] 4. Methods: 4.1. Semen collection: Semen was placed in various preservation solutions, with the ratio of sperm to Siniperca chuatsi sperm cryopreservation diluent being 1:5, and stored at 4°C.

[0040] 4.2. Sperm quality test: After the semen is mixed with the activation solution (ultrapure water or NaCl aqueous solution of appropriate concentration, ultrapure water is used in this embodiment), the sperm quality is observed under a microscope, including the activation rate, activity time and lifespan, where the activation rate refers to the percentage of the number of moving sperm in the same field of view observed under a microscope immediately after the sperm is mixed with the activation solution to the total number of sperm; the movement time refers to the time from the activation of the sperm to the 90% trembling in place; the lifespan refers to the time from the activation of the sperm to the 90% cessation of movement. The experiment was repeated 3 times.

[0041] 4.3. Motility time and lifespan: Use a pipette to draw a small amount of semen into a centrifuge tube with activation solution added in advance, quickly stir it and drop it on a glass slide, and observe the activation rate of sperm under a light microscope; the centrifuge tube is stored at 4°C, and drops are added on a glass slide at regular intervals, and the motility time and lifespan of the sperm are observed under a light microscope.

[0042] 4.4. Ultra-low temperature cryopreservation: Mix the semen and the ultra-low temperature preservation antifreeze solution of Siniperca chuatsi sperm in a ratio of 1:5, balance at 4°C for 10 minutes, and then divide into 0.25mL straws (each containing about 0.2mL). Place the straws containing semen flat on a homemade simple cooling device 4cm above the liquid nitrogen surface for 5 minutes and then quickly transfer them to liquid nitrogen for storage. When thawing, quickly take out the straws from the liquid nitrogen and immediately place them in a 40°C water bath to shake and dissolve. After activation with pure water, detect the activity of the frozen semen under a microscope. Repeat the experiment 3 times.

[0043] 5. Data processing: The experimental data were analyzed using Excel and SPSS 17.0 software, and the differences in sperm motility among the groups were tested by one-way ANOVA, with a significant difference level of P < 0.05. The statistical results were expressed as mean and standard deviation (X ± SD).

[0044] During the research and development process, it was found that fresh semen would be activated in diluents such as Hanks solution and Ren's solution, but would not be activated after adding a certain amount of sodium chloride (0.5g / L), so sodium chloride is necessary. Subsequently, a diluent formula (Hanks solution + sodium chloride 0.5g / L + soy lecithin 0.3g / L + vitamin E 0.5g / L + curcumin 0.1g / L + melatonin 0.01g / L + SOD (superoxide dismutase) 0.01g / L) more suitable for the low temperature (4°C) storage of Siniperca chuatsi semen and an antifreeze formula for ultra-low temperature (-196°C) storage were screened out. The verification results of the diluent and fresh semen groups of the above formulas are shown in Table 1.

[0045] Table 1- Validation results of fresh semen group and fresh semen + diluent group

[0046] / :die.

[0047] The suitable antifreeze for the semen of Siniperca chuatsi is 10% dimethyl sulfoxide (the group with glycerol as antifreeze died, and the group with methanol as antifreeze had a low activation rate of frozen semen). Under the appropriate antifreeze formula, the activation rate of sperm after ultra-low temperature (-196℃) preservation was still 55.67±4.04%, the movement time was 46.33±1.53s, and the life span was 74.67±1.53s; when the antifreeze was only Hanks solution + sodium chloride 0.5g / L + 10% dimethyl sulfoxide, the activation rate of frozen semen was 42.33±2.52%, the movement time was 39.33±1.15s, and the life span was 66.33±1.53s. It can be seen that soybean lecithin, vitamin E, curcumin, melatonin and SOD in the preservation solution all have a positive effect on the effect of ultra-low temperature preservation of sperm in the antifreeze solution.

[0048] As shown in Table 1, the diluent suitable for the low temperature (4°C) storage of Siniperca chuatsi semen (Hanks solution + sodium chloride 0.5 g / L + soy lecithin 0.3 g / L + vitamin E 0.5 g / L + curcumin 0.1 g / L + melatonin 0.01 g / L + SOD 0.01 g / L) can be stored for 240 hours. After 192 hours of storage, the semen still has an activation rate of 21.67±1.53%, a motility time of 12.67±0.58 s, and a lifespan of 24.33±1.15 s.

[0049] The suitable antifreeze agent is 10% dimethyl sulfoxide. Under the suitable antifreeze solution formula (Hanks solution + sodium chloride 0.5g / L + soy lecithin 0.3g / L + vitamin E 0.5g / L + curcumin 0.1g / L + melatonin 0.01g / L + SOD 0.01g / L + 10% dimethyl sulfoxide), after ultra-low temperature (-196℃) storage, the activation rate of sperm is still 55.67±4.04%, the motility time is 46.33±1.53s, and the lifespan is 74.67±1.53s.

[0050] Embodiment 2: On the basis of the above-mentioned embodiment, the formula of the low-temperature preservation diluent for sperm of Siniperca chuatsi was changed to: Hank's solution + sodium chloride 0.01 g / L, soy lecithin 0.01 g / L, vitamin E 0.8 g / L, curcumin 0.5 g / L, melatonin 0.1 g / L, SOD 0.1 g / L and stored in a refrigerator at 4°C for use.

[0051] The ultra-low temperature preservation antifreeze solution for mandarin fish chuatsi sperm is prepared by mandarin fish chuatsi sperm low temperature preservation diluent + antifreeze agent. The antifreeze agent is dimethyl sulfoxide, which is mixed with the mandarin fish chuatsi sperm low temperature preservation diluent to form an antifreeze solution, in which the antifreeze content is 10%. The above antifreeze solution is stored in a 4°C refrigerator for use.

[0052] The method for low-temperature preservation of sperm of Siniperca chuatsi after feed acclimation includes: collecting semen, mixing it with Siniperca chuatsi sperm low-temperature preservation diluent at a volume ratio of 1:5, and storing it at a low temperature of 4°C.

[0053] The method for ultra-low temperature preservation of sperm of Siniperca chuatsi after feed acclimation includes: collecting semen and mixing it with antifreeze solution for ultra-low temperature preservation of Siniperca chuatsi sperm in a volume ratio of 1:5, balancing it at 4°C for 10 minutes, and filling it into straw tubes with a volume of 0.25mL (each tube contains about 0.2mL), placing the straw tubes containing semen flat at 3cm above the liquid nitrogen surface of a homemade simple cooling device for 5 minutes, and then quickly transferring it to liquid nitrogen for storage. When thawing, the straw tubes are quickly taken out of the liquid nitrogen and immediately placed in a 40°C water bath to shake and melt. After activation with pure water, the activity of the frozen sperm is detected under a microscope.

[0054] Embodiment 3: On the basis of the above-mentioned embodiment, the formula of the low-temperature preservation diluent for sperm of Siniperca chuatsi was changed to: Hank's solution + sodium chloride 1g / L, soy lecithin 1g / L, vitamin E 0.01g / L, curcumin 0.01g / L, melatonin 0.0001, SOD 0.0001g / L and stored in a refrigerator at 4°C for use.

[0055] The ultra-low temperature preservation antifreeze solution for mandarin fish chuatsi sperm is prepared by mandarin fish chuatsi sperm low temperature preservation diluent + antifreeze. The antifreeze is dimethyl sulfoxide, which is mixed with the mandarin fish chuatsi sperm low temperature preservation diluent to form an antifreeze solution, in which the antifreeze content is 5%. The above antifreeze solution is stored in a 4°C refrigerator for use.

[0056] The method for low-temperature preservation of sperm of Siniperca chuatsi after feed acclimation includes: collecting semen, mixing it with Siniperca chuatsi sperm low-temperature preservation diluent at a volume ratio of 1:6, and storing it at a low temperature of 4°C.

[0057] The method for ultra-low temperature preservation of sperm of Siniperca chuatsi after feed domestication includes: collecting semen and mixing it with antifreeze solution for ultra-low temperature preservation of Siniperca chuatsi sperm in a volume ratio of 1:6, balancing at 4°C for 5 minutes, and filling it into straw tubes with a volume of 0.25mL (each tube contains about 0.2mL), placing the straw tubes containing semen flat at 4cm above the liquid nitrogen surface of a homemade simple cooling device for 4 minutes, and then quickly transferring them into liquid nitrogen for storage. When thawing, the straw tubes are quickly taken out of the liquid nitrogen and immediately placed in a 40°C water bath to shake and melt. After activation with pure water, the activity of the frozen sperm is detected under a microscope.

[0058] Embodiment 4: On the basis of the above-mentioned embodiment, the formula of the low-temperature preservation diluent for sperm of Siniperca chuatsi was changed to: Hank's solution + sodium chloride 0.5 g / L, soy lecithin 0.5 g / L, vitamin E 1 g / L, curcumin 1 g / L, melatonin 0.05 g / L, SOD 0.05 g / L and stored in a refrigerator at 4°C for use.

[0059] The ultra-low temperature preservation antifreeze solution for mandarin fish chuatsi sperm is prepared by mandarin fish chuatsi sperm low temperature preservation diluent + antifreeze. The antifreeze is dimethyl sulfoxide, which is mixed with the mandarin fish chuatsi sperm low temperature preservation diluent to form an antifreeze solution, in which the antifreeze content is 3%. The above antifreeze solution is stored in a 4°C refrigerator for use.

[0060] The method for low-temperature preservation of sperm of Siniperca chuatsi after feed acclimation includes: collecting semen, mixing it with Siniperca chuatsi sperm low-temperature preservation diluent at a volume ratio of 1:10, and storing it at a low temperature of 4°C.

[0061] The method for ultra-low temperature preservation of sperm of Siniperca chuatsi after feed domestication includes: collecting semen and mixing it with antifreeze solution for ultra-low temperature preservation of Siniperca chuatsi sperm at a volume ratio of 1:10, balancing at 4°C for 8 minutes, and filling it into straw tubes with a volume of 0.25mL (each tube contains about 0.2mL), placing the straw tubes containing semen flat at 3cm above the liquid nitrogen surface of a homemade simple cooling device for 3 minutes, and then quickly transferring them into liquid nitrogen for storage. When thawing, the straw tubes are quickly taken out of the liquid nitrogen and immediately placed in a 40°C water bath to shake and dissolve. After activation with pure water, the activity of the frozen sperm is detected under a microscope.

[0062] Embodiment 5: On the basis of the above-mentioned embodiment, the formula of the diluent for low-temperature storage of sperm of Siniperca chuatsi was changed to: Hank's solution + 0.4 g / L sodium chloride, 0.25 / L soy lecithin, 0.8 g / L vitamin E, 0.3 g / L curcumin, 0.09 g / L melatonin, 0.06 g / L SOD and stored in a refrigerator at 4°C for use.

[0063] The ultra-low temperature preservation antifreeze solution for mandarin fish chuatsi sperm is prepared by mandarin fish chuatsi sperm low temperature preservation diluent + antifreeze. The antifreeze is dimethyl sulfoxide, which is mixed with the mandarin fish chuatsi sperm low temperature preservation diluent to form an antifreeze solution, in which the antifreeze content is 12%. The above antifreeze solution is stored in a 4°C refrigerator for use.

[0064] The method for low-temperature preservation of sperm of Siniperca chuatsi after feed acclimation includes: collecting semen, mixing it with Siniperca chuatsi sperm low-temperature preservation diluent at a volume ratio of 1:4, and storing it at a low temperature of 4°C.

[0065] The method for ultra-low temperature preservation of sperm of Siniperca chuatsi after feed domestication includes: collecting semen and mixing it with antifreeze solution for ultra-low temperature preservation of Siniperca chuatsi sperm in a volume ratio of 1:4, balancing it at 4°C for 10 minutes, and filling it into straw tubes with a volume of 0.25mL (each tube contains about 0.2mL), placing the straw tubes containing semen flat at 3cm above the liquid nitrogen surface of a homemade simple cooling device for 5 minutes, and then quickly transferring it to liquid nitrogen for storage. When thawing, the straw tubes are quickly taken out of the liquid nitrogen and immediately placed in a 40°C water bath to shake and melt. After activation with pure water, the activity of the frozen sperm is detected under a microscope.

[0066] Embodiment 6: On the basis of the above-mentioned Example 1, the formula of the diluent for low-temperature preservation of Siniperca chuatsi sperm was changed to: Hanks solution + sodium chloride 0.5 g / L + vitamin E 0.5 g / L + curcumin 0.1 g / L + melatonin 0.01 g / L + SOD (superoxide dismutase) 0.01 g / L and stored in a refrigerator at 4°C for later use. The Siniperca chuatsi sperm was preserved by the same method as in Example 1.

[0067] Embodiment 7: On the basis of the above-mentioned Example 1, the formula of the low-temperature preservation diluent of Siniperca chuatsi sperm was changed to: Hanks solution + 0.5 g / L sodium chloride + 0.3 g / L soy lecithin + 0.1 g / L curcumin + 0.01 g / L melatonin + 0.01 g / L SOD (superoxide dismutase) and stored in a refrigerator at 4°C for later use. The Siniperca chuatsi sperm was preserved by the same method as in Example 1.

[0068] Embodiment 8: On the basis of the above-mentioned Example 1, the formula of the low-temperature preservation diluent of Siniperca chuatsi sperm was changed to: Hanks solution + 0.5 g / L sodium chloride + 0.3 g / L soy lecithin + 0.5 g / L vitamin E + 0.01 g / L melatonin + 0.01 g / L SOD (superoxide dismutase) and stored in a refrigerator at 4°C for later use. The Siniperca chuatsi sperm was preserved by the same method as in Example 1.

[0069] Embodiment 9: On the basis of the above-mentioned Example 1, the formula of the diluent for low-temperature storage of Siniperca chuatsi sperm was changed to: Hanks solution + 0.5 g / L sodium chloride + 0.3 g / L soy lecithin + 0.5 g / L vitamin E + 0.1 g / L curcumin + 0.01 g / L SOD (superoxide dismutase) and stored in a 4°C refrigerator for later use. The same method as in Example 1 was used to store the Siniperca chuatsi sperm at 4°C and at -196°C.

[0070] Embodiment 10: On the basis of the above-mentioned Example 1, the formula of the diluent for low-temperature storage of Siniperca chuatsi sperm was changed to: Hanks solution + 0.5 g / L sodium chloride + 0.3 g / L soy lecithin + 0.5 g / L vitamin E + 0.1 g / L curcumin + 0.01 g / L melatonin and stored in a refrigerator at 4°C for later use. The same method as in Example 1 was used to store the Siniperca chuatsi sperm at 4°C and at -196°C.

[0071] Experimental Example 1: The activation rate, movement time and life span of sperms after being stored at low temperature (4° C.) for 120 h in Examples 1 to 10 were statistically analyzed. The statistical results are as follows: Figure 1 to Figure 3 shown.

[0072] Depend on Figure 1 to Figure 3 It can be seen that the low-temperature preservation diluents for sperm of Siniperca chuatsi of the preferred embodiments 1 to 5 of the present invention have a good low-temperature preservation effect on sperm of Siniperca chuatsi. After being stored at 4°C for 120 hours, the sperm activation rate is not less than 30%, the motility time is not less than 15 seconds, and the life span is not less than 30 seconds. The durability of the sperm storage time of Siniperca chuatsi can be ensured, and the protection effect on semen is good. After storage, the sperm activation rate is high and the vitality is good.

[0073] from Figure 1 to Figure 3 It can also be known that the diluents in Examples 6 to 8 have poor low-temperature preservation effects on the sperm of Siniperca chuatsi. Specifically, when one of soybean lecithin, curcumin, and vitamin E is missing from the diluent, its low-temperature preservation effect on the sperm of Siniperca chuatsi will be significantly reduced. It is speculated that the regulatory effect of lecithin on cholesterol content and the antioxidant effects of curcumin and vitamin E have an additive effect, which can repair damaged cell membranes and improve cell membrane function, thereby macroscopically improving the low-temperature preservation effect of the diluent on the sperm of Siniperca chuatsi. Melatonin or SOD has little effect on the low-temperature preservation of sperm of Siniperca chuatsi.

[0074] Embodiment 11: On the basis of the above-mentioned Example 1, the cryoprotectant for ultra-low temperature storage of Siniperca chuatsi sperm was changed, specifically, an antifreeze agent was added to the storage solution to prepare the antifreeze solution, the antifreeze agent was selected as methanol, and it was mixed with the storage solution to form an antifreeze solution, wherein the antifreeze agent content was 10%, and the above antifreeze solution was stored in a refrigerator at 4°C for use. The sperm of Siniperca chuatsi was ultra-low temperature stored at -196°C using the same method as in Example 1.

[0075] Embodiment 12: On the basis of the above-mentioned Example 1, the cryopreservation antifreeze solution for the sperm of Siniperca chuatsi was changed, specifically, an antifreeze protectant was added to the preservation solution, and ethylene glycol was selected as the antifreeze protectant, which was mixed with the preservation solution to form an antifreeze solution, wherein the antifreeze protectant content was 10%, and the above antifreeze solution was stored in a refrigerator at 4°C for use. The sperm of Siniperca chuatsi was ultra-low temperature preserved at -196°C using the same method as in Example 1.

[0076] Embodiment 13: On the basis of the above-mentioned Example 1, the cryoprotectant for ultra-low temperature storage of Siniperca chuatsi sperm was changed, specifically, an antifreeze solution was prepared by adding an antifreeze protectant to the storage solution, and glycerol was selected as the antifreeze protectant, which was mixed with the storage solution to form an antifreeze solution, wherein the antifreeze protectant content was 10%, and the above antifreeze solution was stored in a refrigerator at 4°C for use. The sperm of Siniperca chuatsi was ultra-low temperature stored at -196°C using the same method as in Example 1.

[0077] Embodiment 14: On the basis of the above-mentioned Example 1, the cryoprotectant for ultra-low temperature storage of Siniperca chuatsi sperm was changed, specifically, 10% of cryoprotectant was added to the preservation solution, and the cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid, wherein γ-aminobutyric acid accounted for 0.5% of the cryoprotectant; the cryoprotectant was mixed with the preservation solution to form an antifreeze solution, which was stored in a refrigerator at 4°C for later use. The sperm of Siniperca chuatsi was ultra-low temperature stored at -196°C using the same method as in Example 1.

[0078] Embodiment 15: On the basis of the above-mentioned Example 1, the cryopreservation antifreeze solution for the sperm of Siniperca chuatsi was changed, specifically, 10% of the cryoprotectant was added to the preservation solution, and the cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid, wherein the γ-aminobutyric acid accounted for 0.2% of the cryoprotectant; the cryoprotectant was mixed with the preservation solution to form the antifreeze solution, which was stored in a refrigerator at 4°C for use. The sperm of Siniperca chuatsi was ultra-low temperature preserved at -196°C using the same method as in Example 1.

[0079] Embodiment 16: On the basis of the above-mentioned Example 1, the cryoprotectant for ultra-low temperature storage of Siniperca chuatsi sperm was changed, specifically, 10% of cryoprotectant was added to the preservation solution, and the cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid, wherein γ-aminobutyric acid accounted for 1.0% of the cryoprotectant; the cryoprotectant was mixed with the preservation solution to form an antifreeze solution, which was stored in a refrigerator at 4°C for later use. The sperm of Siniperca chuatsi was ultra-low temperature stored at -196°C using the same method as in Example 1.

[0080] Embodiment 17: On the basis of the above-mentioned Example 1, the cryopreservation antifreeze solution for the sperm of Siniperca chuatsi was changed, specifically, 10% of the cryoprotectant was added to the preservation solution, and the cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid, wherein the γ-aminobutyric acid accounted for 0.1% of the cryoprotectant; the cryoprotectant was mixed with the preservation solution to form the antifreeze solution, which was stored in a refrigerator at 4°C for use. The sperm of Siniperca chuatsi was ultra-low temperature preserved at -196°C using the same method as in Example 1.

[0081] Embodiment 18: On the basis of the above-mentioned Example 1, the cryoprotectant for ultra-low temperature storage of Siniperca chuatsi sperm was changed, specifically, 10% of cryoprotectant was added to the preservation solution, and the cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid, wherein γ-aminobutyric acid accounted for 1.5% of the cryoprotectant; the cryoprotectant was mixed with the preservation solution to form an antifreeze solution, which was stored in a refrigerator at 4°C for later use. The sperm of Siniperca chuatsi was ultra-low temperature stored at -196°C using the same method as in Example 1.

[0082] Experimental Example 2: The activation rate, movement time and life span of sperm after ultra-low temperature of -196°C in Example 1 and Example 9 to Example 18 were statistically analyzed. The statistical results are as follows: Figures 4 to 6 shown.

[0083] Combination Figures 4 to 6 By analyzing Example 1 and Example 9 to Example 10, it can be seen that compared with Example 9 and Example 10, adding a trace amount of melatonin and SOD to the sperm cryopreservation diluent of Siniperca chuatsi is beneficial to maintaining sperm motility under ultra-low temperature conditions, and the effect of adding either of them will be significantly reduced. After analyzing Example 1 and Example 11 to Example 13, it was found that compared with other conventional cryoprotectants such as methanol, ethylene glycol, glycerol, etc., using dimethyl sulfoxide as an antifreeze protectant can significantly improve the activation rate and sperm activity of sperm, while the sperm activation rate of the methanol group and the ethylene glycol group was extremely low, and all sperm in the glycerol group died.

[0084] Combination Figures 4 to 6 By analyzing Example 1 and Examples 14 to 18, it can be seen that compared with Example 1 in which only dimethyl sulfoxide is used as an antifreeze protectant, the antifreeze protectant formed by compounding dimethyl sulfoxide with a specific content of γ-aminobutyric acid can further enhance the antifreeze effect of dimethyl sulfoxide, and improve the activation rate of sperm after ultra-low temperature storage. After ultra-low temperature (-196°C) storage, the activation rate of sperm is not less than 65%, the motility time is not less than 60s, and the life span is not less than 80s. The use of the above-mentioned composite antifreeze protectant combined with the low-temperature storage diluent for Siniperca chuatsi sperm provided by the present invention is sufficient to ensure the durability of the storage time of Siniperca chuatsi sperm, and has a good protective effect on semen. After storage, the activation rate of sperm is high, the vitality is good, and the survival time is long, which is beneficial to the improvement of artificial breeding technology and the retention of excellent germplasm resources.

[0085] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0086] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0087] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

[0088] Although the above-mentioned specific embodiments have been shown, described and pointed out the novel features applied to various embodiments, it should be understood that various omissions, substitutions and changes can be made to the form and details of the described device or method without departing from the spirit of the present disclosure. In addition, the above-mentioned various features and methods can be used independently of each other, or can be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the above-mentioned embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the present invention has been disclosed in the context of certain embodiments and examples, it should be understood by those skilled in the art that the present invention can extend beyond the specifically disclosed embodiments to other alternative embodiments and / or applications and obvious modifications and equivalents thereof. Therefore, the present invention is not intended to be limited by the specific disclosure of the preferred embodiments herein.

[0089] Matters not covered in the present invention are all known technologies.

Claims

1. A diluent for the cryopreservation of sperm of Siniperca chuatsi, characterized in that include: Hanks solution, sodium chloride 0.01~1g / L, soy lecithin 0.01~1g / L, vitamin E 0.01~1g / L, curcumin 0.01~1g / L, melatonin 0.0001~0.1g / L, SOD 0.0001~0.1g / L.

2. The diluent for cryopreservation of sperm of Siniperca chuatsi according to claim 1, characterized in that: The low temperature is 1-6°C.

3. The low-temperature preservation diluent for sperm of Siniperca chuatsi according to claim 1, characterized in that: The low-temperature preservation diluent for sperm of Siniperca chuatsi comprises: Hanks solution, 0.05-0.5 g / L sodium chloride, 0.05-0.5 g / L soybean lecithin, 0.05-0.5 g / L vitamin E, 0.05-0.5 g / L curcumin, 0.001-0.05 g / L melatonin, and 0.001-0.05 g / L SOD.

4. The diluent for cryopreservation of sperm of Siniperca chuatsi according to any one of claims 1 to 3, characterized in that: The activation rate of Siniperca chuatsi sperm after cryopreservation for 192 hours with the Siniperca chuatsi sperm cryopreservation diluent is not less than 20%.

5. Use of the Siniperca chuatsi sperm cryopreservation diluent according to any one of claims 1 to 4 in cryopreservation of Siniperca chuatsi sperm.

6. A method for cryopreservation of sperm of Siniperca chuatsi after feed acclimation, characterized in that include: After collecting the semen of Siniperca chuatsi, the semen is mixed with the Siniperca chuatsi sperm cryopreservation diluent according to any one of claims 1 to 4 at a volume ratio of 1:2 to 10, and stored at a low temperature of 1 to 6°C.

7. Antifreeze solution for ultra-low temperature storage of sperm of Siniperca chuatsi, characterized by: The diluent for low-temperature storage of sperm of Siniperca chuatsi according to any one of claims 1 to 4 further comprises an antifreeze protectant, wherein the antifreeze protectant comprises dimethyl sulfoxide.

8. Use of the antifreeze solution for ultra-low temperature preservation of Siniperca chuatsi sperm according to claim 7 in ultra-low temperature preservation of Siniperca chuatsi sperm.

9. A method for cryopreservation of sperm of Siniperca chuatsi after acclimation to feed, characterized in that include: After collecting the semen of Siniperca chuatsi, mix it with the ultra-low temperature preservation antifreeze solution of Siniperca chuatsi sperm described in claim 7 at a volume ratio of 1:2-10, balance it at 4°C for 5-10 minutes, divide it into containers with good thermal conductivity, place it flat at 3-4 cm above the liquid nitrogen surface for 3-5 minutes, and quickly transfer it to liquid nitrogen for storage.

10. The method for cryopreservation of sperm of Siniperca chuatsi after feed acclimation according to claim 9, characterized in that: The process also includes a thawing step, in which the container is quickly taken out of the liquid nitrogen and immediately placed in a 35-45°C water bath to be shaken and melted, and then activated with pure water after the melting is complete.

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