Method for cryopreservation and ultra-low temperature preservation of mandarin fish (mylopharyngodon piceus) sperm after feed domestication

Abstract

The application provides a method for cryopreservation and ultra-cryopreservation of the sperm of feed domesticated mandarin fish, and first provides a cryopreservation diluent for the sperm of the mandarin fish, which comprises Hanks solution, sodium chloride, soybean lecithin, vitamin E, curcumin, melatonin and SOD; an anti-freezing protective agent is added in the cryopreservation diluent to obtain an ultra-cryopreservation anti-freezing solution for the sperm of the mandarin fish; the sperm of the mandarin fish is mixed with the cryopreservation diluent to realize cryopreservation; and the sperm of the mandarin fish is mixed with the ultra-cryopreservation anti-freezing solution to realize ultra-cryopreservation. The cryopreservation diluent for the sperm of the mandarin fish can realize cryopreservation of the sperm of the mandarin fish, and the sperm can be ultra-cryopreserved after the addition of the anti-freezing protective agent, so that the persistence of the preservation time of the sperm of the mandarin fish is ensured, the protection effect on the sperm is good, the activation rate of the sperm after thawing is high, and the sperm has good vitality.

Description

Technical Field

[0001] This invention relates to a technical solution for the preservation of fish genetic resources, specifically to a method for the cryopreservation and ultra-low temperature preservation of sperm from mandarin fish that have been domesticated for feed. Background Technology

[0002] Mandarin fish is a unique and precious species in my country, with an annual production and sales volume of 300,000 to 350,000 tons and an annual output value of 24.5 billion yuan for finished fish, driving an industry worth over 100 billion yuan through upstream and downstream connections. The traditional mandarin fish farming cost is 28-40 yuan / jin (approximately 14-16 yuan / catties), while the cost of feed-fed mandarin fish farming is 16-24 yuan / jin (approximately 14-15 yuan / catties). Therefore, the next 3-5 years will be a period of rapid development for feed-fed mandarin fish. However, research and production of feed-domesticated mandarin fish are still in their initial stages, and the technology is not yet mature. In particular, the reproduction of feed-domesticated mandarin fish requires further research. Compared with undomesticated mandarin fish fed live bait, the reproductive capacity differs due to differences in nutrition and environmental conditions. Therefore, it is necessary to conduct relevant research on their sperm to improve artificial breeding techniques and preserve superior germplasm resources. Furthermore, we have found that the sperm of feed-domesticated mandarin fish is activated in conventional diluents, which increases the difficulty of artificial breeding. Research is needed on low-temperature and ultra-low-temperature preservation methods for the sperm of feed-domesticated mandarin fish.

[0003] The foregoing background information is intended to help those skilled in the art understand prior art that is similar to the present invention, and to facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clearly stated that, in the absence of clear evidence that the above content was disclosed before the filing date of this patent application, the foregoing background information should not be used to evaluate the novelty of the technical solution of this application. Summary of the Invention

[0004] Technical issues

[0005] To address the aforementioned problems, the present invention aims to provide a method for cryopreservation and ultra-low temperature preservation of sperm from mandarin fish after feed domestication. A preferred method is a cryopreservation diluent for mandarin fish sperm, which enables cryopreservation of sperm. The addition of an antifreeze agent allows for ultra-low temperature preservation, ensuring the longevity of sperm preservation time and providing excellent protection for the semen. Furthermore, the preserved sperm exhibits high activation rate and good motility after thawing.

[0006] That is, the present invention is:

[0007] (1) Low-temperature preservation dilution solution for mandarin fish sperm, including: Hanks solution, sodium chloride 0.01~1g / L, soybean lecithin 0.01~1g / L, vitamin E 0.01~1g / L, curcumin 0.01~1g / L, melatonin 0.0001~0.1g / L, and SOD 0.0001~0.1g / L.

[0008] Preferably, the low temperature is 1-6°C, more preferably 2-5°C, and most preferably 4°C.

[0009] Preferably, the cryopreservation diluent for mandarin fish sperm comprises: Hanks' solution, 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 0.05–0.5 g / L, melatonin 0.001–0.05 g / L, and SOD 0.001–0.05 g / L.

[0010] More preferably, the cryopreservation diluent for mandarin fish sperm includes: Hanks' solution, sodium chloride 0.5 g / L, soybean lecithin 0.3 g / L, vitamin E 0.5 g / L, curcumin 0.1 g / L, melatonin 0.01 g / L, and SOD 0.01 g / L.

[0011] Preferably, the activation rate of mandarin fish sperm after cryopreservation in the aforementioned cryopreservation diluent for 192 hours is not less than 20%.

[0012] Soy lecithin is mainly composed of low-density lipoprotein (LDL) and lecithin. LDL can accumulate around the sperm cell membrane to form a protective barrier, protecting frozen sperm and preventing cold shock. Lecithin is a major antioxidant in egg yolk that provides antifreeze protection. Lecithin can regulate cholesterol content in the sperm plasma membrane, making unsaturated fatty acids less susceptible to oxidative damage and ensuring a high sperm plasma membrane integrity rate. Curcumin has a significant protective effect against sperm damage caused by reactive oxygen species, and its mechanism of action is related to reducing cholesterol content and inhibiting the production of lipid peroxides. Vitamin E also has certain antioxidant effects and can neutralize harmful free radicals. The combined use of soy lecithin, curcumin, and vitamin E has a significant synergistic 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 powerful antioxidant and free radical scavenger that can protect the integrity and permeability of the sperm membrane, reduce lipid peroxidation of unsaturated fatty acids in the membrane, and maintain sperm structure. In addition, exogenous melatonin can cross the membrane into sperm mitochondria, directly scavenging free radicals and reducing oxidative stress caused by excess ROS. During free radical scavenging, melatonin produces AMK (N1-acetyl-5-methoxykynurenine) and AFMK (N1-acetyl-N2-methoxykynurenine), both of which are potent antioxidants. Melatonin can also exert its antioxidant effect by upregulating antioxidant enzymes (such as superoxide dismutase) and downregulating prooxidant enzymes (such as nitric oxide synthase). Therefore, adding melatonin to antifreeze is beneficial for maintaining sperm motility under ultra-low temperature conditions. The effect is even more pronounced when melatonin is used in combination with SOD.

[0013] (2) The use of the aforementioned diluent for cryopreservation of mandarin fish sperm in cryopreservation of mandarin fish sperm.

[0014] Preferably, the low temperature is 1-6°C, more preferably 2-5°C, and most preferably 4°C.

[0015] (3) The cryopreservation antifreeze solution for mandarin fish sperm includes an antifreeze agent in addition to the cryopreservation diluent for mandarin fish sperm mentioned above.

[0016] Preferably, the antifreeze agent includes dimethyl sulfoxide.

[0017] Preferably, the content of the antifreeze is 1-15%, more preferably 5-10%, and most preferably 10%.

[0018] More preferably, the antifreeze agent further includes γ-aminobutyric acid (GABA).

[0019] More preferably, the γ-aminobutyric acid accounts for 0.2% to 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, providing extracellular and intracellular protection for sperm, further enhancing the cryoprotective effect of dimethyl sulfoxide, and improving the sperm activation rate after cryopreservation.

[0020] (4) The use of the cryopreservation antifreeze solution for mandarin fish sperm described above in cryopreservation of mandarin fish sperm.

[0021] Preferably, the ultra-low temperature is not higher than -196°C.

[0022] (5) The method for low-temperature preservation of sperm of mandarin fish after feed domestication includes: selecting male mandarin fish that are sexually mature, have well-developed gonads, good health, no injuries or diseases on their body surface, collect semen, mix it with low-temperature preservation diluent for mandarin fish sperm at a volume ratio of 1:2 to 10, and preserve it at low temperature of 1 to 6℃.

[0023] (6) The method for cryopreservation of sperm from mandarin fish after feed domestication includes: selecting male mandarin fish that are sexually mature, have well-developed gonads, good health, no injuries or diseases on their body surface, and are sexually mature after feed domestication; collecting semen and mixing it with cryopreservation antifreeze solution for mandarin fish sperm at a volume ratio of 1:2 to 10; equilibrating at 4°C for 5 to 10 minutes; dispensing into containers with good thermal conductivity; placing them flat 3 to 4 cm above the liquid nitrogen surface and letting them stand for 3 to 5 minutes; and then quickly transferring them into liquid nitrogen for preservation.

[0024] Preferably, the method for cryopreserving sperm of mandarin fish after feed domestication also includes a thawing step, in which the container is quickly removed from liquid nitrogen and immediately placed in a water bath at 35-45°C and shaken to dissolve. After dissolving, the sperm is activated with pure water.

[0025] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined to obtain specific implementation methods.

[0026] The raw materials or reagents involved in this invention are all commercially available products, and the operations involved are all routine operations in the field unless otherwise specified.

[0027] Beneficial effects

[0028] According to the present invention, a preferred cryopreservation diluent for mandarin fish sperm is obtained by compounding Hanks' solution, sodium chloride, soybean lecithin, vitamin E, curcumin, melatonin, and SOD. After cryopreservation at 4°C for 192 hours, the sperm still exhibits an activation rate of 21.67±1.53%, a motility time of 12.67±0.58s, and a lifespan of 24.33±1.15s, thus facilitating the improvement of artificial breeding techniques for mandarin fish and the preservation of superior germplasm resources. Adding a cryoprotectant to the cryopreservation diluent allows for cryopreservation of mandarin fish sperm at -196°C. After cryopreservation, the sperm still exhibits an activation rate of 55.67±4.04%, a motility time of 46.33±1.53s, and a lifespan of 74.67±1.53s. The present invention can ensure the long-term preservation of sperm from mandarin fish, and has a good protective effect on semen, resulting in high sperm activation rate and good motility after preservation.

[0029] The present invention adopts the above-mentioned technical solution to achieve the above objectives, which makes up for the shortcomings of the prior art, is reasonably designed, and is easy to operate. Attached Figure Description

[0030] To make the above and / or other objects, features, advantages and examples of the present invention more apparent and understandable, the accompanying drawings used in the specific embodiments 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 from these drawings without creative effort.

[0031] Figure 1 A schematic diagram illustrating the activation rate of sperm from mandarin fish after cryopreservation.

[0032] Figure 2 A schematic diagram illustrating the motility time of sperm from mandarin fish after cryopreservation;

[0033] Figure 3 A diagram illustrating the lifespan of sperm from mandarin fish after cryopreservation.

[0034] Figure 4 A schematic diagram illustrating the activation rate of sperm from mandarin fish after cryopreservation.

[0035] Figure 5A schematic diagram illustrating the motility time of sperm from mandarin fish after cryopreservation.

[0036] Figure 6 A schematic diagram illustrating the lifespan of mandarin fish sperm after cryopreservation. Detailed Implementation

[0037] Those skilled in the art can refer to the content of this document and appropriately replace and / or modify the process parameters to achieve the desired results. 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 this invention. The products and preparation methods described in this invention have been described through preferred examples, and those skilled in the art can obviously modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0038] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. This 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, patent cases, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the definitions included in this specification shall prevail.

[0039] Unless otherwise stated, all percentages, parts, proportions, etc. are by weight; other statements include, but are not limited to, “%”, “wt%”, “mass%” meaning weight percentage, “mol%” meaning mole percentage, and “vol%” meaning volume percentage.

[0040] When quantities, concentrations, or other numerical values ​​or parameters are given as ranges, preferred ranges, or a series of upper and lower preferred values, it should be understood that they specifically disclose all ranges formed by any pair of values ​​of any larger or preferred range limit and any smaller or preferred range limit, regardless of whether the ranges are disclosed separately. For example, when describing a range of “1 to 5 (1-5)”, the described range should be understood to include ranges such as “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, wherever numerical ranges are described herein, the ranges include the range endpoints as well as all integers and fractions within that range.

[0041] Unless otherwise specified, the materials, methods, and examples described herein are exemplary and not limiting. While similar or equivalent methods and materials can be used to implement or test the invention, suitable methods and materials are described herein.

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Furthermore, in the absence of conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0043] The present invention is described in detail below.

[0044] Example 1:

[0045] This embodiment provides a method for cryopreservation and ultra-low temperature preservation of sperm from mandarin fish that have been domesticated by feed, specifically including the following steps.

[0046] 1. Materials: The male mandarin fish broodstock, after being domesticated with feed, were taken from the Deqing base of Zhejiang Freshwater Fisheries Research Institute. Fish with well-developed gonads, good health, no injuries or diseases on their bodies, and sexual maturity were selected as experimental materials. Semen was collected and prepared for use.

[0047] 2. Prepare a low-temperature preservation solution for mandarin fish sperm, also known as the preservation solution: Hank's solution (D-Hank's solution, free of calcium and magnesium ions and phenol red, Tianjin Haoyang Biological Products Technology Co., Ltd.) + Hank's solution + sodium chloride 0.5g / L + soybean lecithin 0.3g / L + vitamin E 0.5g / L + curcumin 0.1g / L + melatonin 0.01g / L + SOD 0.01g / L. Store in a refrigerator until needed.

[0048] 3. Prepare the cryopreservation antifreeze solution for mandarin fish sperm, which is a mixture of a diluted cryopreservation solution for mandarin fish sperm and an antifreeze agent. The antifreeze agent is dimethyl sulfoxide, which is mixed with the preservation solution to form the antifreeze solution. The antifreeze agent content is 10%. The above antifreeze solution is stored in a 4°C freezer for later use.

[0049] 4. Method:

[0050] 4.1 Semen collection: Semen was placed in various preservation solutions. The ratio of sperm to mandarin fish sperm low-temperature preservation dilution solution was 1:5, and the mixture was preserved at 4℃.

[0051] 4.2 Sperm Quality Testing: After mixing semen with an activation solution (ultrapure water or a NaCl aqueous solution of appropriate concentration; ultrapure water was used in this example), sperm quality was observed under a microscope, including activation rate, motility time, and lifespan. Activation rate refers to the percentage of motile sperm in the same field of view observed immediately after mixing with the activation solution; motility time refers to the time from activation until 90% of the sperm begin to vibrate in place; lifespan refers to the time from activation until 90% of the sperm cease motility. The experiment was repeated three times.

[0052] 4.3. Sperm motility time and lifespan: A small amount of semen was pipetted into a centrifuge tube containing pre-added activation solution, quickly mixed, and then dropped onto a glass slide. The activation rate of sperm was observed under a light microscope. The centrifuge tube was stored at 4°C, and sperm motility time and lifespan were observed under a light microscope at regular intervals.

[0053] 4.4 Cryopreservation: Semen and mandarin fish sperm cryopreservation antifreeze were mixed at a ratio of 1:5 and equilibrated at 4°C for 10 minutes. The mixture was then dispensed into 0.25 mL straws (approximately 0.2 mL per straw). The straws were placed horizontally 4 cm above the liquid nitrogen surface in a homemade simple cooling device for 5 minutes, and then quickly transferred to liquid nitrogen for preservation. During thawing, the straws were quickly removed from the liquid nitrogen and immediately placed in a 40°C water bath with shaking to thaw. After activation with pure water, the sperm motility was examined under a microscope. The experiment was repeated three times.

[0054] 5. Data Processing: Experimental data were processed using Excel and SPSS 17.0 software. One-way ANOVA was used to test the significance of differences in sperm motility among the groups, with a significance level of P < 0.05. Statistical results are expressed as mean ± standard deviation (X ± SD).

[0055] During the research and development process, it was found that fresh semen was activated in diluents such as Hanks' solution and Ringer's solution, but was not activated after adding a certain amount of sodium chloride (0.5 g / L), thus sodium chloride was essential. Subsequently, a diluent formula suitable for low-temperature (4℃) preservation of mandarin fish semen (Hanks' solution + 0.5 g / L sodium chloride + 0.3 g / L soybean lecithin + 0.5 g / L vitamin E + 0.1 g / L curcumin + 0.01 g / L melatonin + 0.01 g / L SOD (superoxide dismutase) 0.01 g / L) and an antifreeze formula for ultra-low temperature (-196℃) preservation were selected. The validation results of the above-mentioned diluent formulas and fresh semen groups are shown in Table 1.

[0056] Table 1 - Validation results of the fresh propolis group and the fresh propolis + diluent group

[0057]

[0058] / :die.

[0059] The suitable cryoprotectant for mandarin fish semen is 10% dimethyl sulfoxide (the group using glycerol as the cryoprotectant all died, and the group using methanol as the cryoprotectant had a lower sperm activation rate). Under the suitable cryoprotectant formulation, after cryopreservation at -196℃, the sperm activation rate was still 55.67±4.04%, the motility time was 46.33±1.53s, and the lifespan was 74.67±1.53s. With only Hanks' solution + 0.5g / L sodium chloride + 10% dimethyl sulfoxide as the cryoprotectant, the sperm activation rate was 42.33±2.52%, the motility time was 39.33±1.15s, and the lifespan was 66.33±1.53s. This indicates that soybean lecithin, vitamin E, curcumin, melatonin, and SOD in the cryopreservation solution all have a positive impact on the cryopreservation effect on sperm.

[0060] As shown in Table 1, the diluent suitable for low-temperature (4℃) preservation of mandarin fish semen (Hanks solution + sodium chloride 0.5g / L + soybean lecithin 0.3g / L + vitamin E 0.5g / L + curcumin 0.1g / L + melatonin 0.01g / L + SOD 0.01g / L) can be used for 240 hours. After 192 hours of preservation, the semen still had an activation rate of 21.67±1.53%, a motility time of 12.67±0.58s, and a lifespan of 24.33±1.15s.

[0061] The suitable cryoprotectant is 10% dimethyl sulfoxide. Under a suitable cryoprotectant formulation (Hanks solution + sodium chloride 0.5g / L + soybean 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 preservation at ultra-low temperature (-196℃), the sperm activation rate is still 55.67±4.04%, the motility time is 46.33±1.53s, and the lifespan is 74.67±1.53s.

[0062] Example 2:

[0063] Based on the aforementioned embodiments, the formula for the low-temperature preservation diluent for mandarin fish sperm was changed to: Hank's solution + sodium chloride 0.01g / L, soybean lecithin 0.01g / L, vitamin E 0.8g / L, curcumin 0.5g / L, melatonin 0.1g / L, and SOD 0.1g / L, and stored in a refrigerator at 4°C for later use.

[0064] The cryopreservation antifreeze solution for mandarin fish sperm is prepared by adding a cryopreservation diluent to the sperm and an antifreeze agent. The antifreeze agent is dimethyl sulfoxide, which is mixed with the cryopreservation diluent to form the antifreeze solution. The antifreeze agent content is 10%. The above antifreeze solution is stored in a 4°C freezer for later use.

[0065] The method for cryopreserving sperm from mandarin fish after feed domestication includes: collecting semen and mixing it with a sperm cryopreservation diluent at a volume ratio of 1:5, and then storing it at 4°C.

[0066] The method for cryopreserving sperm from mandarin fish after feed domestication includes: collecting semen and mixing it with a cryopreservation antifreeze solution at a volume ratio of 1:5, equilibrating at 4°C for 10 minutes, and then dispensing it into 0.25 mL straws (approximately 0.2 mL per straw). The straws containing semen are placed horizontally 3 cm above the liquid nitrogen surface in a homemade simple cooling device for 5 minutes, and then quickly transferred to liquid nitrogen for preservation. During thawing, the straws are quickly removed from the liquid nitrogen and immediately placed in a 40°C water bath to thaw. After activation with pure water, the sperm motility is examined under a microscope.

[0067] Example 3:

[0068] Based on the aforementioned embodiments, the formula for the low-temperature preservation diluent for mandarin fish sperm was changed to: Hank's solution + 1g / L sodium chloride, 1g / L soybean lecithin, 0.01g / L vitamin E, 0.01g / L curcumin, 0.0001g / L melatonin, and 0.0001g / L SOD, and stored in a refrigerator at 4°C for later use.

[0069] The cryopreservation antifreeze solution for mandarin fish sperm is prepared by adding a cryopreservation diluent to the sperm and an antifreeze agent. The antifreeze agent is dimethyl sulfoxide, which is mixed with the cryopreservation diluent to form the antifreeze solution. The antifreeze agent content is 5%. The above antifreeze solution is stored in a 4°C freezer for later use.

[0070] The method for cryopreserving sperm from mandarin fish after feed domestication includes: collecting semen and mixing it with a sperm cryopreservation diluent at a volume ratio of 1:6, and then storing it at 4°C.

[0071] The method for cryopreserving sperm from mandarin fish after feed domestication includes: collecting semen and mixing it with a cryopreservation antifreeze solution at a volume ratio of 1:6, equilibrating at 4°C for 5 minutes, and then dispensing it into 0.25 mL straws (approximately 0.2 mL per straw). The straws containing semen are placed horizontally 4 cm above the liquid nitrogen surface in a homemade simple cooling device for 4 minutes, and then quickly transferred to liquid nitrogen for preservation. During thawing, the straws are quickly removed from the liquid nitrogen and immediately placed in a 40°C water bath and shaken to thaw. After activation with pure water, the sperm motility is examined under a microscope.

[0072] Example 4:

[0073] Based on the aforementioned embodiments, the formula for the low-temperature preservation diluent for mandarin fish sperm was changed to: Hank's solution + sodium chloride 0.5g / L, soybean lecithin 0.5g / L, vitamin E 1g / L, curcumin 1g / L, melatonin 0.05g / L, and SOD 0.05g / L, and stored in a refrigerator at 4°C for later use.

[0074] The cryopreservation antifreeze solution for mandarin fish sperm is prepared by adding a cryopreservation diluent to the sperm and an antifreeze agent. The antifreeze agent is dimethyl sulfoxide, which is mixed with the cryopreservation diluent to form the antifreeze solution. The antifreeze agent content is 3%. The above antifreeze solution is stored in a 4°C freezer for later use.

[0075] The method for cryopreserving sperm from mandarin fish after feed domestication includes: collecting semen and mixing it with a sperm cryopreservation diluent at a volume ratio of 1:10, and then storing it at 4°C.

[0076] The method for cryopreserving sperm from mandarin fish after feed domestication includes: collecting semen and mixing it with a cryopreservation antifreeze solution at a volume ratio of 1:10, equilibrating at 4°C for 8 minutes, and then dispensing it into 0.25 mL straws (approximately 0.2 mL per straw). The straws containing semen are placed horizontally 3 cm above the liquid nitrogen surface in a homemade simple cooling device for 3 minutes, and then quickly transferred to liquid nitrogen for preservation. During thawing, the straws are quickly removed from the liquid nitrogen and immediately placed in a 40°C water bath and shaken to thaw. After activation with pure water, the sperm motility is examined under a microscope.

[0077] Example 5:

[0078] Based on the aforementioned embodiments, the formula for the low-temperature preservation diluent for mandarin fish sperm was changed to: Hank's solution + sodium chloride 0.4 g / L, soybean lecithin 0.25 g / L, vitamin E 0.8 g / L, curcumin 0.3 g / L, melatonin 0.09 g / L, and SOD 0.06 g / L, and stored in a refrigerator at 4°C for later use.

[0079] The cryopreservation antifreeze solution for mandarin fish sperm is prepared by adding a cryopreservation diluent to the sperm and an antifreeze agent. The antifreeze agent is dimethyl sulfoxide, which is mixed with the cryopreservation diluent to form the antifreeze solution. The antifreeze agent content is 12%. The above antifreeze solution is stored in a 4°C freezer for later use.

[0080] The method for cryopreserving sperm from mandarin fish after feed domestication includes: collecting semen and mixing it with a sperm cryopreservation diluent at a volume ratio of 1:4, and then storing it at 4°C.

[0081] The method for cryopreserving sperm from mandarin fish after feed domestication includes: collecting semen and mixing it with a cryopreservation antifreeze solution at a volume ratio of 1:4, equilibrating at 4°C for 10 minutes, and then dispensing it into 0.25 mL straws (approximately 0.2 mL per straw). The straws containing semen are placed horizontally 3 cm above the liquid nitrogen surface in a homemade simple cooling device for 5 minutes, and then quickly transferred to liquid nitrogen for preservation. During thawing, the straws are quickly removed from the liquid nitrogen and immediately placed in a 40°C water bath to thaw. After activation with pure water, the sperm motility is examined under a microscope.

[0082] Example 6:

[0083] Based on Example 1 above, the formula for the cryopreservation diluent for mandarin fish sperm was modified to: Hanks' solution + 0.5 g / L sodium chloride + 0.5 g / L vitamin E + 0.1 g / L curcumin + 0.01 g / L melatonin + 0.01 g / L SOD (superoxide dismutase) and stored at 4°C for later use. The mandarin fish sperm was preserved using the same method as in Example 1.

[0084] Example 7:

[0085] Based on Example 1 above, the formula for the cryopreservation diluent for mandarin fish sperm was modified to: Hanks' solution + 0.5 g / L sodium chloride + 0.3 g / L soybean lecithin + 0.1 g / L curcumin + 0.01 g / L melatonin + 0.01 g / L SOD (superoxide dismutase) and stored at 4°C for later use. The mandarin fish sperm was preserved using the same method as in Example 1.

[0086] Example 8:

[0087] Based on Example 1 above, the formula for the cryopreservation diluent for mandarin fish sperm was modified to: Hanks' solution + 0.5 g / L sodium chloride + 0.3 g / L soybean lecithin + 0.5 g / L vitamin E + 0.01 g / L melatonin + 0.01 g / L SOD (superoxide dismutase) and stored at 4°C for later use. The mandarin fish sperm was preserved using the same method as in Example 1.

[0088] Example 9:

[0089] Based on Example 1 above, the formula for the cryopreservation diluent for mandarin fish sperm was modified to: Hanks' solution + 0.5 g / L sodium chloride + 0.3 g / L soybean lecithin + 0.5 g / L vitamin E + 0.1 g / L curcumin + 0.01 g / L SOD (superoxide dismutase) and stored at 4°C for later use. The same method as in Example 1 was used for cryopreservation of mandarin fish sperm at 4°C and ultra-low temperature preservation at -196°C.

[0090] Example 10:

[0091] Based on Example 1 above, the formula for the cryopreservation diluent for mandarin fish sperm was modified to: Hanks' solution + 0.5 g / L sodium chloride + 0.3 g / L soybean lecithin + 0.5 g / L vitamin E + 0.1 g / L curcumin + 0.01 g / L melatonin, and stored at 4°C for later use. The same method as in Example 1 was used for cryopreservation of mandarin fish sperm at 4°C and ultra-low temperature preservation at -196°C.

[0092] Experimental Example 1:

[0093] The activation rate, motility time, and lifespan of sperm after 120 hours of low-temperature (4°C) preservation in Examples 1 through 10 were statistically analyzed. The results are as follows: Figures 1-3 As shown.

[0094] Depend on Figures 1-3 It can be seen that the low-temperature preservation diluent for mandarin fish sperm in the preferred embodiments 1 to 5 of the present invention has a good effect on the low-temperature preservation of mandarin fish sperm. After preservation 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 lifespan is not less than 30 seconds. It can ensure the durability of the preservation time of mandarin fish sperm and has a good protective effect on semen. After preservation, the sperm activation rate is high and the motility is good.

[0095] from Figures 1-3 It was also found that the diluents in Examples 6 to 8 had poor cryopreservation effects on mandarin fish sperm. Specifically, when one of the following was missing from the diluent—soy lecithin, curcumin, or vitamin E—its cryopreservation effect on mandarin fish sperm was significantly reduced. It is speculated that this may be due to the combined additive effect of lecithin's cholesterol-regulating effect and the antioxidant effects of curcumin and vitamin E, which can repair damaged cell membranes, improve cell membrane function, and macroscopically enhance the cryopreservation effect of the diluent on mandarin fish sperm. Melatonin or SOD had relatively little effect on the cryopreservation effect on mandarin fish sperm.

[0096] Example 11:

[0097] Based on Example 1 above, the cryopreservation antifreeze solution for mandarin fish sperm was modified. Specifically, an antifreeze agent was added to the existing preservation solution. Methanol was selected as the antifreeze agent and mixed with the preservation solution to form the antifreeze solution, wherein the antifreeze agent content was 10%. The above antifreeze solution was stored in a freezer at 4°C for later use. The mandarin fish sperm was then cryopreserved at -196°C using the same method as in Example 1.

[0098] Example 12:

[0099] Based on Example 1 above, the cryopreservation antifreeze solution for mandarin fish sperm was modified. Specifically, an antifreeze agent was added to the existing preservation solution. Ethylene glycol was selected as the antifreeze agent and mixed with the preservation solution to form the antifreeze solution, wherein the antifreeze agent content was 10%. The above antifreeze solution was stored in a freezer at 4°C for later use. The mandarin fish sperm was then cryopreserved at -196°C using the same method as in Example 1.

[0100] Example 13:

[0101] Based on Example 1 above, the cryopreservation antifreeze solution for mandarin fish sperm was modified. Specifically, an antifreeze agent was added to the existing preservation solution. Glycerin was selected as the antifreeze agent and mixed with the preservation solution to form the antifreeze solution, wherein the antifreeze agent content was 10%. The above antifreeze solution was stored in a freezer at 4°C for later use. The mandarin fish sperm was then cryopreserved at -196°C using the same method as in Example 1.

[0102] Example 14:

[0103] Based on Example 1 above, the cryopreservation antifreeze solution for mandarin fish sperm was modified. Specifically, it was prepared by adding 10% cryoprotectant to the preservation solution. The cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid (GABA), with GABA accounting for 0.5% of the cryoprotectant. The cryoprotectant was mixed with the preservation solution to form the antifreeze solution, which was stored in a 4°C freezer for later use. The mandarin fish sperm was then cryopreserved at -196°C using the same method as in Example 1.

[0104] Example 15:

[0105] Based on Example 1 above, the cryopreservation antifreeze solution for mandarin fish sperm was modified. Specifically, it was prepared by adding 10% cryoprotectant to the preservation solution. The cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid (GABA), with GABA accounting for 0.2% of the cryoprotectant. The cryoprotectant was mixed with the preservation solution to form the antifreeze solution, which was stored in a 4°C freezer for later use. The mandarin fish sperm was then cryopreserved at -196°C using the same method as in Example 1.

[0106] Example 16:

[0107] Based on Example 1 above, the cryopreservation antifreeze solution for mandarin fish sperm was modified. Specifically, it was prepared by adding 10% cryoprotectant to the preservation solution. The cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid (GABA), with GABA accounting for 1.0% of the cryoprotectant. The cryoprotectant was mixed with the preservation solution to form the antifreeze solution, which was stored in a 4°C freezer for later use. The mandarin fish sperm was then cryopreserved at -196°C using the same method as in Example 1.

[0108] Example 17:

[0109] Based on Example 1 above, the cryopreservation antifreeze solution for mandarin fish sperm was modified. Specifically, it was prepared by adding 10% cryoprotectant to the preservation solution. The cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid (GABA), with GABA accounting for 0.1% of the cryoprotectant. The cryoprotectant was mixed with the preservation solution to form the antifreeze solution, which was stored in a 4°C freezer for later use. The mandarin fish sperm was then cryopreserved at -196°C using the same method as in Example 1.

[0110] Example 18:

[0111] Based on Example 1 above, the cryopreservation antifreeze solution for mandarin fish sperm was modified. Specifically, it was prepared by adding 10% cryoprotectant to the preservation solution. The cryoprotectant was a mixture of dimethyl sulfoxide and γ-aminobutyric acid (GABA), with GABA accounting for 1.5% of the cryoprotectant. The cryoprotectant was mixed with the preservation solution to form the antifreeze solution, which was stored in a 4°C freezer for later use. The mandarin fish sperm was then cryopreserved at -196°C using the same method as in Example 1.

[0112] Experimental Example 2:

[0113] The activation rate, motility time, and lifespan of sperm after being subjected to ultra-low temperature of -196℃ in Examples 1 and 9-18 were statistically analyzed respectively. The statistical results are as follows: Figures 4-6 As shown.

[0114] Combination Figures 4-6 Analysis of Examples 1 and 9-10 shows that, compared to Examples 9 and 10, adding trace amounts of melatonin and SOD to the cryopreservation diluent for mandarin fish sperm is beneficial for maintaining sperm motility under ultra-low temperature conditions; adding either one results in a significant decrease in effect. Analysis of Examples 1 and 11-13 reveals that, compared to other conventional cryoprotectants such as methanol, ethylene glycol, and glycerol, dimethyl sulfoxide (DMSO) significantly improves sperm activation rate and sperm motility. The methanol and ethylene glycol groups showed extremely low sperm activation rates, while the glycerol group resulted in complete sperm death.

[0115] Combination Figures 4-6Analysis of Examples 1 and 14-18 shows that, compared to Example 1 which only used dimethyl sulfoxide as an antifreeze agent, the antifreeze agent composed of a specific amount of γ-aminobutyric acid (GABA) combined with dimethyl sulfoxide can further enhance the antifreeze effect of dimethyl sulfoxide and improve the sperm activation rate after cryopreservation. After cryopreservation at -196°C, the sperm activation rate is not less than 65%, the motility time is not less than 60 seconds, and the lifespan is not less than 80 seconds. The above-mentioned compound antifreeze agent combined with the low-temperature preservation diluent for mandarin fish sperm provided by this invention is sufficient to ensure the longevity of mandarin fish sperm preservation time, and has a good protective effect on semen. After preservation, the sperm activation rate is high, the motility is good, and the survival time is long, which is conducive to the improvement of artificial breeding technology and the preservation of excellent germplasm resources.

[0116] The conventional techniques described in the above embodiments are existing technologies known to those skilled in the art, and therefore will not be described in detail here.

[0117] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

[0118] Although the present invention has been described in detail and specific embodiments have been cited, it will be apparent to those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the invention.

[0119] While the foregoing detailed descriptions have shown, described, and pointed out novel features applicable to various embodiments, it should be understood that various omissions, substitutions, and changes may be made to the form and details of the described apparatus or methods without departing from the spirit of this disclosure. Furthermore, the various features and methods described above may be used independently of each other or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of this disclosure. Many of the foregoing embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the invention has been disclosed in the context of certain embodiments and examples, those skilled in the art will understand that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or applications, as well as their obvious modifications and equivalents. Therefore, the invention is not intended to be limited to the specific disclosure of the preferred embodiments herein.

[0120] All matters not covered in this invention are common knowledge.

Claims

1. A diluent for low temperature preservation of mandarin fish (Mylopharyngodon piceus) sperm, characterized in that It comprises: Hanks solution, 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 0.05-0.5 g / L, melatonin 0.001-0.05 g / L, SOD 0.001-0.05 g / L.

2. The sperm low-temperature preservation diluent of the mandarin fish according to claim 1, characterized in that: The low temperature is 1-6℃.

3. The sperm low-temperature preservation diluent of the mandarin fish according to claim 1 or 2, characterized in that: The activation rate of the mandarin fish sperm preserved by the sperm low-temperature preservation diluent is not less than 20% after 192 hours.

4. The use of the sperm low-temperature preservation diluent of any one of claims 1-3 in the low-temperature preservation of mandarin fish sperm.

5. A method for cryopreservation of sperm of Mylopharyngodon piceus after feed domestication, characterized in that It comprises: After collecting the mandarin fish sperm, mix it with the sperm low-temperature preservation diluent of any one of claims 1-3 at a volume ratio of 1:2-10, and preserve it at a low temperature of 1-6℃.

6. A cryopreservation medium for the sperm of the mandarin fish (Siniperca chuatsi), characterized in that it comprises: On the basis of the sperm low-temperature preservation diluent of any one of claims 1-5, an antifreeze protective agent is further included, and the antifreeze protective agent comprises dimethyl sulfoxide.

7. The use of the sperm ultra-low-temperature preservation antifreeze solution of claim 6 in the ultra-low-temperature preservation of mandarin fish sperm.

8. A method for cryopreservation of sperm of feed-acclimated Mylopharyngodon piceus, characterized by It comprises: After collecting the mandarin fish sperm and mixing it with the sperm ultra-low-temperature preservation antifreeze solution of claim 6 at a volume ratio of 1:2-10, equilibrate it at 4℃ for 5-10 minutes, divide it into containers with good heat conductivity, and then place the containers at a position 3-4 cm above the surface of liquid nitrogen for 3-5 minutes, and then quickly transfer it into liquid nitrogen for preservation.

9. The sperm ultra-low-temperature preservation method of the mandarin fish after feed domestication according to claim 8, characterized in that: It further comprises a thawing step, in which the container is quickly taken out of the liquid nitrogen, immediately placed in a water bath at 35-45℃ for shaking and melting, and then activated with pure water after the melting is completed.

Images