Low-temperature preservation method for sperms and spermatophores of octopus variabilis

Abstract

The invention discloses a low-temperature preservation method of Octopus variabilis sperms and spermatophore, the Octopus variabilis sperms adopt an ultralow-temperature cryopreservation method, the Octopus variabilis sperms are protected by using a cryoprotectant composed of a 0.6 mol / L glucose solution diluent and 20% glycerol, the diluent contains nutrient substances, and the Octopus variabilis sperms have the characteristics of inactivation of sperms and small damage to sperms. The cryoprotectant has the functions of a permeable antifreeze agent and a non-permeable antifreeze agent, so that the sperms are subjected to dual protection in the freezing process; the octopus variabilis spermatophore is preserved at a low temperature, a 1.0 mol / L glucose solution is used as a spermatophore protection solution, the spermatophore structure can be maintained within one month, and high sperm activity of octopus variabilis sperms can be maintained within a short period (one month). Based on requirements in experiments, two octopus variabilis sperm preservation methods are provided and can be flexibly used, and technical support is provided for preservation of octopus variabilis germplasm resources and overcoming of the problem of asynchronous male and female development.

Description

TECHNICAL FIELD

[0001] The present application relates to a long octopus sperm and sperm acorn cryopreservation method, in particular to a long octopus sperm ultra-low temperature cryopreservation method and a long octopus sperm acorn cryopreservation method, and belongs to the field of marine biological technology. BACKGROUND

[0002] Long octopus (Octopus minor) is also called horse octopus and eight-legged octopus, belonging to cephalopods, octopus, and octopus. It is widely distributed in the coastal areas of north and south China, the Korean Peninsula and Japan. Long octopus has a large individual size, a short life cycle, rich nutrition and delicious meat, with a food rate of 95%. Its fresh and processed food has a broad market at home and abroad, and is an important export octopus in northern China. Due to the bottom dwelling habit of long octopus larvae, natural capture is currently the main method for breeding seedlings, which hinders the industrialization development of long octopus. Natural capture not only consumes time and effort, but also causes great harm to the seedlings and leads to the gradual depletion of natural resources, resulting in genetic problems. Therefore, it is urgent to carry out research on artificial seedling breeding technology of long octopus.

[0003] Sperm ultra-low temperature cryopreservation is a technology for indefinite preservation of sperm and effective protection of biological germplasm resources. This technology can be used for effective management of biological parents, ensuring the availability of active sperm throughout the year, and is the prerequisite for artificial insemination and artificial breeding. In recent years, significant progress has been made in the research of sperm cryopreservation of aquatic animals. Researchers have established a standardized fish sperm cryopreservation technology system and constructed a sperm bank for important economic fish. Although breakthroughs have been made in sperm cryopreservation technology for soft-bodied animals such as long oysters and short octopuses, there are few documents on cephalopod sperm preservation. Ultra-low temperature freezing often requires professional facilities, and the activity of sperm will decrease significantly after thawing. In the Decapoda order, which also has sperm acorns, direct use of sperm acorns for cryopreservation has been proven to be feasible, so we can learn from the experience of Decapoda sperm acorn preservation and obtain a long octopus sperm preservation method with higher sperm activity through sperm acorn preservation.

[0004] In view of the above problems, a complete set of simple, efficient, practical and long-term sperm ultra-low temperature cryopreservation method and a complete set of simple, efficient, practical and short-term sperm acorn cryopreservation method are invented according to the characteristics of long octopus sperm. SUMMARY

[0005] The technical problem to be solved by the present application is to solve the deficiencies in the prior art and fill the gap in long octopus sperm cryopreservation technology. A long octopus sperm and sperm acorn cryopreservation method is provided, in which the long octopus sperm preservation method is ultra-low temperature cryopreservation, and the preservation method is long-term stable. The long octopus sperm acorn preservation method is cryopreservation, and the preservation method is short-term but the sperm activity is high.

[0006] To achieve the above object, the present application provides the following technical solutions: The present application first provides a long squid sperm and sperm acorn cryopreservation method, comprising the following steps: The male long squid with selective gland development and maturation is dissected under the condition of low temperature anesthesia in the laboratory, the sperm acorn is taken out and placed in an ice culture dish, the sperm acorn of each long squid is a group; the sperm activity is detected, the sperm motility reaches more than 90%, and the long squid sperm ultra-low temperature cryopreservation or long squid sperm acorn cryopreservation is carried out; the long squid sperm ultra-low temperature cryopreservation is carried out, the sperm acorn is released first, the released semen is diluted, pre-frozen balanced, cooled and then ultra-low temperature cryopreserved, and thawing and recovery are carried out when the sperm is needed; the long squid sperm acorn cryopreservation needs to be structurally complete, and does not need to release sperm, and is directly cryopreserved, and the sperm is released after the sperm acorn is recovered when the sperm is needed.

[0007] In the above technical solution, the long squid sperm ultra-low temperature cryopreservation specifically comprises the following steps: (1) Collection of long squid sperm acorn: the male long squid with selective gland development and maturation is dissected under the condition of low temperature anesthesia in the laboratory, the sperm acorn is taken out and placed in an ice culture dish, and the sperm acorn of each long squid is a group; (2) Sperm activity detection: microscopic examination is carried out under a 10×20 microscope, 1 sperm acorn in each group is randomly taken into sterile seawater, the sperm acorn is cut with sterile surgical scissors, the sperm in the sperm acorn is activated by seawater, 100 μL of the activated liquid is dropped on a glass slide, and the sperm motility MSR is observed and recorded within 10 min, and the group in which the sperm acorn is located can be used for ultra-low temperature cryopreservation when the detected MSR reaches more than 90%; (3) Sperm release: the sperm acorn is placed in a cryopreservation tube, 1.8 mL of cryoprotective solution and one sperm acorn are contained in each cryopreservation tube, the sperm acorn is cut with sterile surgical scissors, the sperm in the sperm acorn is released, and the sperm is mixed with the cryoprotective solution, the operation process must be completed on the ice surface, and the obtained semen should be completely buried in the ice; (4) Sperm dilution: 500 μL of the semen obtained in step (3) is accurately taken into a new cryopreservation tube, 3 times the volume of the cryoprotective solution is added, so that the dilution ratio of the semen and the cryoprotective solution reaches 1:3; the semen and the cryoprotective solution are mixed to obtain a sperm mixture, which is ready for use; (5) Pre-freezing balance of the semen: the sperm mixture obtained in step (4) is placed in a 4℃ refrigerator for pre-freezing balance; (6) Programmed cooling: the sperm mixture subjected to pre-freezing balance in step (5) is quickly placed in a freezing chamber of a programmed cooling instrument; the programmed cooling starts from 4℃, and is first cooled to -60℃, and then to -140℃; (7) Liquid nitrogen storage: after the procedure cooling in step (6) is completed, the sperm-containing cryopreserved tube is directly taken out and immediately placed in a liquid nitrogen tank for storage at -196℃; if the sperm is needed, the sperm in the cryopreserved tube is thawed and recovered.

[0008] Preferably, in step (1), the used male Octopus variabilis with mature gonadal development weighs more than 100 grams; the low-temperature anesthesia is at 2-3℃.

[0009] Preferably, in steps (3) and (4), the cryoprotective solution is composed of a sperm diluent and an antifreeze agent, the sperm diluent is prepared first, then the antifreeze agent is added to form the cryoprotective solution, and then the cryoprotective solution is pre-cooled in a 4℃ refrigerator for standby; the sperm diluent is a 0.6 mol / L D-glucose solution, and the antifreeze agent is glycerol (Gly), the volume percentage of glycerol in the cryoprotective solution is 20%.

[0010] Preferably, in step (5), the pre-freezing equilibration is for 25-35 minutes.

[0011] Preferably, in step (6), the cooling procedure starts from 4℃, decreases to -60℃ at a speed of 15℃ / min, stays at -60℃ for 2 minutes, then decreases from -60℃ to -140℃ at a speed of 20℃ / min, and finally equilibrates at -140℃ for 5 minutes.

[0012] Preferably, in step (7), the thawing and recovery are specifically performed as follows: the thawing refers to quickly transferring the sperm-containing cryopreserved tube to a 37℃ constant-temperature water bath, gently shaking the cryopreserved tube until the sample is completely melted; the recovery refers to activating the melted sperm using natural filtered seawater (FSW).

[0013] The above technical solution, the low-temperature preservation of Octopus variabilis sperm, specifically includes the following steps: I. Collection of Octopus variabilis sperm: selecting male Octopus variabilis with mature gonadal development, dissecting under the condition of low-temperature anesthesia in the laboratory, taking out the sperm and placing it in an ice-cultured dish, and each Octopus variabilis sperm is a group; II. Sperm motility detection: under a 10x20 microscope, 1 sperm of each group is randomly taken and placed in sterile seawater, the sperm is activated by using sterile surgical scissors to chop the sperm, 100 μL of the activated liquid is dropped on a glass slide, and within 10 minutes, the motility of the sperm is observed and recorded, and the group with a motility of more than 90% is used for low-temperature cryopreservation. III. The sperm pod is placed in a cryogenic tube, 1.8 mL of the preservation solution and one sperm pod are placed in each cryogenic tube, the process must be completed on the ice surface, the cryogenic tube is placed in a 4℃ refrigerator, and the sperm pod is preserved; if the sperm is needed, the sperm pod is thawed and recovered.

[0014] Preferably, in step I, the gonad development mature male Octopus, the weight is more than 100 grams; the low temperature anesthesia, the low temperature condition is 2 ~3℃.

[0015] Preferably, in step III, the preservation solution is a 1.0 mol / L D-glucose solution, and the prepared preservation solution is pre-cooled in a 4℃ refrigerator.

[0016] Preferably, in step III, the thawing and recovery, the specific operation process is: the thawing refers to quickly transferring the cryogenic tube to a 37℃ constant temperature water bath, and gently shaking the cryogenic tube for 30s; the recovery refers to pouring off the preservation solution, adding natural filtered seawater (FSW), cutting the sperm pod with sterile surgical scissors, releasing the sperm therein, and mixing the sperm with the FSW to activate the sperm. The low-temperature preserved sperm pod can only be stored for a short period of time, and the storage effect is best within 1 month.

[0017] Compared with the prior art, the following characteristics are possessed: 1. The freezing protective solution of the present application can make the sperm in a non-activated state when contacting with the sperm, and the sperm can be effectively activated after contacting with natural seawater again; the freezing protective solution contains D-glucose, which can provide a suitable in vitro environment for the sperm, prolong the survival time of the sperm in vitro, and help the sperm to resist freezing damage in a good physiological state.

[0018] 2. The freezing protective solution of the present application is a mixed freezing protective solution, which contains non-permeable D-glucose and permeable glycerol. The combined use of complementary antifreeze not only reduces the toxic effect of glycerol on sperm, but also helps to reduce the damage of ice crystals formed inside and outside the cell membrane to sperm, and also prevents damage to sperm caused by the difference in osmotic pressure between inside and outside.

[0019] 3. In the ultra-low temperature freezing preservation scheme of the present application, the equilibrium temperature is 4℃, and the equilibrium time is 25-35 min. The equilibrium of the Octopus sperm under this condition can make the glycerol fully permeate into the sperm, which is beneficial to enhance the freezing resistance of the sperm, make good physiological preparation for the next step of ultra-low temperature freezing, and reduce the damage of harmful temperature zone ice crystals to the sperm in the freezing process.

[0020] 4. In the ultra-low temperature cryopreservation scheme of the present application, the cooling method is to use a programmed cooling instrument for cooling, and the cooling program uses a two-step program, first to cool the sperm at a stable rate of-15℃ / min to make the sperm safely pass through the harmful temperature zone (-15℃~-60℃), and then to make the sperm enter a stable state at-20℃ / min (-140℃), and finally to be put into liquid nitrogen for cryopreservation; this method can ensure that the cooling rate of the sperm is stable and accurate, and can reduce the freezing damage of the sperm.

[0021] 5. The ultra-low temperature cryopreservation scheme of the present application can maintain a relatively high sperm activity of Octopus variabilis for a long period of time (6 months); the MSR of the sperm preserved for 6 months can reach 26.67±7.5%, and the lifespan is 90.01±10.04 min. The sperm cryopreservation scheme of the present application can maintain a relatively high sperm activity of Octopus variabilis for a short period of time (1 month); the MSR of the sperm preserved for 21 days can reach 89.66%±0.51%. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 To screen the effects of different kinds of sperm diluents in Example 1 on the sperm of Octopus variabilis, wherein: A is the MSR under different glucose concentrations, B is the MSR under different NaCl concentrations, and C is the MSR under different KCl concentrations (● represents the MSR after adding the diluent, and ■ represents the MSR of the sperm activated by filtered seawater after 1 h); Figure 2 To screen the effects of different kinds of sperm diluents in Example 1 at the optimal concentration on the initial MSR and the MSR after 1 h of Octopus variabilis, wherein: G5 is a 0.5 mol / L glucose solution, G6 is a 0.6 mol / L glucose solution, N5 is a 0.5 mol / L NaCl solution, K4 is a 0.4 mol / L KCl solution, K5 is a 0.5 mol / L KCl solution, HA is a Hank's solution, CL is a Cortland solution, and SG is a sodium citrate glycine solution (■ represents the MSR after adding the diluent, and represents the MSR of the sperm activated by filtered seawater after 1 h); Figure 3 To screen the effects of the five diluents screened in Example 1 and the activator (seawater) on the activatable time and lifespan of the sperm of Octopus variabilis, wherein: A is the effect on the activatable time of the sperm of Octopus variabilis, and B is the effect on the lifespan of the sperm of Octopus variabilis (N5 is a 0.5 mol / L NaCl solution, K4 is a 0.4 mol / L KCl solution, K5 is a 0.5 mol / L KCl solution, G5 is a 0.5 mol / L glucose solution, G6 is a 0.6 mol / L glucose solution, and activator is seawater); Figure 4To screen the effects of different dilution concentrations on the MSR and lifespan of *Octopus longiflorus* sperm in Example 3, where A represents the effect on the MSR and B represents the effect on the lifespan of *Octopus longiflorus* sperm; Figure 5 To screen the effects of different freezing procedures in Example 4 on the MSR and lifespan of Octopus japonicus sperm, where A represents the effect on the MSR and B represents the effect on the lifespan of Octopus japonicus sperm.

[0023] The above data are expressed as mean ± standard deviation. Detailed Implementation

[0024] The following describes in detail the specific embodiments of the technical solution of the present invention, but the present invention is not limited to the following description: Screening Example 1: This embodiment is for determining the optimal type of sperm diluent.

[0025] 1. Solution preparation (prepare immediately before use): (1) Artificial seawater with a salinity of 10 PSU: prepared using filtered seawater and ultrapure water.

[0026] (2) Preparation of glucose, sodium chloride and potassium chloride solutions: Glucose, sodium chloride and potassium chloride were weighed using an electronic balance. A linear concentration gradient solution system of glucose, sodium chloride and potassium chloride was constructed using a serial dilution method, with a gradient range of 0.1-1.0 mol∙L⁻¹ and a concentration interval Δc=0.1 mol∙L⁻¹.

[0027] (3) Hank's solution (HA): CaCl2 0.14 g, KCl 0.40 g, KH2PO4 0.06 g, MgCl2⋅6H2O 0.10 g, MgSO4⋅7H2O 0.10 g, NaHCO3 0.35 g, NaCl 8.01 g, Na2HPO4⋅12H2O 0.06 g, dissolved in 1000 ml of solution.

[0028] (4) Cortland solution (Cortland, CL): CaCl2⋅H2O 0.18 g, KCl 0.38 g, MgSO4⋅7H2O 0.23 g, NaCl 7.25 g, NaHCO3 1.00 g, NaH2PO4 0.41 g, glucose 1.00 g, dissolved in 1000 ml of solution.

[0029] (5) Sodium citrate and glycine solution (SG): 29.0 g of sodium citrate and 20.0 g of glycine are dissolved in 1000 mL of solution.

[0030] 2. Sperm collection: The octopus was dissected under low-temperature anesthesia (2-3℃) in the laboratory, the spermatophores were removed and placed in an ice-cold petri dish.

[0031] 3. Sperm motility test: Each octopus is grouped together. When testing motility, one spermatophore is randomly selected from each group, and the sperm in it are tested for motility. If the MSR reaches 90% or more, the group containing that spermatophore can be used for cryopreservation.

[0032] 4. Mixing: Each cryovial (2 mL) contains 1.8 mL of sperm diluent and one sperm pod. Use sterile surgical scissors to cut the sperm pod and release the sperm. Mix thoroughly and then place the cryovial on ice for later use. The sperm diluent is a solution of glucose, sodium chloride, and potassium chloride with a gradient range of 0.1–1.0 mol∙L⁻¹.

[0033] 5. MSR detection: 100 μL of diluted semen was dropped onto a glass slide. Within 10 minutes, a randomly selected area was observed and the MSR was recorded. The results are as follows: Figure 1 As shown in A, B, and C. One hour later, another 100 μL of the diluted solution was dropped onto a glass slide, and filtered seawater was used to activate the sperm. The MSR was observed and recorded, and the results are shown in Figure 1. Figure 1 As shown in Figures A, B, and C. Five diluents with the lowest initial MSR and the highest MSR after 1 hour were selected. These included 0.5 mol∙L⁻¹ glucose solution (G5), 0.6 mol∙L⁻¹ glucose solution (G6), 0.5 mol∙L⁻¹ NaCl solution (N5), 0.4 mol∙L⁻¹ KCl solution (K4), and 0.5 mol∙L⁻¹ KCl solution (K5), as shown in Figures A, B, and C. Figure 2 As shown.

[0034] 6. Activation Time Detection: For these five diluents, sperm were activated using seawater at intervals of 4, 8, 12, 24, 48, 72, 96, and 120 hours, and the MSR (Mean Sequencing Rate) was observed and recorded. The diluent with the longest usable activation time was used for subsequent experiments. Sperm with an MSR > 10% were considered to be activated. Each treatment had three parallel treatments, using filtered seawater with a salinity of 32 PSU as a control. 0.6 mol∙L⁻¹ glucose solution showed the best activation efficacy, reaching 96.00 h ~ 120.00 h (104.00 ± 13.86 h). Therefore, 0.6 mol∙L⁻¹ glucose solution was selected as the sperm diluent, and the results are as follows. Figure 3 As shown.

[0035] Screening Example 2: This embodiment aims to determine the optimal type and concentration of cryoprotectant.

[0036] 1. Solution preparation (prepare immediately before use): (1) Glycerol (Gly), methanol (MeOH), dimethyl sulfoxide (DMSO) and ethylene glycol (EG) were selected as cryoprotectants.

[0037] (2) The cryoprotectant was mixed with the optimal diluent (0.6 mol∙L⁻¹ glucose solution) obtained in Example 1 to obtain a cryoprotectant solution. The ratio was adjusted so that the cryoprotectant accounted for 5%, 10%, 15% and 20% (v / v) of the volume of the diluent solution. At the same time, a diluent without cryoprotectant was used as a control.

[0038] 2. Sperm collection: The octopus was dissected under low-temperature anesthesia (2-3℃) in the laboratory, the spermatophores were removed and placed in an ice-cold petri dish.

[0039] 3. Sperm motility test: Each octopus is grouped together. When testing motility, one spermatophore is randomly selected from each group, and the sperm in it are tested for motility. If the MSR reaches 90% or more, the group containing that spermatophore can be used for cryopreservation.

[0040] 4. Mixing and equilibration: Each cryovial (2 mL) contains 1.8 mL of cryoprotectant and one sperm pod. The sperm pod is cut into pieces with sterile surgical scissors to release the sperm. The cryovial is then inverted to mix the sperm with the cryoprotectant. Finally, it is placed in a freezer at 4°C for 30 min.

[0041] 5. Programmed Cooling and Freezing: The treated sperm was then placed in a computer-controlled programmed cryostat (Kryo-560-16, UK). The cooling program started at 4°C, decreasing to -60°C at a rate of 15°C / minute, holding at -60°C for 2 minutes, then decreasing to -140°C at a rate of 20°C / minute, and finally equilibrating at -140°C for 5 minutes. The treated sperm was then placed in liquid nitrogen for storage.

[0042] 6. Thawing and recovery: After 24 hours, the processed sperm were removed and thawed in a 37°C water bath.

[0043] 7. Sperm motility observation: 50 μL of diluted sperm pellets were absorbed onto a glass slide, and more than four times the volume of filtered seawater was added for activation. The MSR was observed and recorded, as shown in Table 1. Table 1 shows that glycerol (20%) had the highest MSR among all cryoprotectants, reaching 21.00 ± 2.16%. Therefore, glycerol (20%) was selected as the cryoprotectant.

[0044] Table 1. Effects of cryoprotectants on sperm motility and sperm ratio in *Octopus longissima*

[0045] MSR: Motile sperm ratio; GL: Glycerol; MeOH: Methanol; EG: Ethylene glycol; DMSO: Dimethyl sulfoxide. Data are expressed as mean ± standard deviation.

[0046] Screening Example 3: This example is for determining the optimal dilution concentration.

[0047] 1. Solution preparation (prepare immediately before use): (1) Glycerin was selected as the cryoprotectant.

[0048] (2) Glycerol was mixed with the optimal diluent (0.6 mol∙L⁻¹ glucose solution) obtained in Example 1 to obtain a cryoprotectant solution. The ratio was adjusted so that the cryoprotectant accounted for 20% (v / v) of the volume of the diluent solution.

[0049] 2. Sperm collection: The octopus was dissected under low-temperature anesthesia (2-3℃) in the laboratory, the spermatophores were removed and placed in an ice-cold petri dish.

[0050] 3. Sperm motility test: Each octopus is grouped together. When testing motility, one spermatophore is randomly selected from each group, and the sperm in it are tested for motility. If the MSR reaches 90% or more, the group containing that spermatophore can be used for cryopreservation.

[0051] 4. Mixing, Dilution, and Equilibration: Each cryovial (2 mL) contains 1.8 mL of cryoprotectant and one sperm pod. Using sterile surgical scissors, break up the sperm pod to release the sperm. Invert the cryovial to thoroughly mix the sperm with the cryoprotectant. Add 1 mL of this solution to another cryovial, then add another 1 mL of cryoprotectant to dilute, obtaining a 2-fold dilution. Repeat this process with the 2-fold dilution to obtain 2, 4, 8, and 16-fold dilutions. Place the diluted solutions in a freezer at 4°C for 30 minutes.

[0052] 5. Programmed cooling and freezing: The processed sperm is then placed in a computer-controlled programmed freezing machine (Kryo-560-16, UK).

[0053] The cooling process begins at 4°C, decreasing to -60°C at a rate of 15°C / minute, holding at -60°C for 2 minutes, then decreasing to -140°C at a rate of 20°C / minute, and finally equilibrating at -140°C for 5 minutes. The treated sperm is then placed in liquid nitrogen and stored.

[0054] 6. Thawing and recovery: After 24 hours, the processed sperm were removed and thawed in a 37°C water bath.

[0055] 7. Sperm motility observation: 50 μL of diluted sperm pellets were absorbed onto a glass slide, and more than four times the volume of filtered seawater was added for activation. MSR was observed and recorded. The group with the highest MSR was selected. The 4-fold dilution group showed an MSR of 31.00%–40.00% (35.0±4.51%) after thawing, and a lifespan of 79.27 min–91.17 min (85.37±5.96 min), significantly higher than other groups. Therefore, this was determined as the optimal dilution protocol. The results are as follows: Figure 4 As shown.

[0056] Screening Example 4: This embodiment is for determining the optimal freezing procedure.

[0057] 1. Solution preparation (prepare immediately before use): (1) Glycerin was selected as the cryoprotectant.

[0058] (2) Glycerol was mixed with the optimal diluent (0.6 mol∙L⁻¹ glucose solution) obtained in Example 1 to obtain a cryoprotectant solution. The ratio was adjusted so that the cryoprotectant accounted for 20% (v / v) of the volume of the diluent solution.

[0059] 2. Sperm collection: The octopus was dissected under low-temperature anesthesia (2-3℃) in the laboratory, the spermatophores were removed and placed in an ice-cold petri dish.

[0060] 3. Sperm motility test: Each octopus is grouped together. When testing motility, one spermatophore is randomly selected from each group, and the sperm in it are tested for motility. If the MSR reaches 90% or more, the group containing that spermatophore can be used for cryopreservation.

[0061] 4. Mixing, Dilution, and Equilibration: Each cryovial (2 mL) contains 1.8 mL of cryoprotectant and one sperm pod. Using sterile surgical scissors, break up the sperm pod to release the sperm. Invert the cryovial to thoroughly mix the sperm with the cryoprotectant. Accurately measure 500 μL of semen into a new cryovial and add three times its volume of cryoprotectant to achieve a semen-to-preservative dilution ratio of 1:3. Place the diluted solution in a freezer at 4°C for 30 minutes.

[0062] 5. Programmed Cooling and Freezing: The treated sperm was then placed in a computer-controlled programmed cryostat (Kryo-560-16, UK). The cooling program followed four different procedures as shown in Table 1. The treated sperm was then stored in liquid nitrogen.

[0063] Table 2. Details of the freezing process

[0064] 6. Thawing and recovery: After 24 hours, the processed sperm were removed and thawed in a 37°C water bath.

[0065] 7. Sperm motility observation: 50 μL of diluted sperm pellets were absorbed onto a glass slide, and activated with at least four times the volume of filtered seawater. MSR was observed and recorded. The group frozen using procedure C had significantly higher MSR than other groups; therefore, it was determined to be the optimal dilution protocol. The results are as follows: Figure 5 As shown.

[0066] Screening Example 5: This embodiment is for determining the optimal spermatogonial preservation solution.

[0067] 1. Preparation of preservation solution (prepare immediately before use): (1) Artificial seawater with a salinity of 30 PSU: prepared using filtered seawater and ultrapure water.

[0068] (2) Preparation of glucose solution: Glucose was weighed using an electronic balance. A linear concentration gradient solution system of glucose, sodium chloride and potassium chloride was constructed using the serial dilution method, with a gradient range of 0.2-1.4 mol∙L⁻¹ and a concentration interval Δc=0.2mol∙L⁻¹.

[0069] 2. Collection of spermatophores from Octopus: Selectively mature male Octopus species were dissected under low-temperature anesthesia (2-3℃) in the laboratory, and the spermatophores were removed and placed in ice-cold culture dishes. The spermatophores of each Octopus species were considered as a group. 3. Sperm motility test: Under a 10×20x microscope, examine the spermatophore. Randomly select one spermatophore from each group and place it in sterile seawater. Use sterile surgical scissors to cut the spermatophore, activating the sperm with the seawater. Place 100 μL of sperm dilution solution onto a glass slide. Within 10 minutes, randomly select an area and observe and record the sperm motility rate (MSR). If the MSR reaches 90% or higher, the group containing that spermatophore is suitable for cryopreservation. 4. Preservation of sperm pods: Place the sperm pods in cryovials (2.0 ml), each cryovial containing 1.8 mL of preservation solution. The process must be completed on ice, with the cryovials completely submerged in ice. Place the cryovials in a 4°C freezer for preservation for 21 days. 5. Sperm thawing: Quickly transfer the cryovial to a 37°C constant temperature water bath, gently shake the cryovial for 30 seconds, discard the preservation solution, add naturally filtered seawater (FSW), use sterile surgical scissors to cut the sperm pods, release the sperm, mix them with the FSW, and activate the sperm.

[0070] 6. MSR Detection: 100 μL of diluted semen was dropped onto a glass slide. Within 10 minutes, a randomly selected area was observed and the MSR was recorded. One hour later, another 100 μL of diluted solution was dropped onto the slide, and the sperm were activated using filtered seawater. The MSR was observed and recorded again. The preservation solution with the highest MSR was selected. Using a 1.0 mol∙L⁻¹ glucose solution as the preservation medium, the MSR was 89.66% ± 0.51%, significantly higher than other groups. Based on these results, the 1.0 mol∙L⁻¹ glucose solution was determined to be the optimal preservation solution for *Octopus longissima* spermatophores.

[0071] Based on the optimal parameters obtained from the above screening embodiments, the present invention will be further described in detail below with reference to specific embodiments.

[0072] Example 1: A method for cryopreservation of Octopus japonicus sperm includes the following steps: (1) Collection of spermatophores from octopus: Select male octopus with mature glands (weighing more than 100 grams) were dissected in the laboratory under low temperature anesthesia (2~3℃), the spermatophores were removed and placed in an ice-cold culture dish, and the spermatophores of each octopus were considered as a group. (2) Sperm motility test: Under a 10×20x microscope, one sperm pod was randomly selected from each group and placed in sterile seawater. The sperm pod was cut into pieces with sterile surgical scissors to activate the sperm in the seawater. 100μL of activation solution was dropped onto a glass slide. Within 10 minutes, a random area was selected and the sperm motility rate (MSR) was observed and recorded. If the MSR reached 90% or more, the group containing the sperm pod could be used for cryopreservation. (3) Release of sperm: Place the sperm pod in a cryopreservation tube, each cryopreservation tube containing 1.8 mL of cryoprotectant and one sperm pod. Use sterile surgical scissors to cut the sperm pod and release the sperm inside, so that it can be mixed with the cryoprotectant. This operation must be completed on ice, and the obtained semen should be completely buried in ice. The cryoprotectant consists of sperm diluent and cryoprotectant. The sperm diluent is prepared first, then the cryoprotectant is added to form the cryoprotectant, which is then pre-cooled in a 4°C freezer for later use. The sperm diluent is a 0.6 mol / L D-glucose solution, and the cryoprotectant is glycerol (Gly), with a volume percentage of 20% in the cryoprotectant. (4) Sperm dilution: Accurately measure 500 μL of the semen obtained in step (3) into a new cryopreservation tube, add 3 times its volume of cryoprotectant, so that the dilution ratio of semen to cryoprotectant is 1:3; after mixing the semen and cryoprotectant, a sperm mixture is obtained and set aside; the cryoprotectant is the same as in step (3). (5) Pre-freezing equilibration of semen: The sperm obtained after dilution in step (4) was placed in a 4°C freezer for pre-freezing equilibration for 30 minutes. (6) Programmed cooling: The semen that has been balanced before freezing in step (5) is quickly placed into the freezing chamber of the programmed cooling device; the cooling program starts at 4°C and drops to -60°C at a rate of 15°C / minute, stays at -60°C for 2 minutes, then drops from -60°C to -140°C at a rate of 20°C / minute, and finally balances at -140°C for 5 minutes. (7) Liquid nitrogen preservation: After the temperature drop procedure in step (6) is completed, take out the cryopreservation tube containing semen directly and put it into the liquid nitrogen tank immediately for preservation at -196℃; if the semen is to be used, the semen in the cryopreservation tube can be thawed and revived. The thawing and thawing process is as follows: Thawing refers to quickly transferring the cryovial containing semen to a constant temperature water bath at 37°C and gently shaking the cryovial until the sample is completely thawed; thawing refers to activating the thawed sperm using naturally filtered seawater (FSW).

[0073] In this embodiment, the sample cryopreservation volume was 2 mL. After 3 days of cryopreservation, the motile sperm rate (MSR) was 34.67±7.51%, and the sperm lifespan was 95.30±10.21 min. After 6 months of cryopreservation, the motile sperm rate (MSR) was 26.67±7.5%, and the sperm lifespan was 90.01±10.04 min. By cryopreserving Octopus sperm at ultra-low temperatures, the sperm activity of Octopus sperm can be maintained for a long period of time (6 months).

[0074] Example 2: A method for cryopreservation of long octopus spermatophores specifically includes the following steps: I. Collection of spermatophores from Octopus: Select male Octopus species with mature glands (weighing over 100 grams) are dissected under low-temperature anesthesia (2-3℃) in the laboratory, the spermatophores are removed and placed in ice-cold culture dishes, and the spermatophores of each Octopus species are considered as a group. II. Sperm motility test: Under a 10×20x microscope, one spermatophore from each group was randomly selected and placed in sterile seawater. The spermatophore was cut into pieces with sterile surgical scissors to activate the sperm in the seawater. 100μL of the activation solution was dropped onto a glass slide. Within 10 minutes, a random area was selected and the sperm motility rate (MSR) was observed and recorded. If the MSR reached 90% or higher, the group containing the spermatophore could be used for cryopreservation. III. Preservation of sperm pods: Place the sperm pods in cryovials, each containing 1.8 mL of preservation solution (1.0 mol / L D-glucose solution) and one sperm pod. This process must be completed on ice. Place the cryovials in a 4°C freezer for sperm pod preservation. If the semen is to be used, simply thaw and rehydrate the sperm pods. The thawing and thawing process is as follows: Thawing refers to quickly transferring the cryovial to a constant temperature water bath at 37°C and gently shaking the cryovial for 30 seconds; Thawing refers to discarding the preservation solution, adding naturally filtered seawater FSW, using sterile surgical scissors to cut the sperm pods, releasing the sperm, mixing it with the FSW, and activating the sperm.

[0075] In this embodiment, the spermatopod structure can be maintained for one month, and the sperm motility rate is 89.66%±0.51% after being stored at 4°C for 21 days. The low-temperature preservation of the spermatopods allows the octopus sperm to maintain a high sperm activity in a short period (one month).

[0076] The above examples are merely illustrative of the technical concept and features of the present invention and should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the essence of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for cryopreservation of sperm and spermatophores from the long-necked octopus, characterized in that, Includes the following steps: Selected male octopuses with mature glands were dissected under hypothermic anesthesia in the laboratory. The spermatophores were removed and placed in ice-cold culture dishes, with each octopus's spermatophores forming a group. Sperm motility was tested; if the sperm motility rate reached 90% or higher, the octopus sperm was cryopreserved or the spermatophores were cryopreserved. For cryopreservation of octopus sperm, the spermatophores were first released, and the released semen was diluted, pre-freezed, cooled, and then cryopreserved. Thawing and rewarming were performed when the sperm was needed. For cryopreservation of octopus spermatophores, the spermatophores were directly cryopreserved without releasing sperm; the spermatophores were rewarmed before releasing the sperm when needed.

2. The low-temperature preservation method according to claim 1, characterized in that, The cryopreservation of Octopus sperm specifically includes the following steps: (1) Collection of spermatophores from Octopus: Selectively mature male Octopus were dissected under low-temperature anesthesia in the laboratory, and the spermatophores were removed and placed in ice-cold culture dishes. Each Octopus's spermatophores constituted a group. (2) Sperm motility test: Under a 10×20x microscope, one sperm pod was randomly selected from each group and placed in sterile seawater. The sperm pod was cut into pieces with sterile surgical scissors to activate the sperm in the seawater. 100μL of activation solution was dropped onto a glass slide. Within 10 minutes, a random area was selected and the sperm motility rate (MSR) was observed and recorded. If the MSR reached 90% or more, the group containing the sperm pod could be used for cryopreservation. (3) Release of sperm: Place the sperm pod in a cryopreservation tube, each cryopreservation tube containing 1.8 mL of cryoprotectant and one sperm pod. Use sterile surgical scissors to cut the sperm pod and release the sperm inside, so that it can be mixed with the cryoprotectant. This operation must be completed on ice, and the obtained semen should be completely buried in ice. (4) Sperm dilution: Accurately measure 500 μL of the semen obtained in step (3) into a new cryopreservation tube, add 3 times its volume of cryoprotectant, so that the dilution ratio of semen to cryoprotectant is 1:3; after mixing the semen and cryoprotectant, a sperm mixture is obtained and ready for use. (5) Pre-freezing equilibration of semen: The sperm obtained after dilution in step (4) was placed in a 4°C freezer for pre-freezing equilibration; (6) Programmed cooling: The semen that has been balanced before freezing in step (5) is quickly placed into the freezing chamber of the programmed cooling device; the cooling program starts at 4°C, first cooling to -60°C, and then cooling to -140°C. (7) Liquid nitrogen preservation: After the temperature is cooled in step (6), take out the cryopreservation tube containing semen and put it into the liquid nitrogen tank immediately for preservation at -196℃; if semen is needed, the semen in the cryopreservation tube can be thawed and revived.

3. The low-temperature preservation method according to claim 2, characterized in that, In step (1), the gonads of a male long octopus that are fully developed and weigh more than 100 grams are used; the low-temperature anesthesia is performed at a temperature of 2 to 3 degrees Celsius.

4. The low-temperature preservation method according to claim 2, characterized in that, In steps (3) and (4), the cryoprotectant consists of sperm diluent and cryoprotectant. The sperm diluent is prepared first, and then the cryoprotectant is added to it to form the cryoprotectant. After that, it is placed in a 4°C refrigerator for pre-cooling and ready for use. The sperm diluent is a 0.6 mol / L D-glucose solution, and the cryoprotectant is glycerol (Gly). The volume percentage of glycerol in the cryoprotectant is 20%.

5. The low-temperature preservation method according to claim 2, characterized in that, In step (5), the pre-freezing equilibration time is 25 to 35 minutes.

6. The low-temperature preservation method according to claim 2, characterized in that, In step (6), the cooling process starts at 4°C, drops to -60°C at a rate of 15°C / minute, stays at -60°C for 2 minutes, then drops from -60°C to -140°C at a rate of 20°C / minute, and finally balances at -140°C for 5 minutes.

7. The low-temperature preservation method according to claim 2, characterized in that, In step (7), the thawing and revival process is as follows: thawing refers to quickly transferring the cryovial containing semen to a constant temperature water bath at 37°C and gently shaking the cryovial until the sample is completely thawed; revival refers to activating the thawed sperm using naturally filtered seawater FSW.

8. The low-temperature preservation method according to claim 1, characterized in that, The cryopreservation of the spermatophore of the long octopus specifically includes the following steps: I. Collection of spermatophores from Octopus: Selectively mature male Octopus species were dissected under low-temperature anesthesia in the laboratory, and the spermatophores were removed and placed in ice-cold culture dishes. Each Octopus species' spermatophores constituted a group. II. Sperm motility test: Under a 10×20x microscope, one spermatophore from each group was randomly selected and placed in sterile seawater. The spermatophore was cut into pieces with sterile surgical scissors to activate the sperm in the seawater. 100μL of the activation solution was dropped onto a glass slide. Within 10 minutes, a random area was selected and the sperm motility rate (MSR) was observed and recorded. If the MSR reached 90% or higher, the group containing the spermatophore could be used for cryopreservation. III. Preservation of sperm pods: Place the sperm pods in cryovials, each containing 1.8 mL of preservation solution and one sperm pod. This process must be completed on ice. Place the cryovials in a 4°C freezer for sperm pod preservation. If the semen is to be used, simply thaw and rehydrate the sperm pods.

9. The low-temperature preservation method according to claim 8, characterized in that, In step I, the gonadally mature male octopus used weighs more than 100 grams, and the low-temperature anesthesia is performed at a temperature of 2-3°C. In step III, the preservation solution is a 1.0 mol / L D-glucose solution, which is pre-cooled in a 4°C refrigerator after preparation.

10. The low-temperature preservation method according to claim 8, characterized in that, In step III, the thawing and revival processes are as follows: Thawing refers to quickly transferring the cryovial to a constant temperature water bath at 37°C and gently shaking the cryovial for 30 seconds; Revival refers to discarding the preservation solution, adding naturally filtered seawater FSW, using sterile surgical scissors to cut the sperm pods, releasing the sperm, mixing it with the FSW, and activating the sperm.

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