Cyclina sinensis sperm freezing preservation method

Abstract

The invention relates to the technical field of cyclina sinensis sperm preservation, in particular to a cyclina sinensis sperm freezing preservation method. The cyclina sinensis sperm freezing preservation method comprises the following steps: (1) putting parent shellfish into a seawater culture pond, and accelerating ripening for 7-10 days by using a ripening agent; the ripening agent is prepared from a spirulina extract, a radix paeoniae alba extract, a codonopsis pilosula extract, astaxanthin and alginic acid; (2) controlling 0.05-0.15 m / s water flow to scour and stimulate the cyclina sinensis, selecting individuals with sperms discharged after the parent shellfish gametes start to be discharged, and collecting the sperms of the cyclina sinensis; and (3) mixing the cyclina sinensis sperms with a cryoprotectant, pre-cooling, and then cryopreserving for a long time. Research results show that the use of the cryoprotectant can significantly reduce the degree of DNA damage, and the protection effect may be realized through a plurality of mechanisms such as direct combination of trehalose with DNA molecules, free radical removal, maintenance of the stability of a chromatin structure and the like.

Description

Technical Field

[0001] This invention relates to the field of clam sperm preservation technology, and in particular to a method for cryopreservation of clam sperm. Background Technology

[0002] In the shellfish aquaculture industry, artificial seedling cultivation has long been the main source of seedling supply, but the process is complex. After the broodstock are selected, they must first be rinsed with clean water to remove surface dirt, followed by disinfection with a potassium permanganate solution. Then, temperature-controlled cultivation techniques are used, gradually increasing the ambient temperature to build up effective accumulated heat, promoting the development of the broodstock's gonadal tissue, increasing its size, and eventually enclosing its internal organs. Simultaneously, it is crucial to ensure an ample supply of feed, using a variety of feeds to achieve nutritional complementarity, and strictly controlling temperature fluctuations to prevent abortion. Regarding artificial insemination, some shellfish (such as oysters) can be directly inseminated through dissection, while others (such as bay scallops, blue clams, and Manila clams) require artificial induction of spawning through temperature increases and water stimulation. Commonly used artificial induction methods include temperature stimulation, flowing water stimulation, air-drying stimulation, and chemical injection.

[0003] The blue clam (Venereidae) is a marine mollusc with high economic value. Classified as belonging to the Veneridae family and Veneridae order, it is a typical representative of bivalve mollusks. However, in recent years, due to the continuous deterioration of the marine ecological environment, coupled with overfishing, unscientific aquaculture methods, and the invasion of invasive filter-feeding shellfish, the wild population of this species has shown a sharp decline, and the phenomenon of population decline is becoming increasingly prominent. Therefore, the development of artificial breeding technology for blue clams is imperative. Blue clam breeding generally employs fully artificial control technology, effectively overcoming the constraints of natural environmental factors and significantly improving the production efficiency of seedling cultivation. Artificial breeding, conducted under suitable blue clam developmental stages and appropriate natural conditions, typically involves multiple steps, including broodstock selection, induced spawning, selection of superior larvae and larval rearing, fertilization, and hatching. The induced spawning step involves immersing the parent clam in 20 mg / L potassium permanganate for 10 minutes for disinfection, placing it in a seedling bag or on a spawning bed, air-drying it for 2-4 hours, and then stimulating it with airflow and water flow to select suitable individuals for sperm and egg release. Individuals released at the appropriate time are then collected and placed in the same clean water to complete fertilization, thus completing family selection. However, artificial seedling production is significantly limited by the physiological state of the clam itself and environmental factors such as temperature and geographical location during seedling cultivation. Cryopreserved sperm can be used for breeding across time and space, significantly improving the efficiency and accuracy of selection breeding and providing important technical support for clam variety improvement and genetic research.

[0004] However, the low temperature during freezing can cause various types of damage to sperm, leading to decreased sperm motility, plasma membrane rupture, and other impairments. Studies on the mechanisms of cryogenic damage to fish sperm have revealed that sperm damage mainly occurs due to the formation of intracellular and extracellular ice crystals, osmotic stress, and oxidative stress. Sperm cryogenic damage manifests primarily as: physical damage: intracellular ice crystal formation leads to membrane rupture; furthermore, the protein and lipid composition of many fish sperm cells changes after cryopreservation. Biochemical damage: cryogenic-induced oxidative stress triggers lipid peroxidation and DNA breaks. Functional damage: decreased mitochondrial activity and insufficient ATP synthesis lead to loss of motility.

[0005] Meanwhile, thawing is also considered a crucial factor affecting sperm quality during cryopreservation, with thawing temperature and rate influencing sperm viability. Rapid thawing can prevent recrystallization, while slow warming rates may lead to recrystallization, causing cell damage. Warm water thawing at 37–40°C is widely used for the resuscitation of aquatic sperm to ensure sperm reach fertilization temperature and minimize cell damage during thawing. Therefore, by developing cryopreservation technology for clams, hatcheries can obtain a long-term, stable supply of sperm, ensuring seedling cultivation and promoting efficient long-distance breeding, thereby reducing production costs. Summary of the Invention

[0006] The purpose of this invention is to provide a method for cryopreservation of clam sperm.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for cryopreservation of clam sperm, comprising the following steps:

[0009] (1) Release broodstock into a seawater aquaculture pond and use a ripening agent to ripen them for 7-10 days; the ripening agent is: spirulina extract, white peony extract, codonopsis extract, astaxanthin and alginic acid;

[0010] (2) Control the water flow of 0.05 to 0.15 m / s to flush and stimulate the clam. After the parent clam begins to release gametes, select the individuals that release sperm and collect the clam sperm.

[0011] (3) Mix the clam sperm with a cryoprotectant, pre-cool it, and then freeze it for a long time.

[0012] Preferably, the seawater in step (1) has a salinity of 18-20, a pH of 8.0-8.4, and a water temperature of 20-21°C.

[0013] Preferably, the amount of ripening agent in step (1) is 1 / 8 to 1 / 10 of the mass of the clam feed; the clam feed ratio is 5 to 7% of the body weight of the parent clam.

[0014] Preferably, the mass ratio of spirulina extract, white peony extract, codonopsis extract, astaxanthin and alginic acid in step (1) is 12-16:5-9:15-19:6-10:6-10.

[0015] Preferably, the cryoprotectant in step (3) is: 12-14% (v / v) DMSO, 0.1-0.2 mol / L trehalose, 0.05-0.15 mg / mL cod antifreeze protein, 0.1-0.3 mM quercetin, 10-20 μg / mL isoquercitrin, 1-3% (w / v) potassium chloride, and the remainder is a diluent.

[0016] Preferably, the diluent is seawater with a salinity of 35-41‰.

[0017] Preferably, seawater is added to the seawater aquaculture pond in step (1) to a depth of 1.0 to 1.2 m.

[0018] Preferably, the density of parent clams released in step (1) is 20-30 clams / m². 2 .

[0019] Preferably, the volume ratio of clam sperm to cryoprotectant in step (3) is 1:8 to 12.

[0020] Preferably, the pre-cooling temperature in step (3) is 4-6°C and the time is 30-40 min; the long-term freezing temperature is -80 to -85°C.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The ripening agent provided by this invention plays a crucial role in the cryopreservation method of clam sperm. Its main function is to improve sperm quality and yield by promoting the development of the parent clam's gonads and the maturation of gametes. The ripening agent is composed of spirulina extract, white peony extract, codonopsis extract, astaxanthin, and alginic acid. These components work synergistically to achieve the ripening effect. The functions of each component and their synergistic mechanism are as follows:

[0023] Spirulina extract is rich in protein, vitamins, and minerals, providing the nutrients needed for broodstock development and promoting gonadal development. Codonopsis pilosula extract enhances the immunity and metabolic capacity of broodstock, promotes energy reserves, and provides energy support for gamete release. Paeonia lactiflora extract has anti-inflammatory and antioxidant properties, reducing oxidative stress in broodstock during ripening and protecting gonadal cells; together with Codonopsis pilosula extract, it regulates the immune and metabolic functions of broodstock, improving gamete quality.

[0024] Alginic acid regulates the ion balance in the aquatic environment, stabilizes pH, and provides a suitable environment for the maturation of broodstock. Astaxanthin is a powerful antioxidant that protects sperm cell membranes from oxidative damage, maintains sperm motility and plasma membrane integrity; together with alginic acid, it reduces free radical damage to sperm and improves sperm survival rate after cryopreservation.

[0025] It can be seen that the extracts of spirulina, white peony root, and codonopsis root in the ripening agent provide comprehensive nutrition and energy, promoting gonadal development. Astaxanthin and white peony root extract reduce oxidative stress and protect gametes from free radical damage. Through complementary effects, the components significantly improve sperm yield, motility, and survival rate after freezing.

[0026] Under the cryoprotective conditions of this invention, the damage to the flagella of clam sperm was relatively mild, indicating that an appropriate concentration of cryoprotectant can maintain the integrity of the cytoskeleton to a certain extent. This study used FDA-PI dual-fluorescence staining technology to detect the integrity of the plasma membrane of cryopreserved clam sperm. Transitional sperm exhibiting simultaneous red and green fluorescence were observed, a phenomenon also reported in the cryopreservation study of Pinctada martensii sperm, suggesting that plasma membrane damage may exhibit a progressive characteristic. Data on the effect of cryopreservation time on plasma membrane integrity showed that as the cryopreservation time was extended to 96 hours and 1 week, the plasma membrane integrity of clam sperm significantly decreased. This trend is similar to the findings in the cryopreservation study of large yellow croaker sperm, indicating that the main damage occurs in the early stages of freezing. The use of the cryoprotectant of this invention can significantly reduce cryopreservation damage to sperm.

[0027] The experimental results of this invention showed that the plasma membrane integrity of clam sperm remained relatively stable at DMSO concentrations of 12% and 14%, with no significant difference between the two groups (P>0.05). This result is consistent with studies on scallop sperm cryopreservation, suggesting that DMSO concentrations have similar membrane protection effects within a specific range. Studies in oysters found that oyster sperm exhibited the best plasma membrane protection effect in the 10–12% DMSO range, indicating that different shellfish may have specific optimal concentration windows. Furthermore, the addition of trehalose improved plasma membrane integrity. This phenomenon may be related to the mechanism by which trehalose works by stabilizing membrane proteins rather than directly repairing membrane structures.

[0028] The results of this study indicate that the use of the cryoprotectant of this invention can significantly reduce the degree of DNA damage. This protective effect may be achieved through multiple mechanisms, such as trehalose directly binding to DNA molecules, scavenging free radicals, and maintaining the stability of chromatin structure. Attached Figure Description

[0029] Figure 1Ultrastructure of fresh clam sperm in Example 3; Note: a: Sperm population, scanning electron microscope, showing head and tail flagella, ×1000; b: Sperm, scanning electron microscope, showing head and tail flagella, ×15000; c: Longitudinal section of sperm, transmission electron microscope, showing sperm head, nucleus and mid-mitochondria, ×8000; d: Sperm tail, transmission electron microscope, showing nucleus, mid-mitochondria and flagella, ×20000; e: Longitudinal section of sperm head, transmission electron microscope, showing sperm head acrosome, ×20000; f: Sperm tail and mitochondria, transmission electron microscope, showing sperm nucleus and mitochondria, ×20000; g: Sperm mitochondrial group, transmission electron microscope, ×8000; h: Transverse section of sperm tail flagella, transmission electron microscope, ×40000; i: Longitudinal section of sperm tail flagella, transmission electron microscope, ×40000.

[0030] Figure 2 The effect of different concentrations of DMSO cryoprotectant on sperm survival rate.

[0031] Figure 3 To investigate the effect of different DMSO cryoprotectant concentrations on sperm survival rate after the addition of trehalose.

[0032] Figure 4 The effects of different concentrations of DMSO cryoprotectant on sperm motility.

[0033] Figure 5 To investigate the effect of different DMSO cryoprotectant concentrations on sperm motility after the addition of trehalose.

[0034] Figure 6 The effect of different concentrations of DMSO cryoprotectant on total sperm ATPase activity.

[0035] Figure 7 To investigate the effect of different DMSO cryoprotectant concentrations after the addition of trehalose on the total ATPase activity of sperm.

[0036] Figure 8 The effect of different concentrations of DMSO cryoprotectant on sperm LDH enzyme activity.

[0037] Figure 9 To investigate the effect of different DMSO cryoprotectant concentrations after the addition of trehalose on sperm LDH enzyme activity.

[0038] Figure 10 The effect of different concentrations of DMSO cryoprotectant on sperm SDH enzyme activity.

[0039] Figure 11 To investigate the effect of different DMSO cryoprotectant concentrations after the addition of trehalose on sperm SDH enzyme activity.

[0040] Figure 12The effect of different concentrations of DMSO cryoprotectant on sperm SOD activity.

[0041] Figure 13 To investigate the effect of different DMSO cryoprotectant concentrations after the addition of trehalose on sperm SOD enzyme activity.

[0042] Figure 14 The effect of different concentrations of DMSO cryoprotectant on sperm CAT enzyme activity.

[0043] Figure 15 To investigate the effect of different DMSO cryoprotectant concentrations on sperm CAT enzyme activity after the addition of trehalose.

[0044] Figure 16 The effect of different concentrations of DMSO cryoprotectant on sperm GR enzyme activity.

[0045] Figure 17 To investigate the effect of different DMSO cryoprotectant concentrations after the addition of trehalose on sperm GR enzyme activity.

[0046] Figure 18 The effect of different concentrations of DMSO cryoprotectant on sperm fertilization rate.

[0047] Figure 19 To investigate the effect of different DMSO cryoprotectant concentrations on sperm fertilization rate after the addition of trehalose. Detailed Implementation

[0048] This invention provides a method for cryopreservation of clam sperm, comprising the following steps:

[0049] (1) Release broodstock into a seawater aquaculture pond and use a ripening agent to ripen them for 7-10 days; the ripening agent is: spirulina extract, white peony extract, codonopsis extract, astaxanthin and alginic acid;

[0050] (2) Control the water flow of 0.05 to 0.15 m / s to flush and stimulate the clam. After the parent clam begins to release gametes, select the individuals that release sperm and collect the clam sperm.

[0051] (3) Mix the clam sperm with a cryoprotectant, pre-cool it, and then freeze it for a long time.

[0052] In this invention, step (1) preferably involves using a ripening agent to ripen the fruit for 8-9 days; more preferably, 9 days.

[0053] In this invention, step (2) preferably involves controlling a water flow of 0.07 to 0.13 m / s to irritate the clam; more preferably, it involves controlling a water flow of 0.09 to 0.11 m / s to irritate the clam, or controlling a water flow of 0.1 m / s to irritate the clam.

[0054] In this invention, the salinity of the seawater in step (1) is 18-20, pH 8.0-8.4, and water temperature 20-21°C; preferably, the salinity of the seawater is 19, pH 8.1-8.3, and water temperature 20.5°C; more preferably, the salinity of the seawater is 19, pH 8.2, and water temperature 20.5°C.

[0055] In this invention, the amount of ripening agent in step (1) is 1 / 8 to 1 / 10 of the mass of the clam feed; preferably 1 / 9.

[0056] In this invention, the feeding ratio of clam bait is 5-7% of the body weight of the parent clam; preferably 6%.

[0057] In this invention, the mass ratio of spirulina extract, white peony extract, codonopsis extract, astaxanthin and alginic acid in step (1) is 12-16:5-9:15-19:6-10:6-10; preferably 13-15:6-8:16-18:7-9:7-9; and more preferably 14:7:17:8:8.

[0058] In this invention, the cryoprotectant in step (3) is: 12-14% (v / v) DMSO, 0.1-0.2 mol / L trehalose, 0.05-0.15 mg / mL cod antifreeze protein, 0.1-0.3 mM quercetin, 10-20 μg / mL isoquercitrin, 1-3% (w / v) potassium chloride, and the remainder is a diluent; preferably, it is 13% (v / v) DMSO, 0.15 mol / L trehalose, 0.07-0.13 mg / mL cod antifreeze protein, 0.2 mM quercetin, 12-18 μg / mL isoquercitrin, and potassium chloride. The mixture consists of 2% potassium chloride (w / v) and the remainder being a diluent; more preferably, it consists of 13% (v / v) DMSO, 0.15 mol / L trehalose, 0.09–0.11 mg / mL cod antifreeze protein, 0.2 mM quercetin, 14–16 μg / mL isoquercitrin, 2% potassium chloride (w / v) and the remainder being a diluent; more preferably, it consists of 13% (v / v) DMSO, 0.15 mol / L trehalose, 0.1 mg / mL cod antifreeze protein, 0.2 mM quercetin, 15 μg / mL isoquercitrin, 2% potassium chloride (w / v) and the remainder being a diluent.

[0059] In this invention, the diluent is seawater with a salinity of 35-41‰; preferably 36-40‰; more preferably 37-39‰; and even more preferably 38‰.

[0060] In this invention, seawater is added to the seawater aquaculture pond in step (1) to a depth of 1.0 to 1.2 m; preferably 1.1 m.

[0061] In this invention, the density of parent clams released in step (1) is 20-30 clams / m².2 The preferred size is 22-28 pieces / m². 2 Further preferred is 24–26 birds / m² 2 More preferably, 25 pieces / m 2 .

[0062] In this invention, the volume ratio of clam sperm to cryoprotectant in step (3) is 1:8 to 12; preferably 1:9 to 11; and more preferably 1:10.

[0063] In this invention, the pre-cooling temperature in step (3) is 4-6°C and the time is 30-40 min; preferably, the pre-cooling temperature is 5°C and the time is 32-38 min; more preferably, the pre-cooling temperature is 5°C and the time is 34-36 min; more preferably, the pre-cooling temperature is 5°C and the time is 35 min.

[0064] In this invention, the long-term cryopreservation temperature in step (3) is -80 to -85°C; preferably -81 to -84°C; further preferably -82 to -83°C; and more preferably -82°C.

[0065] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] A method for cryopreserving clam sperm, the steps are as follows:

[0068] (1) Select several large, tightly closed blue clams, disinfect them with 0.1% potassium permanganate solution and wash them with clean seawater, then place them in a net rack, lay them flat, and dry them in the shade for 3 hours.

[0069] (2) After air-drying, place the net frame in a recirculating seawater aquaculture tank and add seawater to a depth of 1 meter (salinity 18, pH 8.0, water temperature 20℃). The stocking density of parent shellfish is 20 shellfish / m². 2 The broodstock were matured for 7 days using a ripening agent. The ripening agent consisted of spirulina extract, white peony extract, codonopsis extract, astaxanthin, and alginic acid in a mass ratio of 12:5:15:6:6. The amount of the ripening agent was 1 / 8 of the mass of the clam feed. The clam feed consisted of *Gynostemma pentaphyllum* and *Chaetoceros*, and the feeding ratio was 5% of the broodstock body weight.

[0070] (3) After the parent clams matured, the circulation system was activated, and the water flow was 0.05 m / s to stimulate the clams for 2 hours. Once gamete release was observed, individuals that had released sperm were immediately placed in a small beaker containing 20 mL of seawater, and allowed to continue releasing sperm. After release stopped, the seawater in the beaker was collected into a 20 mL centrifuge tube and centrifuged at 3000 rpm for 5 minutes. The clams were then washed with 2 mL of clean seawater (salinity 28‰, pH 8.0) and transferred to a 2 mL centrifuge tube, centrifuged at 3000 rpm at 4℃ for 10 minutes. This washing process was repeated three times. After the third centrifugation, 1.5 mL of seawater was suspended and placed on ice at 4℃ for later use.

[0071] (4) Prepare a cryoprotectant consisting of the following components: 12% (v / v) DMSO, 0.1 mol / L trehalose, 0.05 mg / mL cod antifreeze protein, 0.1 mM quercetin, 10 μg / mL isoquercitrin, 1% (w / v) potassium chloride, and the remainder is a diluent (seawater with a salinity of 35‰). Mix all components directly until homogeneous.

[0072] (5) Start timing when the semen and cryoprotectant (volume ratio of 1:8) are fully mixed. First, perform a 30-minute pre-cooling equilibration treatment at 4°C. Then, transfer the cryovial containing the mixed sample to a programmed cooling device that has been pre-cooled for 3 hours. Finally, place it in an ultra-low temperature environment of -80°C for long-term preservation.

[0073] Example 2

[0074] A method for cryopreserving clam sperm, the steps are as follows:

[0075] (1) Select several large, tightly closed blue clams, disinfect them with 0.1% potassium permanganate solution and wash them with clean seawater, then place them in a net rack, flatten them, and dry them in the shade for 5 hours.

[0076] (2) After air-drying, place the net frame in a recirculating seawater aquaculture tank and add seawater to a depth of 1 meter (salinity 20, pH 8.0, water temperature 21℃). The stocking density of parent shellfish is 30 shellfish / m². 2 The broodstock were matured for 10 days using a ripening agent. The ripening agent consisted of spirulina extract, white peony extract, codonopsis extract, astaxanthin, and alginic acid in a mass ratio of 16:9:19:10:10. The amount of the ripening agent was 1 / 10 of the mass of the clam feed. The clam feed consisted of *Gynostemma pentaphyllum* and *Chaetoceros*, and the feeding ratio was 7% of the broodstock body weight.

[0077] (3) After the parent clams matured, the circulation was started, and the water flow was 0.15 m / s to stimulate the clams for 3 hours. After observing the release of gametes, individuals that released sperm were immediately selected and placed in a small beaker containing 20 mL of seawater, and allowed to continue releasing sperm. After the release stopped, the seawater in the beaker was collected into a 20 mL centrifuge tube and centrifuged at 3000 rpm for 5 minutes. The clams were washed with 2 mL of clean seawater (salinity 28‰, pH 8.0) and transferred to a 2 mL centrifuge tube, then centrifuged at 3000 rpm at 4℃ for 10 minutes. This washing was repeated three times. After the third centrifugation, 1.5 mL of seawater was suspended and placed on ice at 4℃ for later use.

[0078] (4) Prepare a cryoprotectant consisting of the following components: 14% (v / v) DMSO, 0.2 mol / L trehalose, 0.15 mg / mL cod antifreeze protein, 0.3 mM quercetin, 20 μg / mL isoquercitrin, 3% (w / v) potassium chloride, and the remainder is a diluent (seawater with a salinity of 41‰). Mix all components directly until homogeneous.

[0079] (5) Start timing when the semen and cryoprotectant (volume ratio of 1:12) are fully mixed. First, perform a 30-minute pre-cooling equilibration treatment at 4°C. Then, transfer the cryovial containing the mixed sample to a programmed cooling device that has been pre-cooled for 3 hours. Finally, place it in an ultra-low temperature environment of -80°C for long-term preservation.

[0080] Example 3

[0081] A method for cryopreserving clam sperm, the steps are as follows:

[0082] (1) Select several large, tightly closed blue clams, disinfect them with 0.1% potassium permanganate solution and wash them with clean seawater, then place them in a net rack, flatten them, and dry them in the shade for 4 hours.

[0083] (2) After air-drying, place the net frame in a recirculating seawater aquaculture tank and add seawater to a depth of 1 meter (salinity 19, pH 8.0, water temperature 21℃). The stocking density of parent shellfish is 25 shellfish / m². 2 The broodstock were matured for 8 days using a ripening agent. The ripening agent consisted of Spirulina extract, Paeonia lactiflora extract, Codonopsis pilosula extract, astaxanthin, and alginic acid in a mass ratio of 14:7:17:8:8. The amount of the ripening agent was 1 / 9 of the mass of the clam feed. The clam feed consisted of Chlorella vulgaris and Chaetoceros horn in a comparative ratio of 6% of the broodstock body weight.

[0084] (3) After the parent clams matured, the circulation system was activated, and the water flow was 0.1 m / s to stimulate the clams for 2.5 hours. Once gamete release was observed, individuals that had released sperm were immediately placed in a small beaker containing 20 mL of seawater, and allowed to continue releasing sperm. After release stopped, the seawater in the beaker was collected into a 20 mL centrifuge tube and centrifuged at 3000 rpm for 5 minutes. The clams were then washed with 2 mL of clean seawater (salinity 28‰, pH 8.0) and transferred to a 2 mL centrifuge tube, centrifuged at 3000 rpm at 4℃ for 10 minutes. This washing process was repeated three times. After the third centrifugation, 1.5 mL of seawater was suspended and placed on ice at 4℃ for later use.

[0085] (4) Prepare a cryoprotectant consisting of the following components: 13% (v / v) DMSO, 0.15 mol / L trehalose, 0.1 mg / mL cod antifreeze protein, 0.2 mM quercetin, 15 μg / mL isoquercitrin, 2% (w / v) potassium chloride, and the remainder is a diluent (seawater with a salinity of 38‰). Mix all components directly until homogeneous.

[0086] (5) Start timing when the semen and cryoprotectant (volume ratio of 1:10) are fully mixed. First, perform a 30-minute pre-cooling equilibration treatment at 4°C. Then, transfer the cryovial containing the mixed sample to a programmed cooling device that has been pre-cooled for 3 hours. Finally, place it in an ultra-low temperature environment of -80°C for long-term preservation.

[0087] Comparative Example 1

[0088] The other methods are the same as in Example 3, except that the white peony extract and codonopsis extract in the ripening agent are replaced with an equal amount of Chaetoceros.

[0089] Comparative Example 2

[0090] The other methods are the same as in Example 3, except that the white peony extract and codonopsis extract in the ripening agent are replaced with an equal amount of astragalus extract.

[0091] Comparative Example 3

[0092] The other methods are the same as in Example 3, except that no cod antifreeze protein is added to the cryoprotectant.

[0093] Comparative Example 4

[0094] The other methods are the same as in Example 3, except that quercetin and isoquercitrin are not added to the cryoprotectant.

[0095] Blank control group 1

[0096] The other methods are the same as in Example 3, except that no ripening agent is used.

[0097] Blank control group 2

[0098] The other methods are the same as in Example 3, except that only 13% (v / v) DMSO and 0.15 mol / L trehalose were added to the cryoprotectant, and the rest was a diluent (seawater with a salinity of 38‰).

[0099] Experimental Example 1

[0100] 1. Sperm motility and viability determination

[0101] The experimental procedure is as follows:

[0102] (1) Dilute the obtained semen sample to 1×10⁻⁶. 6 cells / mL;

[0103] (2) Take 100 μL of diluted semen and mix it with an equal volume of 2× trypan blue staining solution (100 μL) in a centrifuge tube and stain at room temperature for 3 minutes;

[0104] (3) Take 10 μL of stained sample and place it in a hemocytometer for detection.

[0105] Live cells, due to their intact membrane structure, resist staining with trypan blue, while dead cells, due to altered membrane permeability, are stained blue. Each sample had a sperm count ≥500, and the sample was repeated three times.

[0106] Sperm survival rate (%) = (Total sperm count - Number of blue sperm) / Total sperm count × 100%

[0107] 15 μL of diluted semen was aspirated into a cell counting chamber and sperm motility was observed under a microscope. The total sperm motility time (the time from when the semen was mixed with seawater until 95% of the sperm stopped motility) and the rapid motility time (the time when the proportion of rapidly motile sperm gradually decreased from the initial 50% to 30%) were recorded. The results were repeated three times.

[0108] Sperm motility (%) = (Time of rapid motility / Total motility time) × 100%

[0109] 2. Sperm fertilization capacity testing

[0110] Six clam individuals that released eggs were selected using an ovulation induction method. Immediately after release, they were placed in a small beaker containing 16 mL of seawater and allowed to continue releasing eggs until the process was complete. The obtained diluted semen (1×10⁻⁶) was then used to further induce ovulation. 6 Three groups of sperm were each mixed with 1 mL of sperm from different beakers, and the mixture was gently stirred with a glass rod to ensure full contact between the sperm and eggs. After incubation at a specific temperature (25±1℃) for a predetermined time, the fertilization rates of the different treatment groups were observed under a microscope and recorded.

[0111] Following the above method, the sperm survival rates of the experimental group, comparative example 1, comparative example 2, and blank control group 1 at different time points were statistically analyzed, and the results are shown in Table 1. The sperm motility at 0h in each experimental group was statistically analyzed, and the fertilization rate of sperm at different time points in each experimental group was statistically analyzed, and the results are shown in Table 3.

[0112] Table 1. Sperm survival rate (%)

[0113] Group 0h 6h 12h 24h 36h experimental group 98.68 86.75 72.94 51.63 37.55 Comparative Example 1 98.41 81.27 65.39 45.24 28.56 Comparative Example 2 97.97 83.58 67.55 47.83 32.78 Blank control group 1 98.52 75.67 61.47 42.71 24.38

[0114] Table 2 Sperm motility

[0115] Group Sperm motility % experimental group 81.3 Comparative Example 1 77.0 Comparative Example 2 79.2 Blank control group 1 74.6

[0116] Table 3. Sperm fertilization rate (%)

[0117] Group 0h 1h 2h 4h 6h experimental group 84.5 78.6 77.2 66.7 54.9 Comparative Example 1 82.3 75.2 69.3 57.8 47.3 Comparative Example 2 83.0 75.9 73.8 59.2 51.6 Blank control group 1 80.1 72.5 66.4 53.7 43.2

[0118] As shown in Tables 1 to 3, the ripening agent prepared by the method of the present invention can improve sperm motility and survival rate, and can significantly improve fertilization rate. The relevant data of the experimental group are significantly better than those of the comparative group and the blank control group.

[0119] Experiment Example 2

[0120] Sperm-related enzyme activity detection

[0121] Two mL of diluted semen was collected, three groups in total, and re-sieved using a 300-mesh cell sieve. The sieved semen was centrifuged at 3000 rpm for 5 min at 4°C, and the precipitate was collected. 1 mL of PBS was added and the mixture was washed by pipetting. The mixture was centrifuged at 3000 rpm for 10 min at 4°C, and this process was repeated twice. The supernatant was discarded, and the mixture was transferred to a round-bottom centrifuge tube and homogenized for 20-30 s with a grinding rod to obtain cell homogenate.

[0122] The enzyme activity assay kits used in the experiment were purchased from Nanjing Jiancheng Biotechnology Co., Ltd., and three replicate assays were performed for each test sample.

[0123] Based on the method of Example 3, the final concentrations of DMSO were made to be 6%, 8%, 10%, 12%, 14%, and 16%, respectively, and denoted as D-6, D-8, D-10, D-12, D-14, and D-16, but without the addition of trehalose.

[0124] This experiment also investigated the effect of trehalose addition on cryopreservation. Based on each of the above cryoprotectants, 0.15 mol / L of trehalose was added, designated as T-6, T-8, T-10, T-12, T-14, and T-16.

[0125] After thoroughly mixing the semen sample with the cryoprotectant at a volume ratio of 1:10, quickly dispense 3.5 mL into 5 mL cryovials. The entire procedure must be performed under ice bath conditions.

[0126] Timing was started when the semen and cryoprotectant were thoroughly mixed. First, a pre-cooling equilibration treatment was performed at 4°C for 30 minutes. Then, the cryovials containing the mixed sample were transferred to a programmed cooling device that had been pre-cooled for 3 hours, and finally placed in an ultra-low temperature environment of -80°C for long-term storage. This experiment studied the changes in various indicators for each treatment group at 1 / 2 / 4 / 6 / 8 / 12 / 18 / 24 / 48 / 96h; 1, 2, 3, 4 weeks; 2, 3, 4, 5 months; and 1 year. The treatment groups were denoted as 1 / 2 / 4 / 6 / 8 / 12 / 18 / 24 / 48 / 96h; 1 / 2 / 3 / 4w; 2 / 3 / 4 / 5m; and 1 year, respectively.

[0127] Results: 1. The cryopreservation effect of clam sperm at different DMSO concentrations showed significant differences (P<0.05). The survival rate of all treatment groups was significantly lower than that of fresh semen (98.05±0.42%), and it decreased with prolonged storage time. The survival rate decreased slowly within 24 hours, then dropped sharply, approaching zero after 4 weeks. The 12% and 14% DMSO groups showed the best preservation effect, significantly better than other concentration groups (P<0.05), with the 10% DMSO group having the lowest survival rate.

[0128] 2. After adding 0.15 mol / L trehalose, the survival rate of all concentration groups improved, but the differences were not significant (P>0.05). Notably, in the 8% DMSO group, trehalose actually led to a decrease in survival rate, indicating that its protective effect is concentration-dependent. The 12% and 14% DMSO + trehalose groups had the highest survival rates, but there was no significant difference between the two groups (P>0.05).

[0129] 3. The sperm motility of clam sperm showed significant time-dependent changes during cryopreservation. Figure 4 Sperm motility (88.42±4.26%) after short-term storage (1–4 hours) was not significantly different from that of fresh samples (P>0.05). Comparison of different DMSO concentration treatment groups showed that the 12% and 14% DMSO groups performed best in maintaining sperm motility, significantly superior to other concentration groups (P<0.05), with the 10% DMSO group showing the worst protective effect. Notably, there was no statistically significant difference between the 12% and 14% DMSO groups (P>0.05). The addition of trehalose improved sperm motility to some extent in all concentration groups, but the differences were not significant (P>0.05). Long-term storage experiments showed that sperm motility was essentially lost (approaching 0) after 4 weeks of cryopreservation, a result highly consistent with the trend in survival rate.

[0130] 4. Analysis of enzyme activity changes during cryopreservation of clam sperm showed that compared with fresh sperm, the activities of five key enzymes—ATP, LDH, SDH, SOD, and CAT—were significantly reduced in cryopreserved sperm (P<0.05), and this reduction continued with prolonged cryopreservation. Among different DMSO concentration treatment groups, 12% and 14% DMSO showed the most significant protective effects on enzyme activity (P<0.05), while the protective effects of 8%, 10%, and 16% DMSO groups were relatively poor, with the 10% DMSO group exhibiting the lowest enzyme activity. Notably, the addition of trehalose specifically reduced the activities of SOD and CAT in the 10% DMSO group (P<0.05), while other concentration groups showed a trend of increasing enzyme activity, but the differences were not statistically significant (P>0.05). During the initial cryopreservation period (within 4 hours), ATPase activity remained relatively stable (P>0.05). However, with prolonged cryopreservation, the differences in protective effects among the treatment groups gradually became significant (P<0.05). The 12% and 14% DMSO groups consistently showed the best protective effects, but there was no significant difference between the two groups (P>0.05). This result is highly consistent with the changing trends of sperm viability and motility, further confirming the optimized effect of the 12–14% DMSO concentration range on the cryopreservation of clam sperm.

[0131] 5. Cryopreservation significantly affected the activity of glutathione reductase (GR) in clam sperm (P<0.05). Compared with fresh sperm (51.45±3.47 U / L), the GR activity of cryopreserved sperm showed a dynamic change of first increasing and then decreasing: reaching a peak during 18–96 hours of cryopreservation, and then slowly declining after one week. Comparison of different DMSO concentration treatment groups showed that the 10% DMSO group had the highest GR activity, significantly higher than other treatment groups (P<0.05); while the 12% and 14% DMSO groups had the lowest GR activity, but there was no significant difference between the two groups (P>0.05). After the addition of trehalose, the GR activity of all treatment groups decreased, but the difference was not statistically significant (P>0.05). This result suggests that the change in GR activity may be related to cryopreservation-induced oxidative stress.

[0132] 6. The fertilization capacity of clam sperm changed significantly after cryopreservation (P<0.05). Compared with fresh sperm (fertilization rate 75.05±11.97%), the fertilization rate of cryopreserved sperm decreased continuously with prolonged storage time: after 96 hours of cryopreservation, it dropped to 50% of that of fresh sperm, and after one week, it essentially lost its fertilization capacity. Among different DMSO concentrations, the 12% and 14% DMSO groups had the highest fertilization rates, significantly better than other concentration groups (P<0.05), but there was no significant difference between the two groups (P>0.05). Notably, the 10% DMSO group had the lowest fertilization rate within 1–12 hours of cryopreservation (P<0.05), while the differences between concentration groups gradually decreased as the cryopreservation time was extended to 18 hours. Although the addition of trehalose could improve the fertilization rate, the effect did not reach a statistically significant level (P>0.05).

[0133] Experimental Example 3

[0134] The diacetylfluorescein (FDA) and propidium iodide (PI) used in this experiment were both purchased from Sigma-Aldrich, Inc., USA.

[0135] Dissolve FDA powder in acetone to prepare a 1 mg / mL FDA stock solution, and store at -20°C protected from light. Prepare a 400 μg / mL PI stock solution using sterile seawater (salinity 28‰, pH 8.0) as the solvent, and store at 4°C protected from light. Dilute the FDA and PI stock solutions to 100 μg / mL using sterile seawater as the diluent. Combine the 100 μg / mL FDA staining solution, PI staining solution, and semen (fresh semen and thawed frozen semen with a cell density of 1 × 10⁻⁶ cells / mL). 6 Mix the samples in a ratio of 1:1:8, incubate the stained solution in the dark for 10 minutes, then take 20 μL of the sample and place it on a glass slide for fluorescence microscopy observation under blue light (450-490 nm) and green light (510-550 nm) excitation wavelengths, respectively.

[0136] Following the above method, the plasma membrane integrity (%) and frozen sperm survival rate (%) of sperm in the experimental group, comparative example 3, comparative example 4 and blank control group at different time points were statistically analyzed. The results are shown in Tables 4 and 5.

[0137] Table 4 Results of sperm cryopreservation after 96 hours

[0138] Group Plasma membrane integrity (%) Frozen sperm survival rate % experimental group 52.67 62.75 Comparative Example 3 50.68 59.62 Comparative Example 4 49.25 58.73 Blank control group 2 45.85 56.47

[0139] Note: Frozen sperm viability percentage is not a FDA-PI test.

[0140] Table 5 Results of sperm cryopreservation after 1 week

[0141]

[0142]

[0143] The results showed that sperm plasma membrane integrity decreased with prolonged cryopreservation time. The use of the cryoprotectant of this invention increased plasma membrane integrity and improved frozen sperm survival rate. The relevant data of the experimental group were significantly better than those of other comparative groups. The data from the comparative groups show that omitting the components of the cryoprotectant of this invention would affect sperm survival rate.

[0144] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for cryopreservation of clam sperm, characterized in that, Includes the following steps: (1) Release broodstock into a seawater aquaculture pond and use a ripening agent to ripen them for 7-10 days; the ripening agent is: spirulina extract, white peony extract, codonopsis extract, astaxanthin and alginic acid; (2) Control the water flow of 0.05 to 0.15 m / s to flush and stimulate the clam. After the parent clam begins to release gametes, select the individuals that release sperm and collect the clam sperm. (3) Mix the clam sperm with a cryoprotectant, pre-cool it, and then freeze it for a long time.

2. The method for cryopreservation of clam sperm according to claim 1, characterized in that, The salinity of the seawater in step (1) is 18-20, pH 8.0-8.4, and water temperature 20-21℃.

3. The method for cryopreservation of clam sperm according to claim 1, characterized in that, The amount of ripening agent in step (1) is 1 / 8 to 1 / 10 of the mass of the clam feed; the clam feed ratio is 5 to 7% of the body weight of the parent clam.

4. The method for cryopreservation of clam sperm according to claim 1, characterized in that, The mass ratio of spirulina extract, white peony extract, codonopsis extract, astaxanthin and alginic acid in step (1) is 12-16:5-9:15-19:6-10:6-10.

5. The method for cryopreservation of clam sperm according to claim 1, characterized in that, The cryoprotectant in step (3) is: 12-14% (v / v) DMSO, 0.1-0.2 mol / L trehalose, 0.05-0.15 mg / mL cod antifreeze protein, 0.1-0.3 mM quercetin, 10-20 μg / mL isoquercitrin, 1-3% (w / v) potassium chloride, and the remainder is a diluent.

6. The method for cryopreservation of clam sperm according to claim 5, characterized in that, The diluent is seawater with a salinity of 35-41‰.

7. The method for cryopreservation of clam sperm according to claim 1, characterized in that, In step (1), seawater is added to the seawater aquaculture pond to a depth of 1.0 to 1.2 m.

8. The method for cryopreservation of clam sperm according to claim 1, characterized in that, The density of parent scallops introduced in step (1) is 20-30 scallops / m². 2 .

9. The method for cryopreservation of clam sperm according to claim 1, characterized in that, The volume ratio of clam sperm to cryoprotectant in step (3) is 1:8 to 12.

10. A method for cryopreservation of clam sperm according to claim 1, characterized in that, The pre-cooling temperature in step (3) is 4-6℃ and the time is 30-40 min; the long-term freezing temperature is -80 to -85℃.

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