A sperm cryopreservation diluent for epinephelus coeruleopunctatus and its use method and application

Abstract

The application provides a low-temperature preservation diluent for sperm of Epinephelus lanceolatus and a use method and application thereof. The diluent is prepared from the following raw materials: glucose, calcium chloride, potassium chloride, sodium chloride, sodium bicarbonate, reduced glutathione, sodium dihydrogen phosphate and fetal bovine serum. The diluent contains the following mass of raw materials per liter: 0.4-0.5 g of glucose, 0.03-0.08 g of calcium chloride, 0.15-0.25 g of potassium chloride, 3-4 g of sodium chloride, 1.5-2.0 g of sodium bicarbonate, 1.5-2.5 g of reduced glutathione, 0.008-0.012 g of sodium dihydrogen phosphate, and 8-12% of the fetal bovine serum in terms of the volume percentage of the diluent. The prepared diluent can better maintain the in-vitro activity of sperm, prolong the sperm preservation time, meet the low-temperature preservation requirements of the sperm of Epinephelus lanceolatus, and especially meet the low-temperature preservation requirements of the sperm of Epinephelus lanceolatus.

Description

Technical Field

[0001] This invention relates to the field of fish semen preservation, and in particular to a low-temperature preservation diluent for blue-bodied large-spotted grouper semen and its usage and application. Background Technology

[0002] The Blue-bodied Large Spotted Grouper, belonging to the order Perciformes, family Serranidae, subfamily Scutellainae, and genus Scutella, is a large grouper species widely distributed in the Indo-Pacific region and southern my country. It is an important marine economic fish due to its rapid growth and delicious flesh. While it boasts a fast growth rate, its seedling production is difficult, resulting in low survival rates and limited market availability. Therefore, it is often used as a paternal parent for hybridization with other groupers. Research on the Blue-bodied Large Spotted Grouper is still in its early stages, with limited data and no systematic descriptions found in domestic or international literature. In practical production, issues such as asynchronous development of male and female gonads or insufficient sperm count in males are frequently encountered. Therefore, a method for short-term sperm storage is needed. Furthermore, once sperm is expelled from the parent fish, it loses its nutrient supply and relies solely on seminal plasma for energy, quickly ceasing motility. Effective preservation methods for isolated sperm could pave the way for artificial insemination and even fully artificial breeding.

[0003] Sperm are capable of motility and require energy from nutrients in their protoplasm. However, sperm do not move when they are in a dense state. In marine fish undergoing in-vitro fertilization, sperm are activated upon contact with seawater and begin to move actively.

[0004] When preserving fish sperm in vitro, contamination should be avoided during sperm retrieval. A suitable diluent can reduce sperm viscosity, maintain internal environmental homeostasis, provide nutrients, reduce the impact of foreign substances, inhibit sperm motility, and mitigate the harmful effects of metabolic products. Further determining the appropriate dilution ratio is crucial. If the dilution ratio is too small, the sperm concentration will be too high and viscous, rendering dilution ineffective and reducing motility. If the dilution ratio is too large, the external morphology of the sperm is prone to change, reducing motility. Storage at low temperatures (0-4℃) or ultra-low temperatures (liquid nitrogen or -80℃) is recommended, as low temperatures can inhibit sperm metabolism and extend preservation time. Furthermore, low temperatures can inhibit bacterial growth, which is also an important reason for extending preservation time. To date, there are no reports or studies on the cryopreservation (0-4℃) of sperm from blue-bodied large-spotted grouper. Only studies on ultra-low temperature sperm preservation have been found. While cryopreservation is suitable, the process is complex. Most fish farms do not have liquid nitrogen and tanks readily available, and operator skill varies. Thawing time is highly subjective, and ultra-low temperature freezing can damage sperm, causing rupture of the sperm head membrane, midpiece detachment, and cell death. Furthermore, the damage to the sperm membrane caused by freezing allows large amounts of enzymes and proteins to enter the seminal plasma, reducing sperm enzyme activity while increasing seminal plasma enzyme activity. The extent of freezing damage increases with freezing time.

[0005] Currently, there is no effective diluent in actual production research that can extend the cryopreservation time of blue-bodied large-spotted grouper semen. This poses a significant obstacle for relevant technicians who need to conduct breeding and artificial reproduction through artificial insemination. Even if cryopreservation technology exists, it cannot meet the short-term needs of general aquaculture farms that do not have liquid nitrogen and only need to preserve the semen for a few hours or even a few days. Furthermore, cryopreservation is a complex process with variations in operation among different personnel. The thawing time of cryopreserved sperm is greatly affected by subjective factors, which directly affects the cryopreservation effect. Even if some sperm are still viable after thawing, their internal and external physiological and biochemical functions are damaged by freezing, rendering them unable to fertilize.

[0006] The formulation of the diluent is crucial for the cryopreservation of semen, but there is currently no unified standard. The general principles are as follows: the marine fish diluent should be isotonic or slightly hypotonic with the semen being preserved; it should have a certain buffering capacity to maintain pH; it should contain certain nutrients to supply the weak metabolic needs of sperm at low temperatures (0-4℃); it should contain substances that inhibit sperm motility, such as K+ which can inhibit the activity of salmonid sperm, and Na+ which can reduce this inhibitory effect; antibiotics can be added in appropriate amounts to prevent the large-scale growth of mold and other microorganisms during preservation; all medicines and utensils used must be strictly sterilized; and it should be prepared and used immediately.

[0007] Low temperatures can slow sperm metabolism, which is beneficial for prolonging sperm lifespan; however, excessively low temperatures can produce ice crystals, damaging sperm. Undiluted sperm can also be stored at low temperatures (0-4℃) for a period of time, but metabolism does not stop, only slows down significantly. During storage, semen produces a large amount of metabolic waste, some of which is toxic. If the concentration is too high, it can kill sperm in the later stages. Metabolism also produces lactic acid and carbon dioxide, lowering the pH of the semen. In addition, undiluted semen is prone to sticking together and drying out. Freshly collected semen is dense and viscous, causing the already limited water content in the seminal plasma to evaporate during storage, making the surface sperm even drier. Furthermore, without a continuous supply of nutrients to maintain metabolism, the preservation effect is greatly reduced. Therefore, there is an urgent need for a diluent that can meet the low-temperature preservation requirements of blue-bodied grouper semen. Summary of the Invention

[0008] In view of this, the present invention proposes a low-temperature preservation diluent for blue-bodied large-spotted grouper semen, which can better maintain sperm viability in vitro, extend preservation time, and meet the low-temperature preservation requirements of blue-bodied large-spotted grouper.

[0009] The technical solution of this invention is implemented as follows:

[0010] A low-temperature preservation diluent for blue-bodied large-spotted grouper semen is prepared from the following raw materials: glucose, reduced glutathione, sodium dihydrogen phosphate, and fetal bovine serum.

[0011] Furthermore, the diluent also contains the following raw materials: calcium chloride, potassium chloride, sodium chloride, and sodium bicarbonate.

[0012] Furthermore, each liter of the diluent contains the following raw materials by mass: 0.4–0.5 g glucose, 0.03–0.08 g calcium chloride, 0.15–0.25 g potassium chloride, 3–4 g sodium chloride, 1.5–2.0 g sodium bicarbonate, 1.5–2.5 g reduced glutathione, 0.008–0.012 g sodium dihydrogen phosphate, and the fetal bovine serum constitutes 8–12% of the volume of the diluent.

[0013] Furthermore, each liter of the diluent contains the following raw materials by mass: 0.45g glucose, 0.05g calcium chloride, 2g potassium chloride, 3.5g sodium chloride, 1.69g sodium bicarbonate, 2g reduced glutathione, 0.01g sodium dihydrogen phosphate, and the fetal bovine serum is 10v / v of the volume percentage of the diluent.

[0014] Furthermore, the method for preparing the preservation diluent includes the following steps: weighing glucose, calcium chloride, potassium chloride, sodium chloride, sodium bicarbonate, reduced glutathione, and sodium dihydrogen phosphate respectively, dissolving them in water to obtain a base solution; before use, taking fetal bovine serum, thawing it, and transferring it into the base solution to obtain the preservation diluent.

[0015] This invention also provides a method for using a low-temperature preservation diluent for blue-bodied large-spotted grouper semen, wherein fresh semen is mixed with the preservation diluent at a volume ratio of 1:1-99, and then stored at low temperature. Further volume ratios are 1:9, 1:29, 1:49, 1:69, or 1:99.

[0016] Furthermore, fresh semen is mixed with the preservation diluent at a volume ratio of 1:49-69, and more preferably at a volume ratio of 1:49 or 1:69.

[0017] Furthermore, the low-temperature storage temperature is 0-4℃.

[0018] Furthermore, the cryogenic preservation time is ≤72h.

[0019] The diluent described in any one of the present invention is used in the preservation of grouper semen.

[0020] Furthermore, the diluent is used in the preservation of semen from blue-bodied grouper.

[0021] This invention also utilizes CASA technology to detect sperm motility. CASA technology is a new technology developed based on multiple techniques and equipment, including photomicrography, microscopic image analysis, and fluorescence microscopy. Initially applied in male reproductive research, it has since been gradually used in sperm motility testing in other mammals. COSSON et al. first used a stroboscopic observation instrument and video recording to describe sperm motility in salmon. With the maturation of this technology, CASA has been widely used in sperm motility testing in fish and invertebrates in recent years. CASA identification technology mainly includes DNA fluorescence identification and grayscale identification. Employing microscopic video recording technology and advanced image processing techniques, it comprehensively quantifies and analyzes dynamic and static sperm images, accurately measuring parameters such as sperm motility rate (MOT), velocity of curvilinear movement (VCL), velocity of linear movement (VSL), whiplash frequency (BCF), and lateral amplitude of sway (ALH).

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] (1) This invention uses glucose, reduced glutathione, sodium dihydrogen phosphate, and fetal bovine serum as raw materials, and selects the optimal ratio to prepare a preservation solution suitable for short-term low-temperature preservation of semen from blue-bodied large-spotted grouper. The diluted solution prepared by this invention greatly extends the external preservation time of blue-bodied large-spotted grouper semen, and can efficiently preserve high-quality male parent sperm, providing technical support for artificial insemination of blue-bodied large-spotted grouper, and effectively meeting the requirements for short-term low-temperature (0-4℃) preservation of sperm during artificial breeding of superior blue-bodied large-spotted grouper.

[0024] (2) The diluent prepared in this invention can inhibit the activation of grouper sperm and provide nutrients and energy to the sperm; it can reduce oxidative damage caused by metabolic products produced during the respiratory metabolism of grouper sperm, and can also reduce cryogenic damage to grouper sperm at low temperatures. The diluent prepared in this invention can reduce sperm metabolic rate, reduce energy consumption, and prolong sperm lifespan. Among them, the reduced glutathione in the formulation of this invention helps to reduce oxidative damage caused by metabolic products produced during the respiratory metabolism of sperm; fetal bovine serum (FBS) not only helps to reduce oxidative damage caused by metabolic products produced during the respiratory metabolism of sperm, but also reduces cryogenic damage to sperm at low temperatures.

[0025] (3) The addition of calcium chloride, potassium chloride, sodium chloride and sodium bicarbonate to the diluent of the present invention helps to maintain the pH value of the system, maintain the osmotic pressure and inhibit the activation of sperm.

[0026] (4) The diluent prepared by the present invention can better meet the requirements of low temperature (0-4℃) preservation of grouper semen, has no toxic effect on sperm, and effectively prolongs the sperm preservation time.

[0027] (5) The preparation and use of the diluent of the present invention is simple and easy. After preparing the low-temperature preservation solution in advance according to the procedural requirements, the sperm can be preserved in the field with an ice box containing ice packs to avoid damage to the sperm caused by ice crystals forming below 0°C. Attached Figure Description

[0028] Figure 1 This is a flowchart of the process of Embodiment 1 of the present invention. Detailed Implementation

[0029] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0030] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0032] The MS-222 used in this embodiment of the invention was purchased from Shanghai Kangting Biotechnology Co., Ltd.

[0033] The reduced glutathione used in the embodiments of the present invention is L-reduced glutathione, brand: McLean, product number: G6268.

[0034] Example 1

[0035] I. Preparation of diluent for low-temperature preservation of semen from blue-bodied large-spotted grouper:

[0036] 1. Weigh 0.45g glucose, 0.05g calcium chloride, 2g potassium chloride, 3.5g sodium chloride, 1.69g sodium bicarbonate, 2g reduced glutathione, and 0.01g sodium dihydrogen phosphate using an electronic balance. Dissolve them in ultrapure water at room temperature and bring the volume to 0.9L to obtain the base solution. Store at 4℃ for 1-3 days.

[0037] 2. Before use, take the fetal bovine serum out at -20℃, dissolve it completely at room temperature, transfer 0.1L to 0.9L of the base solution, mix well, and the low-temperature preservation solution is obtained.

[0038] II. Semen Collection and Dilution

[0039] 1. The semen collection site was selected in Jianjie Village, Huangliu Town, Ledong Li Autonomous County, Hainan Province. During the breeding season, 15 male fish (blue-bodied large-spotted grouper) that had undergone nutritional enhancement, were in good growth, and had mature testes were selected. Semen was collected manually by squeezing. Before collection, the fish were anesthetized with MS-222 and the area around the cloaca was wiped dry with a towel to prevent contamination of the sample by water, urine, etc. The abdomen was gently pressed to the cloaca. After the milky white, clean, and uncontaminated semen flowed out, it was immediately aspirated using a 200μL pipette and gently transferred to the bottom of a 5mL sterile EP tube that was completely dry.

[0040] 2. All collected semen samples are mixed and processed to reduce errors and eliminate individual differences, and the initial motility must be greater than 90%;

[0041] 3. Semen was diluted with cryopreservation solution at ratios of 1:9, 1:29, 1:49, 1:69, and 1:99 and then poured into 5 mL sterile EP tubes. This was the experimental group. The total volume of semen after each dilution was 2.1 mL. The remaining fresh semen without any added reagents served as the control group.

[0042] 4. First, wrap all the EP tubes from step 3 with aluminum foil to protect them from light and prevent photoactivation. Then wrap them with absorbent cotton, and finally wrap them tightly with gauze. Place them in an ice box containing ice packs at a temperature of about 4°C.

[0043] III. Sperm motility testing

[0044] 1. Fresh semen motility test: According to step 3 in section 2, the fresh semen was tested at 10 min, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after being extracted from the parent male fish. The specific operation steps for each test are as follows: 1 μL of fresh semen was drawn onto a glass slide using a 2.5 μL pipette, 2 μL of filtered natural seawater was added to activate the sperm, and the focus was adjusted until the image on the display screen was clear. After the final semen was diluted 300 times, the sperm motility test system (CASA) was used to test the sperm motility, mainly recording the motility rate (MOT).

[0045] 2. Semen analysis at different dilution ratios:

[0046] 1) When the semen to cryopreservation solution dilution ratio v / v is 1:9 (equivalent to a 10-fold dilution), the specific operation is as follows: Sperm is taken from the parent male fish, diluted at a volume ratio of 1:9, and stored at 4℃. Testing is performed at 10 min, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after removal from the parent male fish: 9 μL of leopard gill spiny perch semen cryopreservation solution is added to a 200 μL EP tube using a 10 μL pipette. 1 μL of the diluted semen (1:9 dilution ratio as described in section 2) is then pipetted in using a 2.5 μL pipette and gently mixed. 1 μL of the mixture is transferred to a glass slide, the focus is adjusted until the image on the display is clear, and 2 μL of filtered natural seawater is added to activate the sperm. The final semen is diluted 300 times. Sperm motility is then detected using a sperm motility assay system (CASA), primarily recording motility rate (MOT).

[0047] 2) When the semen to cryopreservation solution dilution ratio v / v is 1:29 (equivalent to a 30-fold dilution), the specific operation is as follows: Semen is extracted from the parent male fish, diluted at a ratio of 1:29, and stored at 4℃. Testing is performed at 10 min, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after extraction from the parent male fish: 2.8 μL of blue-spotted grouper semen cryopreservation solution is added to a 200 μL EP tube using a 2.5 μL pipette. 1.2 μL of the diluted semen (1:29 dilution ratio as described in section 2) is then pipetted and gently mixed. 1 μL of the mixture is transferred to a glass slide, and 2 μL of filtered natural seawater is added to activate the sperm. The focus is adjusted until the image on the display screen is clear, indicating a final semen dilution of 300 times. Sperm motility is then detected using a sperm motility assay (CASA) system, primarily recording motility rate (MOT).

[0048] 3) When the semen to cryopreservation solution dilution ratio v / v is 1:49 (equivalent to a 50-fold dilution), the specific operation is as follows: Semen is extracted from the parent male fish, diluted at a volume ratio of 1:49, and stored at 4℃. Testing is performed at 10 min, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after extraction from the parent male fish: 1 μL of blue-spotted grouper semen cryopreservation solution is added to a 200 μL EP tube using a 2.5 μL pipette. 1 μL of the diluted semen (1:49 dilution ratio as described in section 2) is then pipetted and gently mixed. 1 μL of the mixture is transferred to a glass slide, and 2 μL of filtered natural seawater is added to activate the sperm. The focus is adjusted until the image on the display screen is clear. After the final semen dilution is 300 times, sperm motility is detected using a sperm motility assay (CASA) system, primarily recording motility rate (MOT).

[0049] 4) When the semen and cryopreservation solution are diluted at a ratio of 1:69 (equivalent to a 70-fold dilution), the specific procedure is as follows: Semen is extracted from the parent male fish, diluted at a volume ratio of 1:69, and stored at 4°C. Testing is performed at 10 min, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after extraction from the parent male fish. 0.6 μL of blue-spotted grouper semen cryopreservation solution is added to a 200 μL EP tube using a 2.5 μL pipette. 1.4 μL of the diluted semen (1:69 as described in section 2) is then pipetted and gently mixed. 1 μL of the mixture is transferred to a glass slide, and 2 μL of filtered natural seawater is added to activate the sperm. The focus is adjusted until the image on the display screen is clear. This represents a final semen dilution of 300 times. Sperm motility is then detected using a sperm motility assay (CASA) system, primarily recording motility rate (MOT).

[0050] 5) When the semen and cryopreservation solution dilution ratio v / v is 1:99 (equivalent to a 100-fold dilution), the specific operation is as follows: Sperm is taken from the parent male fish, diluted at a volume ratio of 1:99, and stored at 4℃. The test is performed at 10 min, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h after being taken from the parent male fish: 1 μL of the diluted semen at the 1:99 dilution ratio described in section 2 is taken with a 2.5 μL pipette and placed on a glass slide. 2 μL of filtered natural seawater is added to activate the sperm. The focus is adjusted until the image on the display screen is clear. After the final semen is diluted 300 times, sperm motility is detected using a sperm motility detection system (CASA), mainly recording the motility rate (MOT).

[0051] After detecting sperm motility according to the method described in section 3, the data obtained are shown in Table (1). It can be seen that the sperm motility is highest at dilution ratios of 1:49 and 1:69, with 47.56% and 44.53% respectively at 24h, which is better than the motility of fresh sperm. The motility is still more than 10% at 72h, indicating that the maximum preservation time of sperm is 3 days, i.e., 72h, when diluted 50 times and 70 times. At 96h, the motility rate of all experimental groups and control groups is 0, which can be understood as all of them dying.

[0052] Table (1) Sperm motility of blue-bodied grouper under different storage times and dilution ratios

[0053]

[0054] Note: "-" indicates inactive, with a movement rate of 0, which means death.

[0055] IV. Artificial Insemination

[0056] Based on the results obtained from the three studies, it can be seen that the semen of the blue-bodied large-spotted grouper diluted with the cryopreservation solution at a ratio of 1:49 can be preserved for approximately 72 hours, and 47.56% of the sperm can still be activated up to 24 hours later. Therefore, in the artificial insemination experiment, the semen diluted 50 times was used as the experimental group, and fresh semen was used as the control group. Wet fertilization was employed. In the experimental group, 50 μL of diluted semen was activated with seawater, and immediately 2 mL of eggs were poured onto a culture dish containing activated sperm and spread evenly for several minutes, stirring continuously, before being transferred to clean seawater at 28°C for incubation. In the control group, 1 μL of fresh semen was used instead of 50 μL of diluted semen. Fertilization was performed on the eggs at 2 hours, 6 hours, 12 hours, and 24 hours after preservation, and the fertilization rate and hatching rate were recorded.

[0057] Fertilization rate = (Number of embryos developed to the 4-16 cell stage / Total number of eggs) × 100%;

[0058] Hatching rate = (Number of newly hatched fry / Number of fertilized embryos) × 100%.

[0059] It should be noted that the eggs squeezed out from the parent female fish must be in good condition, spherical, homogeneous and transparent in appearance, and most of them float on the surface of the seawater.

[0060] Based on the above artificial insemination steps, the experimental data in Table (2) were obtained. According to the results, when the semen was stored at low temperature for 2 hours, the fertilization ability of the diluted semen was slightly better than that of the fresh semen. The fertilization rate of the fresh semen of the blue-bodied large-spotted grouper decreased rapidly after 6 hours. The diluted semen still had a high fertilization rate and hatching rate of 70.03% and 64.78% respectively when stored at low temperature for 24 hours, which could still meet the actual production needs.

[0061] Table (2) Fertilization rate and hatching rate after artificial insemination at different time periods and dilution ratios

[0062]

[0063] Note: "-" indicates a fertilization rate or hatching rate of 0.

[0064] Example 2

[0065] Based on Example 1, the amounts of each raw material were adjusted. Preparation of low-temperature preservation diluent for blue-bodied large-spotted grouper semen:

[0066] Weigh 0.40g glucose, 0.05g calcium chloride, 1.5g potassium chloride, 3.7g sodium chloride, 1.54g sodium bicarbonate, 2.5g reduced glutathione, and 0.01g sodium dihydrogen phosphate using an electronic balance. Dissolve them in ultrapure water at room temperature and bring the volume to 0.9L to obtain a base solution, which is then stored at 4℃ for later use. Before use, remove fetal bovine serum from -20℃, dissolve it completely at room temperature, and transfer 0.1L to the 0.9L base solution. Mix thoroughly to obtain the cryopreservation solution. This cryopreservation solution meets the requirements for short-term preservation of semen from blue-bodied large-spotted grouper.

[0067] Example 3

[0068] Based on Example 1, the amounts of each raw material were adjusted. Preparation of low-temperature preservation diluent for blue-bodied large-spotted grouper semen:

[0069] Weigh 0.50g glucose, 0.04g calcium chloride, 2.5g potassium chloride, 3.4g sodium chloride, 1.75g ​​sodium bicarbonate, 1.5g reduced glutathione, and 0.01g sodium dihydrogen phosphate using an electronic balance. Dissolve them in ultrapure water at room temperature and bring the volume to 0.9L to obtain a base solution, which is then stored at 4℃ for later use. Before use, remove fetal bovine serum from -20℃, completely dissolve it at room temperature, and then transfer 0.1L to the 0.9L base solution. Mix thoroughly to obtain the cryopreservation solution. This cryopreservation solution meets the requirements for short-term preservation of semen from blue-bodied large-spotted grouper.

[0070] Example 4

[0071] Based on Example 1, the raw materials were adjusted and reduced to exclude calcium chloride, potassium chloride, sodium chloride, and sodium bicarbonate. The preparation of the low-temperature preservation diluent for blue-bodied large-spotted grouper semen was as follows: 0.45g of glucose, 2g of reduced glutathione, and 0.01g of sodium dihydrogen phosphate were weighed using an electronic balance and dissolved in ultrapure water at room temperature. The volume was adjusted to 0.9L to obtain the base solution, which was stored at 4°C until use. Before use, fetal bovine serum was removed from -20°C, completely dissolved at room temperature, and 0.1L was transferred to the 0.9L base solution and mixed thoroughly to obtain the low-temperature preservation solution. Compared with Example 1, the preservation effect of this low-temperature preservation solution on blue-bodied large-spotted grouper semen was somewhat reduced, but it still met the requirements for short-term preservation of blue-bodied large-spotted grouper semen.

[0072] Comparative Example 1

[0073] Based on Example 1, reduced glutathione was replaced with soybean polypeptide (brand: Huayuanshengtai). Preparation of low-temperature preservation diluent for blue-bodied large-spotted grouper semen: Weigh 0.45g glucose, 0.05g calcium chloride, 2g potassium chloride, 3.5g sodium chloride, 1.69g sodium bicarbonate, 2g soybean polypeptide, and 0.01g sodium dihydrogen phosphate using an electronic balance. Dissolve in ultrapure water at room temperature and bring to a final volume of 0.9L to obtain the base solution. Store at 4℃ until use. Before use, remove fetal bovine serum from -20℃, completely dissolve at room temperature, and transfer 0.1L to the 0.9L base solution. Mix thoroughly to obtain the preservation solution.

[0074] Fresh semen (motility greater than 90%) was diluted with preservation solution at ratios of 1:9, 1:29, 1:49, 1:69, and 1:99 and then placed into 5 mL sterile EP tubes, designated as control groups 1-5. The total volume of semen after each dilution was 2.1 mL. Results showed that the sperm motility rate in all groups was below 20% after 24 hours of preservation. The preservation effect on blue-bodied grouper semen was significantly lower than that of the control group.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A diluent for cryopreservation of blue marlin (Makaira indica) spermatozoa, characterized by comprising, The diluent is prepared from the following raw materials: glucose, reduced glutathione, sodium dihydrogen phosphate, fetal bovine serum, calcium chloride, potassium chloride, sodium chloride, sodium bicarbonate and water; the fetal bovine serum is 10% of the volume percentage of the diluent; The diluent contains the following mass of raw materials per liter: glucose 0.45 g, calcium chloride 0.05 g, potassium chloride 2 g, sodium chloride 3.5 g, sodium bicarbonate 1.69 g, reduced glutathione 2 g, sodium dihydrogen phosphate 0.01 g; Or the diluent contains the following mass of raw materials per liter: glucose 0.40 g, calcium chloride 0.05 g, potassium chloride 1.5 g, sodium chloride 3.7 g, sodium bicarbonate 1.54 g, reduced glutathione 2.5 g, sodium dihydrogen phosphate 0.01 g; Or the diluent contains the following mass of raw materials per liter: glucose 0.50 g, calcium chloride 0.04 g, potassium chloride 2.5 g, sodium chloride 3.4 g, sodium bicarbonate 1.75 g, reduced glutathione 1.5 g, sodium dihydrogen phosphate 0.01 g.

2. The P. bluehead wrasse cryopreservation diluent of claim 1, wherein, The preparation method of the preservation diluent comprises the following steps: weighing glucose, calcium chloride, potassium chloride, sodium chloride, sodium bicarbonate, reduced glutathione and sodium dihydrogen phosphate respectively, dissolving in water to obtain a base solution; before use, take fetal bovine serum, thaw it, and then transfer it to the base solution to prepare a preservation diluent.

3. The method of claim 1, wherein the method is for the use of a cryopreservation diluent for blue-banded sea perch spermatozoa, characterized in that, The fresh sperm is mixed with the preservation diluent at a volume ratio of 1:1-99, and then stored at low temperature after mixing.

4. The method of claim 3, wherein the method is for the use of a cryopreservation diluent for blue-banded sea perch spermatozoa, characterized in that, The fresh sperm is mixed with the preservation diluent at a volume ratio of 1:49-69.

5. The method of claim 1, wherein the method is for the use of a cryopreservation diluent for blue-banded sea perch spermatozoa, characterized in that, The low-temperature storage temperature is 0-4°C.

6. The diluent of any one of claims 1-2 is used for preserving blue body large spotted grouper sperm.

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