Pufferfish sperm cryopreservation solution, and preparation method and application method thereof

Abstract

Anchovy sperm freezing preservation solution, and a preparation method and application method thereof, belong to the technical field of aquatic germplasm resource protection and breeding, and particularly relate to a method for collecting, preserving and recovering sperm of anchovy fish. The present application is to solve the problems of low efficiency, short preservation time and low sperm activity of the existing anchovy sperm collection, and the anchovy sperm freezing preservation solution is composed of methanol, lecithin, resveratrol and glucose, and is diluted with double distilled water to 1L. The application method is as follows: 1. sperm collection and quality evaluation; 2. preparation of anchovy sperm freezing preservation solution; 3. mixing the sperm with the pre-cooled 4 DEG C anchovy sperm freezing preservation solution, dispensing, and adopting a four-step method for programmed cooling; 4. oocyte collection; 5. sperm warming and activity detection; 6. artificial insemination and hatching. The sperm preservation time is prolonged from several hours to more than several years, and through rapid thawing, the sperm activity can reach more than 40%, and the fertilization rate can reach more than 57%.

Description

Technical Field

[0001] This invention belongs to the field of aquatic germplasm resource protection and breeding technology, specifically relating to a method for collecting, preserving and reconstituted semen from fish of the genus *Amanita*. Background Technology

[0002] The long-term preservation and efficient restoration of semen germplasm resources are among the core technologies for aquaculture, genetic breeding, and the protection of endangered fish species. Establishing reliable sperm cryopreservation banks can provide a crucial material foundation and technical guarantee for the cross-temporal and spatial utilization of superior parent lines, the long-term preservation of genetic diversity, and the restoration and reconstruction of endangered populations.

[0003] Thimallus are typical cold-water stream fish, mainly distributed in the Heilongjiang and Yalu River basins in Northeast China and the Ertis River basin in Northwest China, possessing significant economic and ecological value. Due to overfishing and habitat change, their natural resources are severely threatened, making them endangered species. For example, the short-headed thrush (T. brevicephalus) (formerly known as the Arctic thrush, T. arcticus) and the lower Heilongjiang thrush (T. tugarinae) have been listed as Class II protected wild animals in China. To effectively protect the germplasm resources of thrushes and maintain their genetic diversity, establishing an efficient system for semen collection, preservation, and reconstitution is crucial.

[0004] Traditional methods for preserving fennel semen primarily involve short-term cryopreservation at 4°C. However, tests have shown that fennel semen can only be preserved at 4°C for about 6 hours before losing its rapid motility, with an activation rate below 10%, rendering it virtually ineffective for fertilization. Currently, there is no systematic technology for the long-term preservation and reconstitution of fennel semen. While cryopreservation technology for fish semen has been applied to some commercially important fish species such as salmon and cyprinids, directly applying existing methods for model fish to fennel presents significant technical incompatibility issues. This is mainly because the physiological characteristics of fennel sperm differ from those of other fish. Existing general-purpose or other-fish-specific cryoprotectant compositions, pH values, and types / concentrations of cryoprotectants are insufficient to meet the specific protective requirements of fennel sperm during cryopreservation. After thawing, sperm often exhibit low motility and a short effective motility time, failing to meet the practical operational window requirements for large-scale artificial insemination, severely hindering the practical application of cryopreserved semen.

[0005] Therefore, current technology lacks a standardized, efficient, and stable technical system specifically for grayling, encompassing the entire chain from semen collection to cryopreservation and thawing. This makes it difficult to achieve long-term safe preservation of grayling's superior germplasm resources, limiting its application in artificial breeding, genetic improvement, and the revitalization of endangered populations. Summary of the Invention

[0006] The purpose of this invention is to solve the technical problems of low collection efficiency, short preservation time and low reconstituted sperm motility of existing anchovy semen, and to provide an anchovy semen cryopreservation solution, its preparation method and application method.

[0007] The cryopreservation solution for fennel semen consisted of 80 mL methanol, 8 g lecithin, 5 mg resveratrol, and 27 g glucose, and was diluted to 1 L with double-distilled water.

[0008] The anchovies mentioned include Heilongjiang anchovies (T. grubii), downstream Heilongjiang anchovies, and short-headed anchovies.

[0009] The preparation method of the anchovy semen cryopreservation solution is as follows: Add 600 mL of double-distilled water to a clean beaker, add 80 mL of methanol and mix well, then dissolve 27 g of glucose, then add 5 mg of resveratrol and stir thoroughly to dissolve, then add 8 g of lecithin and stir to form a uniform emulsion, and finally transfer to a volumetric flask and make up to 1 L with double-distilled water.

[0010] The application method of the anchovy semen cryopreservation solution is as follows:

[0011] I. Semen Collection and Quality Assessment:

[0012] Select robust and mature male fennel fish aged 2 to 4 years, collect semen, and select those with a dark white color and no pollution for microscopic examination to assess sperm motility.

[0013] II. Preparation of cryopreservation solution for anise semen: Add 600 mL of double-distilled water to a clean beaker, add 80 mL of methanol and mix well, then dissolve 27 g of glucose, then add 5 mg of resveratrol and stir thoroughly to dissolve, then add 8 g of lecithin and stir to form a uniform emulsion, finally transfer to a volumetric flask and make up to 1 L with double-distilled water, then store temporarily in a 4℃ refrigerator.

[0014] 3. Mix the semen with pre-cooled (4°C) anchovy semen cryopreservation solution at a volume ratio of 1:5, dispense into 250 μL frozen wheat tubes and seal quickly, then perform programmed cooling using a four-step method:

[0015] Step 1: Equilibrate on ice for 15 minutes; Step 2: Equilibrate 6 cm above liquid nitrogen for 10 minutes; Step 3: Float on liquid nitrogen for 5 minutes; Step 4: Immerse in liquid nitrogen for long-term preservation.

[0016] IV. Egg Collection

[0017] The following breeding season, select healthy, mature female fennel fish aged 3 to 4 years and collect their eggs;

[0018] V. Semen rewarming and motility testing

[0019] Remove the straw from liquid nitrogen and immediately place it in a 37°C water bath for rapid thawing for 10 seconds until the semen is just completely thawed. After thawing, transfer the semen to a 4°C refrigerator for storage and then use a microscope to detect sperm motility.

[0020] VI. Artificial insemination and incubation

[0021] Dry insemination is used. The thawed semen and eggs are gently mixed evenly, and water is added to activate the sperm. After gently stirring for 10 seconds, the mixture is left to stand in the dark for 5 minutes to complete the fertilization process. The fertilized eggs are then transferred to an incubator and left to incubate in the dark. After an effective accumulated temperature of 120℃•d, the fertilized eggs will develop into eyed eggs. At this time, the fertilized eggs need to be removed from the incubator, and dead eggs are picked out. The eyed eggs are then placed in a parallel tank for centralized hatching. After an effective accumulated temperature of 40℃•d, the recovered fry can be obtained.

[0022] The present invention aims to overcome the defects and deficiencies of the prior art and provide a complete method from collection, preservation to restoration, so as to achieve the goal of long-term preservation and high-activity restoration of fennel semen germplasm resources, thereby laying a reliable technical foundation for the protection and sustainable utilization of fennel germplasm resources.

[0023] The beneficial effects of this invention are:

[0024] 1. Significantly extended preservation time: Through optimized cryopreservation solution and cooling procedures, the semen preservation time has been extended from several hours to several years or more, realizing the long-term preservation of germplasm resources.

[0025] 2. High recovery activity: Through rapid thawing, sperm motility can reach over 40%, and fertilization rate can reach over 57%, ensuring the efficiency of semen recovery.

[0026] 3. Simple composition and easy operation: The raw materials used in this invention are simple and readily available, and a standardized preparation method is provided, which improves the scientific nature and reproducibility of the management of anchovy semen germplasm resources. Attached Figure Description

[0027] Figure 1 This is a flowchart of the application method of the anchovy semen cryopreservation solution in this invention;

[0028] Figure 2 This is a graph showing the results of fertilized egg detection in Experiment 1;

[0029] Figure 3 This is a graph showing the results of the unfertilized egg test in Experiment 1;

[0030] Figure 4 This is a diagram of the eyed egg from Experiment 1;

[0031] Figure 5This is a picture of newly hatched fry restored from Experiment 1. Detailed Implementation

[0032] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0033] Specific Implementation Method 1: The composition of the cryopreservation solution for anchovy semen in this implementation method is 80 mL methanol, 8 g lecithin, 5 mg resveratrol, and 27 g glucose, and is adjusted to 1 L with double-distilled water.

[0034] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the anchovies mentioned include Heilongjiang anchovies (T. grubii), downstream Heilongjiang anchovies, and short-headed anchovies. Everything else is the same as in Specific Implementation Method One.

[0035] Specific Implementation Method 3: The preparation method of the anchovy semen cryopreservation solution described in this implementation method and Specific Implementation Method 1 or 2 is as follows: Add 600 mL of double-distilled water to a clean beaker, add 80 mL of methanol and mix well, then dissolve 27 g of glucose, then add 5 mg of resveratrol and stir thoroughly to dissolve, then add 8 g of lecithin and stir to form a uniform emulsion, and finally transfer to a volumetric flask and make up to 1 L with double-distilled water.

[0036] Specific Implementation Method Four: The application method of the anchovy semen cryopreservation solution described in this implementation method and in one of the specific implementation methods one to three is as follows:

[0037] I. Semen Collection and Quality Assessment:

[0038] Select robust and mature male fennel fish aged 2 to 4 years, collect semen, and select those with a dark white color and no pollution for microscopic examination to assess sperm motility.

[0039] II. Preparation of cryopreservation solution for anise semen: Add 600 mL of double-distilled water to a clean beaker, add 80 mL of methanol and mix well, then dissolve 27 g of glucose, then add 5 mg of resveratrol and stir thoroughly to dissolve, then add 8 g of lecithin and stir to form a uniform emulsion, finally transfer to a volumetric flask and make up to 1 L with double-distilled water, then store temporarily in a 4℃ refrigerator.

[0040] 3. Mix the semen with pre-cooled (4°C) anchovy semen cryopreservation solution at a volume ratio of 1:5, dispense into 250 μL frozen wheat tubes and seal quickly, then perform programmed cooling using a four-step method:

[0041] Step 1: Equilibrate on ice for 15 minutes; Step 2: Equilibrate 6 cm above liquid nitrogen for 10 minutes; Step 3: Float on liquid nitrogen for 5 minutes; Step 4: Immerse in liquid nitrogen for long-term preservation.

[0042] IV. Egg Collection

[0043] The following breeding season, select healthy, mature female fennel fish aged 3 to 4 years and collect their eggs;

[0044] V. Semen rewarming and motility testing

[0045] Remove the straw from liquid nitrogen and immediately place it in a 37°C water bath for rapid thawing for 10 seconds until the semen is just completely thawed. After thawing, transfer the semen to a 4°C refrigerator for storage and then use a microscope to detect sperm motility.

[0046] VI. Artificial insemination and incubation

[0047] Dry insemination is used. The thawed semen and eggs are gently mixed evenly, and water is added to activate the sperm. After gently stirring for 10 seconds, the mixture is left to stand in the dark for 5 minutes to complete the fertilization process. The fertilized eggs are then transferred to an incubator and left to incubate in the dark. After an effective accumulated temperature of 120℃•d, the fertilized eggs will develop into eyed eggs. At this time, the fertilized eggs need to be removed from the incubator, and dead eggs are picked out. The eyed eggs are then placed in a parallel tank for centralized hatching. After an effective accumulated temperature of 40℃•d, the recovered fry can be obtained.

[0048] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that step one involves selecting healthy, mature male graylings aged 3 years. Everything else is the same as in Specific Implementation Methods One to Four.

[0049] The following experiments were used to verify the effectiveness of the invention:

[0050] Experiment 1:

[0051] Application methods of fennel semen cryopreservation solution:

[0052] Collection, preservation and restoration of fennel semen

[0053] 1. Semen collection and quality assessment

[0054] In the first spring, after the water temperature rose above 10℃, 10 healthy, uninjured male Heilongjiang graylings, 10 downstream Heilongjiang graylings, and 10 short-headed graylings aged 2 to 4 years were selected. After anesthetizing with a 500 mg / L 2-phenoxyethanol solution, estrus-inducing hormones were injected into the abdominal cavity from the base of the pectoral fin. The hormone combination was human chorionic gonadotropin (HCG) + luteinizing hormone-releasing hormone A2 (LRH-A2), at a dose of 800 IU / kg HCG + 5 μg / kg LRH-A2. Five days later, sampling examination revealed abundant, bright white semen in the male fish. After anesthetizing with 2-phenoxyethanol again, the fish were dried with a towel. One person gently pressed the abdomen to squeeze out the semen, while another person used a 5 mL syringe to extract the semen from the cloaca.

[0055] After collecting all the semen and numbering it, place the syringe containing the semen in a sterile container pre-cooled at 4°C. Select semen that is thick, white, and free from contamination, and immediately evaluate the semen quality according to the following method: Drop 1 μL of semen onto a glass slide, add 50 μL of clear water, mix quickly, immediately cover with a coverslip, and perform microscopic examination. Sperm motility ≥ 90%, rapid movement time ≥ 45 s, and total lifespan ≥ 80 s are considered qualified. Avoid direct sunlight throughout the operation.

[0056] 2. Preparation of cryopreservation solution and cryopreservation of semen

[0057] First, add 600 mL of double-distilled water to a clean beaker, add 80 mL of methanol, mix well, then dissolve 27 g of glucose, then add 5 mg of resveratrol and stir until fully dissolved, then add 8 g of lecithin and stir to form a uniform emulsion. Finally, transfer it to a volumetric flask, make up to 1 L with double-distilled water, and pre-cool it in a 4°C refrigerator.

[0058] Mix the semen with the cryopreservation solution pre-cooled at 4°C in a volume ratio of 1:5, fill it into 250 μL cryotubes, and quickly seal them. Use the four-step method for programmed cooling: First step: Equilibrate on ice for 15 min; second step: Equilibrate 6 cm above liquid nitrogen for 10 min; third step: Float on liquid nitrogen for 5 min; fourth step: Immerse in liquid nitrogen for long-term storage.

[0059] 3. Semen recovery

[0060] 3.1 Inducing spawning of female fish and collecting eggs

[0061] In the second breeding season, select 10 healthy and mature female Amur graylings, downstream Amur graylings, and blunthead graylings aged 3 - 4 years each. Inject the ovulation-inducing hormone intraperitoneally. The drugs are domperidone (DOM) + human chorionic gonadotropin (HCG) + luteinizing hormone-releasing hormone A2 (LRH-A2), with a dosage of 2 mg / kg DOM + 1200 IU / kg HCG + 10 μg / kg LRH-A2. To make the female fish ovulate集中, adjust the injection amount appropriately according to the maturity of the broodstock during injection, and stop feeding after injection. 5 days later, after sampling inspection, it is found that all female fish can lay eggs. Then, anesthetize the female fish deeply with a 500 mg / L 2-phenoxyethanol solution, dry the body surface moisture with a dry towel, and one person gently presses the abdomen to squeeze out the eggs, while another person catches the eggs with a clean and dry small basin.

[0062] 3.2 Semen rewarming and artificial insemination

[0063] After removing the straws from liquid nitrogen and identifying the semen type and number, they were immediately placed in a 37°C water bath for rapid thawing until just completely thawed. The thawed semen was then temporarily stored in a 4°C freezer. Microscopic examination revealed that after one year of cryopreservation, the sperm motility of the fennel was consistently above 40%. Specifically, the sperm motility of Heilongjiang fennel ranged from 41.23% to 52.34%, that of downstream Heilongjiang fennel ranged from 40.59% to 54.82%, and that of short-headed fennel ranged from 45.28% to 57.61%.

[0064] Table 1. Comparison of sperm motility before and after freezing (%)

[0065]

[0066] Dry insemination was used. Thawed semen of the appropriate species was gently mixed with the eggs, and then a suitable amount of oxygenated and temperature-balanced water was added to activate the sperm. After gently stirring for 10 seconds, the mixture was left to stand in the dark for 5 minutes to complete the fertilization process. Finally, excess semen was washed away with clean water, and the fertilized eggs were left to stand in the dark for 30 minutes to allow them to absorb water and swell. The water was changed 2-3 times during this period to prevent oxygen deprivation.

[0067] 3.3 Hatching of fertilized eggs and detection of fertilization rate

[0068] The fully expanded fertilized eggs were transferred to a vertical incubator and placed in the dark for static incubation. The incubation water source was spring water, the temperature was 7℃~12℃, the dissolved oxygen was greater than 8mg / L, and the water flow was controlled to avoid disturbing the fertilized eggs. During incubation, the fertilized eggs were disinfected daily with a 300 mg / L formaldehyde solution for 30 min using a running water disinfection method. When the accumulated temperature of the fertilized eggs reached 90℃•d, the fertilization rate was tested using a fertilization egg identification solution (7g / L NaCl + 50 mL / L CH3COOH). It was found that the fertilization rate of the fennel was higher than 57%. Figure 2 , Figure 3 The fertilization rates of Heilongjiang anchovies were 60.27%–68.42%, those of downstream Heilongjiang anchovies were 57.81%–75.22%, and those of short-headed anchovies were 61.46%–65.88%.

[0069] Table 2 Comparison of fertilization rates before and after freezing (%)

[0070]

[0071] After 120℃•d, the fertilized eggs were removed from the incubator, and dead eggs were manually picked out before the eyed eggs were removed. Figure 3 They are placed in parallel tanks for centralized membrane breaking. After about 40℃•d, the fry that have been rehydrated from frozen sperm break through the membrane and hatch.

Claims

1. A cryopreservation solution for anchovy semen, characterized in that... The cryopreservation solution for fennel semen consisted of 80 mL methanol, 8 g lecithin, 5 mg resveratrol, and 27 g glucose, and was diluted to 1 L with double-distilled water.

2. The anchovy semen cryopreservation solution according to claim 1, characterized in that... The anchovies mentioned include Heilongjiang anchovies, downstream Heilongjiang anchovies, and short-headed anchovies.

3. The method for preparing the anchovy semen cryopreservation solution according to claim 1, characterized in that... The preparation method of the anchovy semen cryopreservation solution is as follows: Add 600 mL of double-distilled water to a clean beaker, add 80 mL of methanol and mix well, then dissolve 27 g of glucose, then add 5 mg of resveratrol and stir thoroughly to dissolve, then add 8 g of lecithin and stir to form a uniform emulsion, and finally transfer to a volumetric flask and make up to 1 L with double-distilled water.

4. The method of applying the anchovy semen cryopreservation solution according to claim 1, characterized in that... The application method of the anchovy semen cryopreservation solution is as follows: I. Semen Collection and Quality Assessment: Select robust and mature male fennel fish aged 2 to 4 years, collect semen, and select those with a dark white color and no pollution for microscopic examination to assess sperm motility. II. Preparation of cryopreservation solution for anise semen: Add 600 mL of double-distilled water to a clean beaker, add 80 mL of methanol and mix well, then dissolve 27 g of glucose, then add 5 mg of resveratrol and stir thoroughly to dissolve, then add 8 g of lecithin and stir to form a uniform emulsion, finally transfer to a volumetric flask and make up to 1 L with double-distilled water, then store temporarily in a 4℃ refrigerator.

3. Mix the semen with pre-cooled (4°C) anchovy semen cryopreservation solution at a volume ratio of 1:5, dispense into 250 μL frozen wheat tubes and seal quickly, then perform programmed cooling using a four-step method: Step 1: Equilibrate on ice for 15 minutes; Step 2: Equilibrate 6 cm above liquid nitrogen for 10 minutes; Step 3: Float on liquid nitrogen for 5 minutes; Step 4: Immerse in liquid nitrogen for long-term preservation. IV. Egg Collection The following breeding season, select healthy, mature female fennel fish aged 3 to 4 years and collect their eggs; V. Semen rewarming and motility testing Remove the straw from liquid nitrogen and immediately place it in a 37°C water bath for rapid thawing for 10 seconds until the semen is just completely thawed. After thawing, transfer the semen to a 4°C refrigerator for storage and then use a microscope to detect sperm motility. VI. Artificial insemination and incubation Dry insemination is used. The thawed semen and eggs are gently mixed evenly, and water is added to activate the sperm. After gently stirring for 10 seconds, the mixture is left to stand in the dark for 5 minutes to complete the fertilization process. The fertilized eggs are then transferred to an incubator and left to incubate in the dark. After an effective accumulated temperature of 120℃•d, the fertilized eggs will develop into eyed eggs. At this time, the fertilized eggs need to be removed from the incubator, and dead eggs are picked out. The eyed eggs are then placed in a parallel tank for centralized hatching. After an effective accumulated temperature of 40℃•d, the recovered fry can be obtained.

5. The method of applying the anchovy semen cryopreservation solution according to claim 4, characterized in that... Step 1: Select a healthy, mature male fennel fish that is 3 years old.

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