A cryopreservation solution for improving sperm survival rate in Penaeus monodon and its cryopreservation method

Abstract

This patent discloses a cryopreservation solution and method for improving the sperm survival rate of Penaeus monodon, comprising a base solution and combined cryoprotectants. The base solution is a Penaeus monodon sperm cell culture medium containing Penaeus monodon serum; the combined cryoprotectants include permeable and non-permeable cryoprotectants. By using Penaeus monodon sperm cell culture medium instead of traditional calcium-free saline as the base solution for cryopreservation, an optimized environmental basis is established for cryopreservation, significantly improving cryoprotection efficiency. The cryopreservation method provided in this application enables Penaeus monodon sperm to achieve a survival rate of over 89% after 10 months of cryopreservation, achieving long-term stable preservation of sperm under ultra-low temperature conditions; its key biological activity indicators show no significant difference compared to before cryopreservation, successfully achieving artificial insemination. This provides key technical support for the long-term preservation of Penaeus monodon germplasm resources and lays the foundation for the construction of germplasm banks.

Description

Technical Field

[0001] This invention belongs to the field of shrimp sperm cryopreservation technology, specifically relating to a cryopreservation solution and method for improving the sperm survival rate of Penaeus monodon. Background Technology

[0002] The tiger prawn (Penaeus monodon), also known as the grass prawn or tiger shrimp, belongs to the phylum Arthropoda, class Malacostraca, order Decapoda, family Penaeidae, and genus Penaeus. It is one of the world's three major farmed shrimp species. It is a large shrimp, with adults reaching up to 33 cm in length. Its carapace is thick and has prominent horizontal stripes. Its body color is dark green or blackish-green with dark brown to black horizontal bands. The shrimp meat is rich in inosinic acid, a flavorful compound, and has a high meat yield, making it extremely valuable economically.

[0003] However, shrimp farming is significantly affected by seasonal and climate changes, which limits shrimp production. Therefore, finding ways to overcome seasonal limitations, improve reproductive efficiency, and preserve superior genetic resources has become particularly important. Ultra-low temperature cryopreservation technology for shrimp sperm can be a key solution to these problems, helping to protect superior germplasm resources, improve reproductive efficiency, and reduce production costs.

[0004] Currently, the cryopreservation technology for sperm (-196℃ liquid nitrogen preservation) in marine animal germplasm preservation research mainly focuses on a few species of marine organisms, including fish and shellfish. Differences in sperm size and structure, as well as seminal plasma ion concentration and osmotic pressure, among different species result in their own adaptive optimal cryopreservation solution ratios, optimal freezing and cooling rates, and optimal activation methods. Therefore, sperm cryopreservation techniques are not universally applicable across different species. For example, patent document CN113455497A discloses a cryopreservation method for sperm from *Litopenaeus vannamei*, achieving a sperm survival rate of over 85%. However, *Litopenaeus vannamei* belongs to the genus *Litopenaeus* of the family Penaeidae, a different genus from *Penaeus monodon*, exhibiting significant species differentiation and genetic differences. Therefore, this method is not applicable to *Penaeus monodon*.

[0005] Furthermore, although research on cryopreservation of Penaeus monodon sperm has been conducted for decades, it still faces a dual bottleneck: firstly, the existing cryoprotectants are not effective enough, resulting in generally low sperm survival rates after thawing, which severely limits the application of artificial insemination; secondly, the embryo hatching rate after artificial insemination with frozen-thawed sperm is extremely low, making it impossible to produce effective reproductive output. Meanwhile, calcium-free saline, as the mainstream base solution, is widely used for diluting sperm cryoprotectants, but it performs poorly in the cryopreservation of Penaeus monodon sperm, specifically exhibiting low sperm survival rates.

[0006] Therefore, further improving the sperm survival rate of frozen Penaeus monodon sperm, achieving long-term stable preservation of Penaeus monodon sperm under ultra-low temperature conditions, ensuring that key biological activity indicators (such as enzyme activity and DNA integrity) are not significantly different from those before freezing, and successfully achieving artificial insemination with frozen and thawed sperm are of great value and significance for Penaeus monodon aquaculture. This can provide key technical support for the long-term preservation of Penaeus monodon germplasm resources and lay the foundation for the construction of germplasm banks. Summary of the Invention

[0007] The purpose of this invention is to further improve the sperm survival rate of Penaeus monodon after freezing, so as to achieve long-term stable preservation of Penaeus monodon sperm under ultra-low temperature conditions and successfully realize artificial insemination, thereby providing key technical support for the long-term preservation of Penaeus monodon germplasm resources and laying the foundation for the construction of germplasm banks.

[0008] To solve the above-mentioned technical problems, this application provides the following technical solution:

[0009] A cryopreservation solution for cryopreserving Penaeus monodon sperm includes a base solution and a combined cryoprotectant. The base solution is a Penaeus monodon sperm cell culture medium containing Penaeus monodon serum. The combined cryoprotectant includes a permeable cryoprotectant and a non-permeable cryoprotectant.

[0010] Furthermore, the concentration of Penaeus monodon serum in the Penaeus sperm cell culture medium is 3-5%, 5-10%, 10-15%, or 15-20%.

[0011] Furthermore, the permeability protectant includes one or more combinations of dimethyl sulfoxide, propylene glycol, glycerol, methanol, and ethylene glycol.

[0012] Furthermore, non-permeable protective agents include one or more combinations of polyethylene glycol, bovine serum albumin, glucose, mannitol, sucrose, fetal bovine serum, and tiger prawn serum.

[0013] Furthermore, the amount of base solution added is 93.0~95.0%, 95.0~97.0%, 97.0~99.0%, or 99.0~99.9% of the frozen storage liquid volume ratio; the amount of tiger prawn serum added is 1~3%, 3~5%, or 5~15% of the base solution volume ratio.

[0014] Furthermore, the amount of permeability protectant added is 0.1~0.3%, 0.3~0.5%, 0.5~1.0%, 1.0~3.0% or 3.0~7.0% of the volume ratio of the cryopreservation liquid.

[0015] Furthermore, the amount of non-permeable preservative added is 0.1~1.5%, 1.5~4.0%, 4.0~6.0%, 6.0~8.0% or 8.0~10.0% of the volume ratio of the cryopreservation liquid.

[0016] Another aspect of the present invention provides a cryopreservation solution for cryopreservation of Penaeus monodon sperm for the application of improving the survival rate of cryopreserved Penaeus monodon sperm or in breeding.

[0017] Another aspect of the present invention provides a Penaeus monodon sperm cryopreservation kit, comprising the cryopreservation solution for Penaeus monodon sperm cryopreservation described above.

[0018] Another aspect of the present invention provides a method for cryopreservation to improve the sperm survival rate of Penaeus monodon, comprising the following steps:

[0019] Step A: Obtain discrete sperm or spermatophores from male tiger prawns, and pre-treat the discrete sperm or spermatophores to obtain pre-treated discrete sperm or pre-treated spermatophores.

[0020] Step B: Place the pretreated discrete sperm or pretreated spermatophores into the cryopreservation solution for frozen Penaeus monodon sperm to obtain a sperm suspension;

[0021] Step C: The sperm suspension is initially frozen and then placed in liquid nitrogen for cryopreservation at ultra-low temperatures.

[0022] Further, step A specifically involves: disinfecting and anesthetizing the male tiger prawns, dissecting the dorsal shell under aseptic conditions to remove the vas deferens, placing the vas deferens in a shrimp sperm cell culture medium, mechanically breaking the vas deferens to obtain a sperm mixture, centrifuging and filtering to obtain a sperm precipitate, and resuspending the sperm precipitate in the aforementioned shrimp sperm cell culture medium to obtain pretreated discrete sperm.

[0023] If chosen, step A specifically involves using an electroporation method to extract the spermatophores from male tiger prawns, placing the spermatophores in PBS buffer for preservation, and obtaining pretreated spermatophores.

[0024] Furthermore, in step B, the sperm density in the sperm suspension is 10. 6 ~10 8 per mL.

[0025] Furthermore, step C specifically involves:

[0026] Step C-1: Place the sperm suspension in a cryovial in a programmed freezing box, and place the programmed freezing box in a -80 ℃ freezer overnight for initial freezing; the initial freezing cooling rate is 1 ℃ per minute.

[0027] Step C-2: Place the cryovial containing tiger prawn sperm after the initial freezing in step C-1 into liquid nitrogen for cryopreservation at -196°C for at least 7 days.

[0028] Furthermore, the long-term cryopreservation time for Penaeus monodon sperm is at least 3 months, 6 months or 10 months, and the sperm revival survival rate after 10 months of cryopreservation is ≥89%.

[0029] Furthermore, the fertilization rate of artificial insemination using spermatophores of Penaeus monodon frozen for 10 months is ≥20%, and the hatching rate of hatching after artificial insemination using spermatophores of Penaeus monodon frozen for 10 months is ≥5.5%.

[0030] Another aspect of this patent provides the application of Penaeus monodon sperm or Penaeus monodon spermatophores preserved using the above-mentioned cryopreservation method for improving the sperm survival rate in breeding.

[0031] This invention utilizes shrimp sperm cell culture medium instead of traditional calcium-free saline as the base solution for cryopreservation, establishing an optimized environmental foundation for cryopreservation. It also systematically screens the optimal concentration ratio and synergistic combination of permeable and non-permeable cryoprotectants, significantly improving cryopreservation efficiency. Using the cryopreservation method provided in this application, the survival rate of frozen-thawed sperm from *Litopenaeus monodon* stored for 10 months is greater than 89%, while achieving long-term stable preservation of sperm under ultra-low temperature conditions. Key biological activity indicators (such as enzyme activity and DNA integrity) show no significant difference compared to before cryopreservation, and artificial insemination with the frozen-thawed sperm is successfully achieved. This not only demonstrates the high efficiency of this method in maintaining sperm activity and function but also establishes a stable and reproducible complete technical system, providing key technical support for the long-term preservation of *Litopenaeus monodon* germplasm resources and laying the foundation for germplasm bank construction. Attached Figure Description

[0032] The above description of the present invention and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0033] Figure 1 The above are the screening and comparison results of shrimp cell culture media for in vitro culture of Penaeus monodon sperm cells in the examples (where A1~A4 are without SP; B1~B4 are with 1% SP; C1-C5 are with 3% SP; D1-D4 are with 5% SP; E1-E4 are with 10% SP; F1-F4 are with 15% SP, and the scale bar is 100 μm).

[0034] Figure 2The following is a comparison of the cryopreservation results of Penaeus monodon sperm cells in artificial seawater and shrimp culture medium in the examples (where A is a comparison of the preservation effects of Penaeus monodon sperm cells in artificial seawater and cell culture medium, scale bar 50 μm; B is the recovery effect of Penaeus monodon sperm cells after cryopreservation in artificial seawater and culture medium for 1 week, 400× is the result at 400x magnification, scale bar 20 μm; 1000× is the result at 1000x magnification, scale bar 5 μm; C is the statistical result of the integrity of the recovered sperm cell membrane in B, different superscript letters in Figure C indicate significant differences between data, P < 0.05).

[0035] Figure 3 The images show the microscopic observation results of the effects of cryopreservation solutions containing different osmotic protectants on ultra-low temperature cryopreserved sperm cells of Penaeus monodon in the examples (where 400× is the result at 400x magnification, scale bar is 25 μm; 1000× is the result at 1000x magnification, scale bar is 10 μm; A is fresh sperm cells, and B~F are the results of thawing after 7 days of liquid nitrogen cryopreservation in cryopreservation solutions containing PAⅠ, PBⅢ, PCⅠ, PDⅠ and PEⅡ, respectively).

[0036] Figure 4 for Figure 3 Survival rate of sperm cells from *Litopenaeus chinensis* cryopreserved in liquid nitrogen for 7 days after thawing (where a, a', a'' represent the difference between the intra-group level and the baseline, and A, A', A'' represent the difference between the groups, P < 0.05).

[0037] Figure 5 The following are microscopic observations of the effects of cryopreservation solutions containing different non-permeable cryoprotectants on ultra-low temperature cryopreserved sperm cells of Penaeus monodon (where 400× represents 400x magnification, scale bar 25 μm; 1000× represents 1000x magnification, scale bar 10 μm; A represents fresh sperm cells, and B~F represent the thawing results after 7 days of liquid nitrogen cryopreservation with cryopreservation solutions containing PFⅠ, PGⅠ, PHⅠ, PIⅢ and PJⅢ, respectively).

[0038] Figure 6 for Figure 5 Survival rate of sperm cells from *Litopenaeus chinensis* cryopreserved in liquid nitrogen for 7 days after thawing (where a, a', a'' represent the difference between the intra-group level and the baseline, and A, A', A'' represent the difference between the groups, P < 0.05).

[0039] Figure 7The following are microscopic observations of the effects of cryopreservation solutions containing the combined cryoprotectant on ultra-low temperature cryopreserved sperm cells of Penaeus monodon (where 400× represents 400x magnification, scale bar 20 μm; 1000× represents 1000x magnification, scale bar 5 μm; A represents fresh sperm cells, and B~R represent the thawing results after 7 days of cryopreservation in liquid nitrogen containing PKⅠ~PKXVI, respectively).

[0040] Figure 8 for Figure 7 Survival rate of sperm cells from Penaeus monodon cryopreserved under liquid nitrogen for 7 days after cryopreservation (where a and b represent differences between letter-labeled groups, P < 0.05).

[0041] Figure 9 The images shown are statistical microscopy observations of the liquid nitrogen cryopreservation results of Penaeus monodon sperm cells in the examples (where 400× represents 400x magnification, scale bar 20 μm; 1000× represents 1000x magnification, scale bar 5 μm; A~E are morphological images of Penaeus monodon sperm cells after 7 d, 30 d, 90 d, 180 d and 300 d of revival after liquid nitrogen cryopreservation).

[0042] Figure 10 for Figure 9 Survival rate of sperm cells from ultra-low temperature cryopreserved in liquid nitrogen after thawing at various stages (where x-axis is cryopreservation time, y-axis is survival rate, in %; a is significance, P>0.05).

[0043] Figure 11 The images shown are comparisons of scanning electron microscopy results of Penaeus monodon sperm cells before and after cryopreservation in the example (where A1 is before cryopreservation, A2~A4 are after cryopreservation, and the scale bar is 1.5 μm).

[0044] Figure 12 The images shown are transmission electron microscopy comparisons of sperm cells from Penaeus monodon before and after cryopreservation in the examples (where B1 is before cryopreservation, B2-B4 are after cryopreservation, scale bar is 2 μm; SP is the spine; AC is the acrosome; CP is the cytoplasmic band of the ring nucleus).

[0045] Figure 13 The image shows the comparison results of sperm cell genomic DNA integrity before and after cryopreservation in the example (where A is fresh sperm genomic DNA of Penaeus monodon, and B is sperm genomic DNA of Penaeus monodon cryopreserved at ultra-low temperature after one week of liquid nitrogen freezing).

[0046] Figure 14 This is a morphological comparison of the effects of cryopreservation of sperm cells from Penaeus monodon on embryonic development in the examples (scale bar: 125 μm).

[0047] Figure 15 This is a morphological comparison of the effects of cryopreservation of Penaeus monodon sperm cells on post-embryonic larval development in the examples (scale bar: 250 μm). Detailed Implementation

[0048] The following detailed description of the features and advantages of the present invention is sufficient to enable any person skilled in the art to understand the technical content of the present invention and implement it accordingly. Furthermore, based on the specification, claims and drawings disclosed herein, those skilled in the art can easily understand the related objects and advantages of the present invention.

[0049] It should be noted that in this specification, similar reference numerals and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0050] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0051] (1) Source of sample materials

[0052] The tiger prawns used in this embodiment were from two sources: first, male commercial tiger prawns (body length 15.48±1.26 cm, weight 34.95±1.87 g) purchased from the Yazhou Central Agricultural Trade Market in Hainan Province, used to obtain discrete tiger prawn sperm; second, broodstock (breeding shrimp) purchased from Hainan Zhongzheng Aquatic Technology Co., Ltd., of which female broodstock had a body length of 29.22±1.37 cm and a weight of 150.65±6.31 g; and male broodstock had a body length of 20.32±0.56 cm and a weight of 100.95±5.02 g, used to obtain tiger prawn spermatophores. The above-mentioned tiger prawns were temporarily held in artificial seawater, with the water quality adjusted to control the temperature at 28~29℃, salinity at 32~35‰, and oxygen content (OD) ≥6.0 mg / L.

[0053] (2) Sources of reagents and consumables

[0054] Ophthalmic scissors and forceps were purchased from Jiangxi Yuyuan Medical Equipment Co., Ltd.; sterilizable needle filters were purchased from Qingdao Shibei District Feimate Laboratory Supplies Store; potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, sodium hydroxide, manganese chloride, and glucose were purchased from Sinopharm Chemical Reagent Co., Ltd.; Leibovitz's L-15 was purchased from Gibco (Thermo Fisher Scientific); L-lysine, β-alanine, and γ-aminobutyric acid were purchased from Shanghai Maclean Biotechnology Co., Ltd.; taurine, L-proline, L-aspartic acid, L-glutamic acid, hydroxyproline, ornithine, cysteine, glutamine, trehalose, sucrose, ribose, arabinose, lactose, fucose, xylose, fructose, propylene glycol, glycerol, methanol, ethylene glycol, polyethylene glycol, and mannitol were purchased from Shanghai Aladdin Biotechnology Co., Ltd.; gentiobiose was purchased from Bailingwei Technology Co., Ltd.; dimethyl sulfoxide was purchased from Sigma-Aldrich; fetal bovine serum was purchased from BI; epidermal growth factor, basic fibroblast growth factor, and [other ingredients] were purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.; 50×TAE electrophoresis buffer, 100× penicillin-streptomycin antibiotic stock solution, and 0.4% trypan blue staining solution were purchased from Beijing Solarbio Science & Technology Co., Ltd.; apoptosis-DNA [other ingredients] were also purchased. The Ladder extraction kit, superoxide dismutase assay kit, lactate dehydrogenase assay kit, catalase assay kit, succinate dehydrogenase test kit, ultra-micro Na+ / K+–ATPase test kit, and protein quantification kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0055] (3) Source of instruments and equipment

[0056] Pipettes and refrigerated centrifuges were purchased from Eppendorf GmbH, Germany; vertical pressure steam sterilizers were purchased from Shanghai Shenan Medical Instrument Factory; clean benches were purchased from Suzhou Antai Air Technology Co., Ltd.; pH test strips were purchased from Shanghai Sanaisi Reagent Co., Ltd.; ultrapure water systems were purchased from Jinan Taipingma Equipment Co., Ltd.; electric thermostatic drying ovens were purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.; electronic balances were purchased from Ruderweireld GmbH, Poland; refrigerators were purchased from Qingdao Haier Co., Ltd.; thermostatic water baths were purchased from Jiangsu Shuangjie Experimental Instrument Factory; snowflake ice makers were purchased from Changshu Xueke Electric Appliance Co., Ltd.; and double-headed oxygen pumps were purchased from Guangdong Minjiang. The following equipment was purchased from various suppliers: Aquarium Industry Co., Ltd.; Freezing point osmometer from Shanghai Medical University Instrument Factory; JY-C electrophoresis apparatus and JY04S-3C gel imaging analysis system from Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.; Microplate reader from Thermo Fisher Scientific Co., Ltd.; Metal bath and mini low-speed centrifuge from Beijing Tiangen Biochemical Technology Co., Ltd.; Metal bath, mini low-speed centrifuge and hemocytometer from BioSharp; Adjustable DC regulated power supply from Maisheng Medical Equipment Co., Ltd.; Liquid nitrogen tank from Qingdao Haier Co., Ltd.; Fluorescence upright microscope from Leica GmbH, Germany; Cell filter (40 μm pore size, individually packaged in paper and plastic, sterile), microscope slides and microscope coverslips from Shanghai Beyotime Biotechnology Co., Ltd.; Disposable needle filter from Sartorius GmbH, Germany; Disposable syringe from Shaanxi Longkangxin Medical Device Co., Ltd.

[0057] (4) Preparation method of experimental reagents

[0058] 1) 2.5×PBS buffer: PBS refers to phosphate buffer saline (PBS). The preparation method is as follows: Weigh 10.0 g of sodium chloride, 0.25 g of potassium chloride, 3.75 g of disodium hydrogen phosphate and 0.25 g of potassium dihydrogen phosphate into volumetric flasks, dissolve them completely with ddH2O, and bring the volume up to 500 mL. Sterilize at 121℃ for 20 min. Then, in a clean bench, aseptically dispense the cooled PBS into 100 mL reagent bottles and store at 4℃ for later use.

[0059] 2) Aseptic preparation of serum from Penaeus monodon

[0060] Anesthetize the tiger prawns by placing them in crushed ice for about 10 minutes. Then, immerse the anesthetized prawns in 75% ethanol for 10 minutes for deep anesthesia and disinfection, and fix the prawns with alcohol swabs. Using a 2.5 mL syringe pre-filled with about 1 mL of 2.5×PBS, insert it into the area below the base of the 5th peroneal leg and above the 1st abdominal segment, and slowly aspirate the hemolymph. Mix the hemolymph with 2.5×PBS in a 1:1 ratio, invert and mix well. Centrifuge at 1000 ×g for 5 minutes at 4°C, and collect the supernatant as a crude mixture of serum and 2.5×PBS. Incubate the crude mixture in a 56°C water bath for 30 minutes, and centrifuge at 5000 ×g for 20 minutes at 4°C to remove the precipitate. The supernatant obtained is the prawn serum, which is aliquoted and stored at -20°C for later use.

[0061] 3) Preparation of 1% agarose gel

[0062] Weigh 0.2 g of agarose powder into a 50 mL Erlenmeyer flask, add 20 mL of 1×TAE solution, heat in a microwave oven until completely dissolved, pour into a mold, and use after cooling and solidification.

[0063] Example

[0064] A cryopreservation solution for improving sperm survival rate in Penaeus monodon and a method for cryopreservation thereof, comprising the following steps:

[0065] S1. Preparation of shrimp sperm cell culture medium, the specific steps are as follows:

[0066] Using artificial seawater as a control, and based on shrimp cell culture medium (refer to Zhao et al., 2023), the modified OSGM culture medium was obtained by adding 1%, 3%, 5%, 10% and 15% shrimp serum (hereinafter referred to as SP) to the shrimp cell culture medium.

[0067] The modified OSGM medium is prepared as follows: Based on 1.5×L⁻¹⁵ medium, add the following to a final concentration: 665 mg / L taurine, 1213 mg / L proline, 43 mg / L aspartic acid, 79 mg / L glutamic acid, 136 mg / L lysine, 50 mg / L hydroxyproline, 15 mg / L ornithine, 4 mg / L β-alanine, 3 mg / L γ-aminobutyric acid, 20 mg / L cysteine, 300 mg / L glutamine, 18 mg / L arabinose, 4 mg / L xylose, 28 mg / L ribose, 8 mg / L fucose, 2905 mg / L glucose, 4 mg / L fructose, 224 mg / L trehalose, 63-69 mg / L sucrose, 11 mg / L lactose, 81 mg / L gentiobiose, 1... The solution consists of g / L sodium bicarbonate, 3% shrimp serum (v / v), 15% fetal bovine serum (v / v), 20 μg / L epidermal growth factor, 20 μg / L basic fibroblast growth factor, and 10000 U / mL penicillin-streptomycin. The osmotic pressure is adjusted to 720±20 mOsm / kg using sodium chloride, and the pH is then adjusted to 7.2±0.2 using 1 M sodium hydroxide or 1 M hydrochloric acid.

[0068] In some other specific embodiments, the final concentrations of taurine, proline, aspartic acid, glutamic acid, lysine, hydroxyproline, ornithine, β-alanine, cysteine, arabinose, xylose, ribose, glucose, fructose, and trehalose can be adjusted as follows: taurine 665-829 mg / L; proline 1213-1518 mg / L; aspartic acid 43-54 mg / L; glutamic acid 79-98 mg / L; lysine 136-171 mg / L; hydroxyproline 50-63 mg / L; ornithine 15-18 mg / L; β-alanine 4-5 mg / L; cysteine ​​20-23 mg / L; arabinose 18-19 mg / L; xylose 4-6 mg / L; ribose 28-32 mg / L; glucose 2905-2936 mg / L; fructose 4-6 mg / L; and trehalose 224-234 mg / L. Adjusting the final concentration of sucrose to 63-69 mg / L, the final concentration of lactose to 11-18 mg / L, and the final concentration of gentiobiose to 81-88 mg / L can achieve the same technical effect as the modified OSGM medium.

[0069] S2. Screening and optimization of sperm cell culture medium for Penaeus monodon, the specific steps are as follows:

[0070] (1) Male shrimp (purchased from Yazhou Central Agricultural Market in Hainan Province) were temporarily kept in pre-prepared artificial seawater to adapt to the experimental environment.

[0071] (2) Take out the tiger prawns that have been temporarily held in step (1) above, and soak them in 75% ethanol for 2 min to disinfect and anesthetize them. Then place the disinfected tiger prawns in a clean bench, carefully cut open the dorsal shell of the prawns with dissecting scissors to expose the internal organs, identify the location of the vas deferens, and gently separate the vas deferens with forceps. Place the separated vas deferens in 2.5×PBS buffer to wash it to remove surface impurities, and then transfer the washed vas deferens to a centrifuge tube containing modified OSGM medium. Use scissors to mechanically break up the vas deferens to release the sperm and obtain a sperm mixture.

[0072] (3) Shrimp sperm cells were cultured in vitro for a long period using a modified OSGM medium to screen for suitable shrimp sperm cell culture media. The results are as follows: Figure 1 As shown, the modified OSGM medium containing 3% SP (hereinafter referred to as OSGM medium) exhibited the best preservation effect, maintaining the normal morphology of shrimp sperm cells for up to 30 days.

[0073] (4) Shrimp sperm cells were then preserved in vitro using sterile artificial seawater and OSGM culture medium. The results are as follows: Figure 2 As shown, shrimp sperm cells exhibited obvious aggregation and fragmentation after being stored in artificial seawater for one day; however, in OSGM medium, the morphological characteristics of Penaeus monodon sperm cells showed no significant changes. Figure 2 In section A), sperm cells were cryopreserved directly using artificial seawater and OSGM medium. After one week (7 days) of cryopreservation, the percentage of shrimp sperm cells with intact cell membranes in the OSGM medium group (4.763±1.16%) was significantly higher than that in the artificial seawater group (1.380±0.46%) after thawing. Figure 2 B and Figure 2 The results (C) indicate that SP plays an important role in the in vitro culture of shrimp sperm cells and provides crucial experimental evidence for subsequent cryopreservation research.

[0074] S3. Obtaining sperm from tiger prawns, the specific steps are as follows:

[0075] (1) The sperm mixture of tiger prawns was obtained in the same way as (1) and (2) in step S2 above. The obtained sperm mixture was then filtered in a cell filter (40 μm pore size, individually packaged in paper and plastic, sterile) to obtain the first sperm suspension. The sperm suspension was evenly distributed into centrifuge tubes and centrifuged at 800 × g for 8 min to remove the supernatant and retain the precipitate, which is the sperm precipitate.

[0076] (2) Add OSGM culture medium in equal multiples according to the volume of sperm precipitate obtained in step (1) above, and resuspend the sperm precipitate to obtain a second sperm suspension.

[0077] (3) The second sperm suspension was stained with a 0.4% trypan blue solution and the number of live sperm was examined under an optical microscope by blood cell counting. The results showed that the collected sperm were of good quality.

[0078] S4. Obtaining spermatophores from Penaeus monodon: The specific steps are as follows:

[0079] Select sexually mature male Penaeus monodon with a body length ≥19 cm and full spermatophores (purchased from Hainan Zhongzheng Aquatic Technology Co., Ltd., hereinafter referred to as male shrimp). Wrap the male shrimp in damp gauze and fix it ventrally upwards. Place the electrode head of an adjustable DC regulated power supply (double copper needles, 6 mm apart) vertically and firmly above and below the spermatophores. Select the electroshock parameters according to the shrimp size (000mA, 7.60 V, 2-3 shocks, each lasting 1-2 seconds). Within 2-10 seconds after electroshock, gently pinch the white, translucent, ribbon-like spermatophores exposed at the genital opening with pointed forceps and slowly pull them out; these are the Penaeus monodon spermatophores. Place the obtained Penaeus monodon spermatophores in centrifuge tubes containing sterile 2.5×PBS buffer and store at 4°C for a short period (≤48 hours). After the procedure, place the male shrimp in sterile artificial seawater with a salinity of 32-35‰ for recovery.

[0080] S5. Ultra-low temperature cryopreservation of Penaeus monodon sperm and Penaeus monodon spermatophores, the specific steps are as follows:

[0081] Using the shrimp cell culture medium (OSGM medium) containing 3% SP selected in step S1 as the basal medium, the optimal cryopreservation conditions (including cryoprotectants, freezing program, and thawing temperature, etc.) were screened using Penaeus monodon sperm (also known as discrete sperm cells). The optimal scheme was then applied to mature male Penaeus monodon spermatophores provided by Hainan Zhongzheng Aquatic Technology Co., Ltd., and these spermatophores were cryopreserved for subsequent artificial insemination.

[0082] 1. Screening and optimization of permeability protectants, the specific steps are as follows:

[0083] As shown in Table 1, ultra-low temperature cryoprotective solutions (hereinafter referred to as the first cryoprotective solution) were prepared using OSGM medium as the base solution and different concentrations of dimethyl sulfoxide (DMSO), propylene glycol, glycerol, methanol and ethylene glycol as cryoprotectants.

[0084] The sperm precipitate obtained in step S3 (1) was resuspended in the above cryopreservation solution to prepare a third sperm suspension. The third sperm suspension was evenly distributed into cryovials, sealed, and symmetrically placed in a programmed cryopreservation box (cooling rate -1℃ / min). The programmed cryopreservation box was then placed in a -80℃ freezer overnight for initial freezing. After 15 h, the cryovials were quickly transferred to liquid nitrogen (-196℃) for ultra-low temperature cryopreservation for 7 days. After 7 days of cryopreservation, the cryovials were removed and quickly placed in a 37℃ water bath for thawing. After thawing, 0.4% trypan blue solution was used for staining, and the thawing rate and cryopreservation quality were determined by blood cell counting. Finally, by comparing the thawing rate and cryopreservation quality of different permeability protectants, the permeability protectant with the best cryopreservation effect was selected.

[0085] Table 1 Screening scheme for first cryopreservation solution of Penaeus monodon sperm

[0086] ;

[0087] Shrimp sperm cells were thawed after being frozen in liquid nitrogen for 7 days. After staining with 0.4% trypan blue solution, the survival status of shrimp sperm cells was observed under a 10x eyepiece, a 40x objective lens, and a 100x oil immersion lens. The survival rate of shrimp sperm cells (hereinafter referred to as cell survival rate or survival rate) was calculated.

[0088] The results are as follows Figure 3 and Figure 4 As shown, cell cryopreservation solution PA was used. Ⅰ PA Ⅱ PA Ⅲ and PA Ⅳ The survival rates of shrimp sperm cells after resuscitation were 32.41±3.15%, 24.27±0.96%, 10.29±1.41%, and 3.23±1.67%, respectively; using PB cell cryopreservation medium... Ⅰ PB Ⅱ PB Ⅲ and PB Ⅳ The survival rates of shrimp sperm cells after resuscitation were 8.29±1.38%, 2.76±0.47%, 51.92±2.86%, and 23.35±1.86%, respectively; using PC cell cryopreservation medium... Ⅰ PC Ⅱ PC Ⅲ and PC Ⅳ The survival rates of shrimp sperm cells after resuscitation were 64.82±1.16%, 12.29±1.82%, 4.30±1.16%, and 4.61±1.22%, respectively; using PD cell cryopreservation medium... Ⅰ PD Ⅱ PD Ⅲ and PD ⅣThe survival rates of shrimp sperm cells after resuscitation were 22.43±2.20%, 19.66±1.16%, 14.59±0.71%, and 10.29±1.16%, respectively; using PE cell cryopreservation medium... Ⅰ PE Ⅱ PE Ⅲ and PE Ⅳ The survival rates of shrimp sperm cells after resuscitation were 11.37±1.66%, 19.82±0.93%, 2.77±0.80%, and 2.76±0.47%, respectively. The results indicate that the PC cell cryopreservation solution, composed of 5% glycerol and 95% OSGM medium (v / v), is effective. Ⅰ The effect is significantly higher than that of other cell cryopreservation solutions regulated by osmotic substances. Figure 3 (The red box in the middle).

[0089] 2. Screening and optimization of non-permeable protective agents, the specific steps are as follows:

[0090] As shown in Table 2, ultra-low temperature cryopreservation solutions (hereinafter referred to as second cryopreservation solutions) were prepared using OSGM medium as the base solution and different concentrations of polyethylene glycol, bovine serum albumin, fetal bovine serum, sugar mixture, and shrimp serum as cryoprotectants.

[0091] The sperm precipitate obtained in step S3 (1) was resuspended in the second cryopreservation solution to prepare a fourth sperm suspension. The fourth sperm suspension was evenly distributed into cryovials. After sealing the cryovials, they were symmetrically placed in a programmed cryopreservation box (-1 ℃ / min). The programmed cryopreservation box was then placed in a -80℃ freezer overnight for initial freezing. After 15 hours, the cryovials were quickly transferred to liquid nitrogen (-196℃) for ultra-low temperature cryopreservation for 7 days. After 7 days of cryopreservation, the cryovials were removed and quickly placed in a 37℃ water bath for thawing. After thawing, the cryovials were stained with 0.4% trypan blue solution, and the thawing rate and cryopreservation quality were determined by blood cell counting. Finally, by comparing the thawing rate and cryopreservation quality of different permeable cryopreservatives, the non-permeable cryopreservative with the best cryopreservation effect was selected.

[0092] Table 2 Screening scheme for second cryopreservation solution of Penaeus monodon sperm

[0093] ;

[0094] The results are as follows Figure 5 and Figure 6 As shown, shrimp sperm cells were thawed after being cryopreserved in liquid nitrogen for 7 days. Cell viability was observed under 10x eyepiece, 40x objective lens, and 100x oil immersion lens after staining with 0.4% trypan blue solution, and the cell viability was calculated. The cell cryopreservation solution PF was used. Ⅰ PF Ⅱ PFⅢ and PF Ⅳ The survival rates of shrimp sperm cells after resuscitation were 91.55±2.16%, 82.49±0.92%, 64.98±3.02%, and 58.83±1.94%, respectively; using PG cell cryopreservation medium... Ⅰ PG Ⅱ and PG Ⅲ The survival rates of shrimp sperm cells after resuscitation were 58.52±3.18%, 39.63±0.46%, and 23.81±1.62%, respectively; the cell cryopreservation solution pH was adjusted accordingly. Ⅰ pH Ⅱ and PH Ⅲ The survival rates of shrimp sperm cells after resuscitation were 72.04±1.74%, 63.90±0.96%, and 24.11±0.96%, respectively; using cell cryopreservation medium PI... Ⅰ PI Ⅱ and PI Ⅲ The survival rates of shrimp sperm cells after resuscitation were 2.61±0.96%, 2.76±1.39%, and 2.92±0.96%, respectively; using PJ cell cryopreservation medium... Ⅰ PJ Ⅱ PJ Ⅲ and PJ Ⅳ The survival rates of shrimp sperm cells after resuscitation were 57.50±1.16%, 62.41±1.73%, 82.34±1.62%, and 40.40±3.15%, respectively.

[0095] The results showed that the cell cryopreservation solution PF, composed of 99% OSGM medium and 1% polyethylene glycol, was effective. Ⅰ The effect is significantly higher than that of other cell cryopreservation solutions regulated by non-permeable substances. Figure 5 (See the red box in the middle). Furthermore, microscopic observation revealed that the pH of the cell cryopreservation solution... Ⅰ Although the cryopreservation-thawing effect is not as good as PF Ⅰ The effect was significant, but the morphology and integrity of the spermatocyte spikes were better than those of PF. Ⅰ ( Figure 5 (The blue box in the middle).

[0096] 3. Screening and optimization of combined protective agents, the specific steps are as follows:

[0097] As shown in Table 3, a cryopreservation solution was prepared with OSGM medium as the base solution, glycerol as the permeability protectant, and polyethylene glycol and mannitol as the non-permeability protectants. This solution is the third cryopreservation solution.

[0098] The sperm precipitate obtained in step S3 (1) was suspended in the third cryopreservation solution to prepare the fifth sperm suspension. The fifth sperm suspension was evenly distributed into cryovials. After sealing the cryovials, they were placed symmetrically in a programmed cryopreservation box (-1℃ / min). The programmed cryopreservation box was then placed in a -80℃ freezer overnight for initial freezing. After 15 hours, the cryovials were quickly transferred to liquid nitrogen (-196℃) for ultra-low temperature cryopreservation for 7 days. After 7 days of cryopreservation, the cryovials were removed and quickly placed in a 37℃ water bath for thawing. After thawing, the cryovials were stained with a 0.4% trypan blue solution, and the thawing rate and cryopreservation quality were determined by blood cell counting. Finally, by comparing the thawing rate and cryopreservation quality of different cryopreservation solutions, the combined cryoprotectant with the best cryopreservation effect was screened (formulations shown in Table 4).

[0099] Table 3 Screening scheme for third cryopreservation solution of Penaeus monodon sperm

[0100] ;

[0101] Table 4. Formula for cryopreservation solution of Penaeus monodon sperm

[0102] ;

[0103] The results are as follows Figure 7 and Figure 8 As shown, shrimp sperm cells were thawed after being cryopreserved in liquid nitrogen for 7 days. Cell viability was observed under a 10x eyepiece, 40x objective lens, and 100x oil immersion lens after staining with 0.4% trypan blue solution, and the cell viability was calculated. The combined cell cryopreservation solution PK was used. Ⅰ ~PK XVII The survival rates of shrimp sperm cells after resuscitation were 81.67±1.74%, 84.87±2.60%, 86.04±1.65%, 88.56±1.28%, 85.98±1.61%, 90.49±0.84%, 75.73±0.89%, 92.96±1.88%, 87.72±1.62%, 85.62±2.34%, 90.09±1.70%, 86.36±0.44%, 80.29±1.14%, 77.05±0.51%, 70.00±0.50%, 69.31±1.05%, and 80.32±2.27%, respectively. The results indicate that the combined cell cryopreservation solution PK... VI PK VIII and PK XI The cryopreservation effect of PK is significantly higher than that of other cell cryopreservation solutions. VI PK VIII and PK XI There were no significant differences among the three. Figure 8(See the red box in the image), and a glycerol concentration between 1% and 3% is the optimal range for cryopreservation of shrimp sperm. Furthermore, the optimal concentrations of polyethylene glycol and mannitol, two non-permeable cryoprotectants, are mutually restrictive in the cryopreservation of shrimp sperm. When the glycerol concentration is the same, a lower concentration of one non-permeable cryoprotectant necessitates a relatively higher concentration of the other to achieve the best cryopreservation effect.

[0104] S6. Statistical analysis of sperm physiological indicators in ultra-low temperature frozen and thawed Litopenaeus monodon, the specific steps are as follows:

[0105] (1) Frozen-thaw survival rate statistics: Frozen sperm were thawed at 37°C, and the morphology and survival rate of sperm after different time periods (7 days (1 week), 30 days (1 month), 90 days (3 months), 180 days (6 months), 300 days (10 months)) in liquid nitrogen were detected by trypan blue staining and blood cell counting to assess the effect of long-term cryopreservation on sperm motility.

[0106] The results are as follows Figure 9 and Figure 10 As shown, the survival rate after cryopreservation for 7 days was 92.96±1.88%, for 30 days it was 91.11±1.35%, for 90 days it was 89.55±2.36%, for 180 days it was 89.24±1.13%, and for 300 days it was 89.20±1.62%. Using the optimized cryopreservation solution for cryopreservation of shrimp sperm, there was no significant difference in survival rate after cryopreservation in liquid nitrogen from 7 to 300 days. This preliminarily verifies the feasibility and effectiveness of the optimized cryopreservation solution in cryopreservation of shrimp sperm.

[0107] (2) The morphological and ultrastructural changes of Penaeus monodon sperm before and after cryopreservation were observed using scanning electron microscopy (SEM). The operation process is as follows: First, the sperm samples of Penaeus monodon before and after cryopreservation were obtained and immediately chemically fixed with 2.5% glutaraldehyde solution to maintain the original morphology of the sperm. Then, the fixed sperm samples were dehydrated stepwise with 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol aqueous solutions. After dehydration, the critical point drying method was used to remove the liquid in the sperm samples to avoid the formation of ice crystals that would damage the sample structure. Then, the dried sperm samples were sputtered with a layer of gold or platinum with a thickness of 10~20 nm under vacuum conditions to enhance their conductivity. Finally, the treated sperm samples were loaded onto the SEM sample stage to observe the surface morphology and structural characteristics of the sperm and record any changes before and after cryopreservation.

[0108] The results are as follows Figure 11As shown, live sperm cells remained largely unchanged after cryopreservation compared to before cryopreservation, while the structure of dead sperm cells underwent significant changes after cryopreservation, with some sperm cell spinous processes detaching and varying degrees of wrinkling appearing on the surface of the sperm cell membrane.

[0109] (3) The procedure for observing the morphological and ultrastructural changes of Penaeus monodon sperm before and after cryopreservation using transmission electron microscopy (TEM) is as follows: First, obtain the sperm samples with the highest recovery rate and before cryopreservation, and immediately perform chemical fixation with 2.5% glutaraldehyde solution to maintain the original structure of the sperm; then wash the sperm samples with PBS buffer, and then perform post-fixation with 1% osmium tetroxide; then dehydrate the samples with ethanol aqueous solutions of 30%, 50%, 70%, 80%, 90%, 95% and 100% concentration gradients, and transition with acetone; after dehydration, use epoxy resin to impregnate, replace and embed the samples; after the resin has solidified, use an ultramicrotome to cut the samples into slices of about 60~100 nm thick; then place the slices on a copper grid and double stain with uranium acetate and lead citrate to enhance the contrast; finally, load the processed samples onto the TEM sample stage, observe the internal ultrastructure of the sperm, and record any changes before and after cryopreservation.

[0110] The results are as follows Figure 12 As shown, the morphology and structure of live sperm cells remained basically unchanged after cryopreservation compared to before cryopreservation, while dead sperm cells were damaged after cryopreservation, with the sperm spikes falling off and, in severe cases, the acrosomes falling off.

[0111] (4) The enzyme activity of shrimp sperm before and after cryopreservation was evaluated using a kit for lactate dehydrogenase, catalase, succinate dehydrogenase, ultra-micro Na+ / K+–ATPase and superoxide dismutase. Protein quantification was performed using a protein quantification kit during the enzyme activity assay. Each assay step was performed according to the instruction manual. The specific steps are as follows: Collect 4 million shrimp sperm cells before and after cryopreservation and thawing. After washing with pre-cooled 2.5×PBS buffer, lyse on ice for 30-40 min using 1% Triton X-100 lysis buffer. Then centrifuge at 4℃ and 4000 rpm for 10 min, collect the supernatant and determine the protein concentration.

[0112] ① Lactate dehydrogenase (LDH): After determining the protein concentration, prepare reaction systems for blank wells, standard wells, assay wells, and control wells respectively. Gently shake the plate to mix and let it stand at room temperature for 5 min. After the reaction is complete, take 200 μL of reaction solution into a 96-well plate, use an ELISA reader to detect the absorbance at a wavelength of 440 nm and calculate the enzyme activity.

[0113] ② Catalase (CAT): After determining the protein concentration, reaction systems for the assay group and the control group were prepared respectively; after the reaction was completed, 200 μL of reaction solution was transferred to a 96-well plate, and the absorbance was detected at a wavelength of 405 nm using an ELISA reader to calculate the enzyme activity.

[0114] ③ Succinate dehydrogenase (SDH): After determining the protein concentration, prepare the working solution, preheat it at 37°C for at least 5 minutes in the dark, add 100 μL of the sample to be tested to the test tube, quickly add 2.6 mL of the preheated working solution, mix immediately and start timing. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 600 nm wavelength at 10 seconds and 1 minute and 10 seconds after the start of the reaction and calculate the SDH enzyme activity.

[0115] ④ Trace amounts of Na + / K + -ATPase: After determining the protein concentration, prepare a chromogenic solution for the enzymatic reaction and phosphorus determination reaction. After the reaction is complete, transfer 200 μL of the reaction solution to a 96-well plate, and use a microplate reader to detect the absorbance at a wavelength of 636 nm and calculate the enzyme activity.

[0116] ⑤ Superoxide dismutase (SOD): After determining the protein concentration, prepare the substrate application solution and enzyme working solution, and at the same time prepare the reaction system. Mix the reaction system well and incubate at 37℃ for 20 min. After the reaction is completed, take 200 μL of the reaction solution into a 96-well plate and use a microplate reader to detect the absorbance at a wavelength of 450 nm. Finally, calculate the enzyme activity according to the formula in the instruction manual.

[0117] The results showed that the enzyme activity of lactate dehydrogenase was 0.72±0.01 U / gprot before freezing and 0.64±0.06 U / gprot after freezing; the enzyme activity of catalase was 1.50±0.11 U / gprot before freezing and 1.40±0.03 U / gprot after freezing; and the enzyme activity of succinate dehydrogenase was 26.73±1.86 U / gprot before freezing. The activity of Na⁺ / K⁺-ATPase was 23.02±1.71 U / gprot before cryopreservation and 2.53±0.29 U / gprot after cryopreservation; the activity of superoxide dismutase was 15.38±1.81 U / gprot before cryopreservation and 14.50±0.35 U / gprot after cryopreservation. There were no significant differences in the enzyme activities among the 4 million shrimp sperm cell samples before and after cryopreservation using the optimized cryopreservation solution.

[0118] (5) Genomic DNA was extracted and analyzed from shrimp sperm cells before and after cryopreservation using an apoptosis-DNA ladder extraction kit. The procedure was as follows: Shrimp sperm cells were collected before and after cryopreservation, centrifuged (800 × g, 8 min), and the supernatant was removed, while the precipitate was retained. The cell precipitate was fully lysed using lysis buffer (containing proteinase K), and then the genomic DNA was purified by phenol / chloroform extraction and ammonium acetate / ethanol precipitation. The obtained DNA precipitate was dissolved in TE buffer, and the DNA fragmentation pattern was finally analyzed by 1% agarose gel electrophoresis to detect the characteristic DNA ladder bands as an indicator of apoptosis.

[0119] The results are as follows Figure 13 As shown, the genomic DNA of shrimp sperm cells remained intact before and after cryopreservation, with no significant differences observed. This indicates that the optimized cryopreservation solution can effectively protect the genomic DNA of shrimp sperm cells from damage during ultra-low temperature preservation. This further verifies the reliability and effectiveness of the cryopreservation method and cryopreservation solution formulation used, ensuring the genetic stability of sperm cells during freezing and thawing.

[0120] S7. Artificial insemination of spermatophores in Penaeus monodon and statistics on fertilization and hatching rates, the specific steps are as follows:

[0121] (1) Female Penaeus monodon have a closed spermatheca, therefore the artificial insemination method is as follows: First, the frozen spermatheca is quickly thawed and washed repeatedly with 2.5×PBS buffer to remove residual cryopreservation solution. Then, the spermatheca is manually peeled off, and finally, the sperm clump inside is carefully aspirated and collected using a 5 mL syringe. Females that have just completed molting 12-24 hours ago and are sexually mature are selected. Using a syringe with the needle removed, the sperm clump is precisely injected into the female shrimp's spermatheca. After artificial insemination, the shrimp are temporarily kept in a rearing pond for 4-7 days until they spawn naturally.

[0122] (2) Nine to 11 hours after artificial insemination and oviposition, 200 ovipositors (n=5) were randomly selected from both the frozen-thawed sperm cell group and the fresh sperm cell group and observed under a light microscope. The results are as follows: Figure 14 As shown, there was no significant difference in the developmental morphology of embryos and larvae at different stages between the frozen-thawed sperm cell group and the fresh sperm cell group.

[0123] Embryos exhibiting the morphological structures of the cleavage stage, blastocyst stage, gastrula stage, limb bud stage, and intramembranous nauplius stage are considered successfully fertilized eggs, and the fertilization rate is calculated according to the following formula 1:

[0124] Fertilization rate (%) = (Number of fertilized eggs / 200) × 100% (Formula 1)

[0125] After waiting for 2-4 hours for hatching, observe under a microscope. Individuals that have successfully hatched by breaking through the membrane of the nauplius larvae are recorded as having successfully hatched, and the statistics are compiled according to the following formula 2:

[0126] Hatching rate = (Total number of hatched individuals / Total number of fertilized eggs) × 100 (Formula 2)

[0127] The results are shown in Table 5, using the optimized formulation for PK. VIII The spermatophores of Penaeus monodon were preserved in cryopreservation solution. After thawing, the sperm motility rate was measured to be 72.39±1.25%, still maintaining a certain fertilization capacity. Artificial insemination techniques allowed for successful fertilization of female shrimp, enabling embryonic development and larval hatching. The fertilization rate of cryopreserved sperm cells was 20.83±2.51%, and the hatching rate was 5.78±0.58%.

[0128] Table 5. Statistics on sperm motility, fertilization rate, and hatching rate

[0129] ;

[0130] (3) Further comparison of the development of Penaeus monodon after artificial insemination with cryopreserved spermatophores and Penaeus monodon after artificial insemination with fresh spermatophores was conducted, and the results are as follows: Figure 15 As shown, there were no significant differences in morphology between the two nauplius, zoea, mysid, and larval stages.

[0131] Comparative Example

[0132] The cryopreservation of fresh sperm from *Litopenaeus monodon* was performed using the cryopreservation solution and method disclosed in patent document CN113455497A. The cryopreservation solution used sterilized natural seawater as a base, glycerol as an antifreeze agent, and bovine serum albumin (BSA) or trehalose as a non-permeable antifreeze agent. The volume fraction of the antifreeze agent in the cryopreservation solution was 5-10%, the concentration of BSA or trehalose was 0.25 mol / L, and the volume ratio of *Litopenaeus monodon* sperm to the cryopreservation solution was 1:5. The programmed cooling for cryopreservation was as follows: equilibration at 0°C for 20 min, cooling at a rate of -5°C / min to -20°C for 5 min, cooling at a rate of -10°C / min to -80°C for 5 min, cooling at a rate of -20°C / min to -180°C, and then storage in liquid nitrogen.

[0133] The results showed that the survival rate of Penaeus monodon sperm preserved using the comparative method dropped to below 30% after 7 days. In stark contrast, using the preservation solution and cryopreservation method provided by this invention, Penaeus monodon sperm maintained a high survival rate of >89% after 10 months of cryopreservation. Therefore, the cryopreservation solution and method provided in patent document CN113455497A are not applicable to Penaeus monodon, as the two species are different, and the cryopreservation solution and method are not universally applicable.

[0134] Therefore, it can be concluded that this invention provides a cryopreservation solution and method for improving the sperm survival rate of Penaeus monodon. By using shrimp sperm cell culture medium instead of traditional calcium-free saline as the base solution for cryopreservation, an optimized environmental foundation is established for cryopreservation. Furthermore, the optimal concentration ratio and synergistic combination of permeable and non-permeable cryoprotectants are systematically screened, significantly improving cryoprotection efficacy. Using the cryopreservation method provided in this application, the survival rate of Penaeus monodon sperm frozen and thawed for 10 months is greater than 89%, achieving long-term stable preservation of sperm under ultra-low temperature conditions. Key bioactivity indicators (such as enzyme activity and DNA integrity) show no significant difference compared to before cryopreservation, and artificial insemination has been successfully achieved with frozen and thawed spermatophores. There are no significant morphological differences between Penaeus monodon artificially inseminated with cryopreserved spermatophores and those artificially inseminated with fresh spermatophores in the nauplius, zoea, mysid, and postlarval stages. This not only confirms the high efficiency of this method in maintaining sperm activity and function, but also establishes a stable and reproducible complete technical system, providing key technical support for the long-term preservation of Penaeus monodon germplasm resources and laying the foundation for the construction of a germplasm bank.

[0135] The terminology and expressions used herein are for descriptive purposes only, and the invention should not be limited to these terms and expressions. The use of these terms and expressions does not imply the exclusion of any illustrative and descriptive equivalents (or parts thereof), and it should be recognized that various modifications that may exist should also be included within the scope of the claims. Other modifications, variations, and substitutions may also exist. Accordingly, the claims should be considered to cover all such equivalents.

[0136] Similarly, it should be noted that although the present invention has been described with reference to the specific embodiments described above, those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions can be made without departing from the spirit of the present invention. Therefore, any changes or modifications to the above embodiments within the scope of the essential spirit of the present invention will fall within the scope of the claims of the present invention.

Claims

1. A cryopreservation solution for the cryopreservation of Penaeus monodon spermatozoa, characterized in that it comprises: The freezing solution comprises a base solution and a combined protective agent, The base solution is a penaeid sperm cell culture medium, the penaeid sperm cell culture medium comprises Marsupenaeusensole serum; the adding amount of the base solution is 93.0-95.0% of the volume ratio of the freezing solution; the penaeid sperm cell culture medium is based on 1.5xL-15 culture medium, and further comprises taurine with a final concentration of 665-829 mg / L, proline with a final concentration of 1213-1518 mg / L, aspartic acid with a final concentration of 43-54 mg / L, glutamic acid with a final concentration of 79-98 mg / L, lysine with a final concentration of 136-171 mg / L, hydroxyproline with a final concentration of 50-63 mg / L, ornithine with a final concentration of 15-18 mg / L, beta-alanine with a final concentration of 4-5 mg / L, gamma-aminobutyric acid with a final concentration of 3 mg / L, cystine with a final concentration of 20-23 mg / L, glutamine with a final concentration of 300 mg / L, arabinose with a final concentration of 18-19 mg / L, xylose with a final concentration of 4-6 mg / L, ribose with a final concentration of 28-32 mg / L, fucose with a final concentration of 8 mg / L, glucose with a final concentration of 2905-2936 mg / L, fructose with a final concentration of 4-6 mg / L, trehalose with a final concentration of 224-234 mg / L, sucrose with a final concentration of 63-69 mg / L, lactose with a final concentration of 11-18 mg / L, gentiobiose with a final concentration of 81-88 mg / L, sodium bicarbonate with a final concentration of 1 g / L, 3v / v% of the Marsupenaeusensole serum, 15v / v% of fetal bovine serum, epidermal growth factor with a final concentration of 20 μg / L, basic fibroblast growth factor with a final concentration of 20 μg / L, and penicillin-streptomycin with a final concentration of 10000 U / mL; the osmotic pressure of the penaeid sperm cell culture medium is adjusted to 720±20 mOsm / kg by using sodium chloride; and the pH value of the penaeid sperm cell culture medium is adjusted to 7.2±0.2 by using 1 M sodium hydroxide or 1 M hydrochloric acid; The combined protective agent comprises an osmotic protective agent and a non-osmotic protective agent; the osmotic protective agent is glycerol, and the adding amount of the osmotic protective agent is 1.0-3.0% of the volume ratio of the freezing solution; and the non-osmotic protective agent is polyethylene glycol and mannitol, and the adding amount of the non-osmotic protective agent is 4.0-6.0% of the volume ratio of the freezing solution.

2. Application of the freezing solution for freezing and preserving Marsupenaeusensole sperm according to claim 1 in improving the survival rate of Marsupenaeusensole sperm freezing and preservation.

3. Application of the freezing solution for freezing and preserving Marsupenaeusensole sperm according to claim 1 in breeding.

4. A penaeid shrimp sperm cryopreservation kit, characterized by, The freezing solution for freezing and preserving Marsupenaeusensole sperm according to claim 1.

5. A method for improving the survival rate of frozen Penaeus monodon sperms, characterized by, The method comprises the following steps: Step A: obtaining discrete sperm or spermatophores of male Marsupenaeusensole, and pre-treating the discrete sperm or the spermatophores to obtain pre-treated discrete sperm or pre-treated spermatophores; Step B: the pre-processed discrete sperm or the pre-processed spermatophore is suspended in the cryopreservation solution of the frozen sperm of the prawn Penaeus monodon as claimed in claim 1 to obtain a sperm suspension; Step C: the sperm suspension is subjected to primary freezing and then ultra-low temperature freezing in liquid nitrogen.

6. The method for improving the cryopreservation of Penaeus monodon sperms according to claim 5, characterized in that, The step A is specifically: The male prawn Penaeus monodon is sterilized and anesthetized, the back shell is dissected under sterile conditions to take out the vas deferens, the vas deferens is placed in the prawn sperm cell culture medium, the vas deferens is mechanically broken to obtain a sperm mixture, the sperm mixture is centrifuged and filtered to obtain a sperm precipitate, and the sperm precipitate is resuspended in the prawn sperm cell culture medium to obtain pre-processed discrete sperm; Alternatively, The spermatophore of the male prawn Penaeus monodon is taken out by electric shock method, and the spermatophore is placed in PBS buffer for preservation to obtain pre-processed spermatophore.

7. The method for improving the cryopreservation of Penaeus monodon sperms according to claim 5, characterized in that, The step C is specifically: Step C-1: the sperm suspension is placed in a cryopreservation tube in a programmed cooling box, and the programmed cooling box is placed in a-80℃ refrigerator overnight for primary freezing; the primary freezing rate is 1℃ per minute; Step C-2: the cryopreservation tube containing prawn sperm after primary freezing in step C-1 is placed in liquid nitrogen for ultra-low temperature freezing preservation, and the ultra-low temperature freezing preservation condition is-196℃ for at least 7 days.

8. The method for improving the survival rate of prawn Penaeus monodon sperm according to claim 5, wherein the long-term cryopreservation time of the prawn Penaeus monodon sperm is at least 10 months, the recovery survival rate of the prawn Penaeus monodon sperm cryopreserved for 10 months is ≥ 89%, the fertilization rate of artificial insemination using the prawn Penaeus monodon spermatophore cryopreserved for 10 months is ≥ 20%, and the hatching rate of the prawn Penaeus monodon after artificial insemination using the prawn Penaeus monodon spermatophore cryopreserved for 10 months is ≥ 5.5%.

9. The prawn Penaeus monodon sperm or the prawn Penaeus monodon spermatophore preserved by the method for improving the survival rate of prawn Penaeus monodon sperm according to claim 8 is used in breeding. ​ ​

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