Deep hypothermia preservation method of black carp eggs

Abstract

The invention relates to the field of aquatic products, in particular to a deep hypothermia preservation method for black carp eggs. By microinjection of saccharides such as trehalose or polysaccharide or antifreeze protein (AFP) extracted from deep-sea fishes, formation of ice crystals in the freezing process is reduced, and the survival rate of ova and embryos is increased. The ovum and the embryo are encapsulated by using sodium alginate hydrogel to form a cell spherical structure, so that the problems of mechanical damage and dehydration in the freezing process are reduced. In addition, DMSO or other low-temperature protective agents with different concentrations are added step by step, damage of osmotic pressure to the ovum and the embryo is reduced, a transparent glassy state is formed in liquid nitrogen, formation of ice crystals is avoided, and the preservation effect is improved. The ova preserved by the deep hypothermia preservation method of the fish ova or embryo is complete in form, and the proportion of the ova with water absorption capacity reaches 82.3%.

Description

Technical Field

[0001] The invention relates to the field of aquatic products, and in particular to a deep-low temperature preservation method for black carp eggs. Background Art

[0002] Aquaculture occupies an important position in my country's national economy and is one of the important agricultural pillar industries in my country, with freshwater aquaculture as the main form. At present, through the gynogenetic technology, the disease resistance of black carp germplasm resources is being continuously optimized. However, for the optimized black carp germplasm resources, they can only be continuously propagated through continuous feeding of mature fish, which requires a lot of economic cost and time. Deep cryopreservation technology can preserve only the eggs and embryos of gynogenetic black carp, and realize the preservation of a large number of black carp embryos. At the same time, the pairing of fish germplasm resources from different regions and the artificial hybridization breeding of different paternal and maternal parents over a long distance require the collection of the paternal and maternal parents at different specific times, which sometimes presents certain difficulties. Preserving the eggs and embryos and thawing and hatching them when needed can effectively solve the problems of long-distance hybridization breeding and continuous optimization of germplasm resources.

[0003] Existing black carp eggs and embryos used for gynogenesis are difficult to preserve for a long time through cryopreservation, and there are almost no practical methods. During the cooling process, fish eggs and embryos are easily damaged by frozen ice crystals or due to rapid dehydration. In addition, when using traditional cryopreservation methods or human oocyte preservation methods, eggs and embryos are easily damaged by the internal salt environment or the toxicity of the added cryoprotectants, and it is very difficult to preserve their integrity. There are two difficulties in cryopreserving fish eggs compared to human oocytes. The first is that they are larger in size, and the temperature difference between the inside and outside of the eggs during freezing and thawing is greater; the second is that the large yolk of fish eggs makes it difficult to form a glassy state without ice crystals during the cooling process, while human eggs do not have this problem. Summary of the Invention

[0004] In light of this, the present invention aims to address the poor preservation effects of existing technologies due to ice crystal damage, dehydration, and cryoprotectant toxicity, thereby conserving high-quality freshwater fish germplasm and promoting long-distance, cross-regional breeding of high-quality species. The present invention provides a method for deep cryopreservation of black carp eggs. Experiments have shown that, compared to conventional cryopreservation methods (28.1% integrity), the present method improves egg morphological integrity to 82.3%.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a cryoprotectant, comprising a low-temperature protectant, trehalose and an ovarian protective solution; the low-temperature protectant comprises one or more of DMSO, ethylene glycol or isopropanol; and the ovarian protective solution comprises BSA, Na2HPO4, NaCl, KCl, MgCl2·6H2O, CaCl2, tyrosine and glycine.

[0007] In some specific embodiments of the present invention, the volume fraction of the cryoprotectant is 10% to 40%.

[0008] In some specific embodiments of the present invention, based on 1 L of system, the ovarian protective solution includes: 4.11 g / LBSA, 3.8 mmol Na2HPO4, 118.0 mmol NaCl, 12.7 mmol KCl, 0.7 mmol MgCl2·6H2O, 2.7 mmol CaCl2, 5.5 mmol tyrosine and 5.5 mmol glycine, and the pH value is adjusted to 8.14 with NaHCO3.

[0009] In some specific embodiments of the present invention, the volume ratio of the cryoprotectant, trehalose solution, and ovarian protective solution includes (1-4):5:(1-4).

[0010] In some specific embodiments of the present invention, the concentration of the trehalose solution is 1 mol / L.

[0011] The present invention also provides a composition for deep cryopreservation of fish eggs or embryos, comprising a biocompatible antifreeze substance, a sodium alginate solution and the cryoprotectant;

[0012] The biocompatible antifreeze material includes one or more of trehalose, polysaccharide or antifreeze protein AFP.

[0013] The present invention also provides the use of any of the following in cryopreservation of fish eggs or embryos:

[0014] (I), the cryoprotectant; and / or

[0015] (II), the composition.

[0016] The present invention also provides a method for cryopreservation of fish eggs or embryos, comprising the following steps:

[0017] Step 1: introducing the biocompatible antifreeze substance in the composition into fish eggs or embryos to obtain fish eggs or embryos introduced with the biocompatible antifreeze substance, and placing the fish eggs or embryos in the ovary protection solution in the cryoprotectant to prepare an egg or embryo suspension;

[0018] Step 2: mixing the egg or embryo suspension described in step 1 with the sodium alginate solution in the composition, and then dripping the mixture into a calcium chloride solution to form cell microspheres;

[0019] Step 3, equilibrating the cell microspheres described in step 2 in the cryoprotectant to obtain equilibrated cell microspheres;

[0020] Step 4: Storing the balanced cell microspheres in liquid nitrogen to obtain frozen vitreous fish eggs or embryos.

[0021] In some embodiments of the present invention, the fish comprises black carp.

[0022] In some specific embodiments of the present invention, the dosage of the biocompatible antifreeze substance introduced in step 1 includes: 1-10 μL.

[0023] In some specific embodiments of the present invention, the concentration of the egg or embryo suspension in step 1 is 10 / mL.

[0024] In some specific embodiments of the present invention, the mass volume fraction of the sodium alginate solution is 2%.

[0025] In some specific embodiments of the present invention, the volume ratio of the egg or embryo suspension and the sodium alginate solution in step 2 is 1:1.

[0026] In some specific embodiments of the present invention, step 3 specifically comprises: equilibrating the cell microspheres of step 2 in the cryoprotectant containing 10%, 20% or 40% of the cryoprotectant by volume.

[0027] In some specific embodiments of the present invention, the equilibration time comprises 1 min.

[0028] In some specific embodiments of the present invention, the volume of the storage unit for the frozen vitreous fish eggs or embryos in step 4 is less than 10 mL.

[0029] In some specific embodiments of the present invention, the cryopreservation method further comprises: after obtaining the frozen glassy fish eggs or embryos in step 4, heating them at 20°C to 30°C for thawing.

[0030] In some specific embodiments of the present invention, the heating method includes water bath heating, single-frequency resonant microwave heating or laser and water bath combined heating.

[0031] In some specific embodiments of the present invention, the cryopreservation method further comprises: after the frozen vitreous fish eggs or embryos are thawed by the cryopreservation method, using a sodium citrate solution to remove the sodium alginate on the surface of the cell microspheres to release the fish eggs or embryos.

[0032] In some specific embodiments of the present invention, the concentration of the sodium citrate solution in step 2 is 0.1 mol / L to 0.5 mol / L.

[0033] The present invention includes but is not limited to providing the following beneficial effects:

[0034] The present invention reduces ice crystal formation during freezing and improves the survival rate of eggs and embryos by microinjecting sugars such as trehalose or polysaccharides, or using antifreeze proteins (AFPs) extracted from deep-sea fish. Sodium alginate hydrogel is used to encapsulate eggs and embryos, forming a spherical cell structure that reduces mechanical damage and dehydration during freezing. Furthermore, the present invention gradually adds varying concentrations of DMSO or other cryoprotectants to reduce osmotic damage to eggs and embryos, forming a transparent glassy state in liquid nitrogen, avoiding ice crystal formation and improving preservation effectiveness. The proportion of eggs preserved with morphological integrity and water absorption capacity using the deep-cryopreservation method for fish eggs or embryos described herein reached 82.3%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0036] Figure 1 A flow chart showing a method for cryopreservation of gynogenetic black carp eggs or embryos;

[0037] Figure 2 Shows the morphology of the egg. DETAILED DESCRIPTION

[0038] The present invention discloses a method for cryopreservation of black carp roe. Those skilled in the art may refer to the contents herein and appropriately modify the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described using preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0039] Explanation of terms:

[0040] Biocompatible antifreeze substances include sugars, antifreeze proteins and other substances with ice crystal inhibition function.

[0041] The present invention provides a method for cryopreserving gynogenetic black carp eggs or embryos at low temperatures, thereby achieving the purpose of protecting high-quality freshwater fish germplasm resources and conducting long-distance, cross-regional breeding of high-quality species. The method specifically comprises:

[0042] 1. Inject sugars such as trehalose and polysaccharides into black carp eggs and embryos through microinjection. Sugars such as trehalose can be replaced with antifreeze protein AFP (Anti-Freezing Protein) extracted from deep-sea fish.

[0043] 2. Place the eggs and female nucleus embryos in a sodium alginate solution to form a cell spherical structure. The spherical shell is a gel solid and the inside is the egg or embryo to be preserved.

[0044] 3. Pass the sodium alginate cell sphere through the mesh tube and place it in 10% (v / v) DMSO (dimethyl sulfoxide) + ovarian protective solution, 20% (v / v) DMSO + ovarian protective solution, and 40% (v / v) DMSO + ovarian protective solution in sequence. DMSO can be replaced with other permeable cryoprotectants such as ethylene glycol and isopropanol.

[0045] 4. Seal the sodium alginate cell spheres, cryoprotectant, and ovarian protection solution into a cryovial with a volume of less than 10 mL. Place the cryovial in liquid nitrogen to form a transparent glassy state.

[0046] 5. Store in liquid nitrogen.

[0047] 6. Heat the cell spheroids in a 30°C water bath (the water bath temperature can be between 20°C and 30°C). Heating methods include: single-frequency resonant microwave heating, laser and water bath combined heating.

[0048] 7. Use sodium citrate solution (concentration 0.1 mol / L~0.5 mol / L) to remove the cell microspheres formed by sodium alginate and remove the eggs.

[0049] Unless otherwise specified, the raw materials and reagents used in the method for cryopreservation of black carp eggs provided by the present invention can be purchased from the market.

[0050] The present invention will be further described below in conjunction with the embodiments:

[0051] Example

[0052] 1. Collect black carp eggs and inject 10 μL of 0.5 mol / L trehalose into the eggs by microinjection. Place the eggs in ovarian fluid (4.11 g / L BSA, 3.8 mmol Na2HPO4, 118.0 mmol NaCl, 12.7 mmol KCl, 0.7 mmol MgCl2·6H2O, 2.7 mmol CaCl2, 5.5 mmol tyrosine, 5.5 mmol glycine, and adjust the pH to 8.14 with NaHCO3) to prepare an egg suspension (approximately 10 black carp eggs / mL).

[0053] 2. Mix the egg suspension with 2% sodium alginate in a 1:1 volume ratio. Use a plastic dropper to draw up the egg mixture and drop it into a 1 mol / L calcium chloride solution. Once the reaction is complete, the eggs will be encapsulated in the sodium alginate hydrogel. Wash the encapsulated hydrogel two to three times with 1x PBS to minimize damage to the eggs caused by the calcium chloride.

[0054] 3. Encapsulated eggs were placed on a homemade grid tool (1 mm mesh size) and cryoprotectant was added in three balanced steps: first, immersing in 10% (v / v) DMSO solution for 1 minute, then transferring to 20% (v / v) DMSO solution for 1 minute, then transferring to 40% (v / v) DMSO solution for 1 minute. Finally, the embryos were transferred to a cryovial and directly plunged into liquid nitrogen. DMSO was prepared using ovarian fluid and 1 mol / L trehalose (Table 1).

[0055] Table 1 Solution preparation

[0056]

[0057] 4. After storage in liquid nitrogen, thaw the cryovials in a 30°C water bath.

[0058] 5. After thawing, immerse the eggs in 40% DMSO solution, 20% DMSO solution, and 10% DMSO solution in the reverse order of DMSO addition for 1 minute each. Then, wash the hydrogel twice with 1× PBS to elute the DMSO.

[0059] 6. Transfer the encapsulated eggs to a 3% (w / v) trisodium citrate solution (0.14 mol / L) to remove calcium alginate and release the eggs. Finally, transfer the eggs to natural water for culture and observation to check the egg integrity and water absorption capacity.

[0060] Comparative Example 1

[0061] Sodium alginate hydrogel was used to encapsulate the eggs, but no sugar or antifreeze protein microinjection was performed. The remaining steps were the same as the preservation method in the embodiment.

[0062] Comparative Example 2

[0063] The black carp eggs were preserved by conventional cryopreservation methods, i.e., sodium alginate hydrogel was not used, sugar microinjection was not performed, 10% (v / v) DMSO cryoprotectant solution was used, and the remaining steps such as liquid nitrogen cooling and water bath heating were the same as those in the embodiment.

[0064] Comparative Example 3

[0065] The sodium alginate hydrogel was not used, and the sugar injection was performed. The other steps were the same as the conventional low-temperature preservation method in Comparative Example 2.

[0066] Comparative Example 4

[0067] Antifreeze protein injection (A / F Protein Canada Inc., Canada) model: antifreeze protein type I AFP-I, dosage: 1 mg) was performed without using sodium alginate hydrogel. Other steps were the same as the conventional cryopreservation method in Comparative Example 2.

[0068] Effect Examples

[0069] The eggs obtained by the methods of Examples and Comparative Examples 1 to 4 were transferred to natural water bodies for culture and observation to check the egg integrity and water absorption capacity. The results are shown in Table 2.

[0070] Table 2 Proportion of eggs with integrity and water absorption capacity

[0071]

[0072] The results showed that 14 were intact and 3 were dehydrated. The proportion of eggs preserved by the method of the embodiment of the present invention with morphological integrity and water absorption capacity reached 82.3% ( Figure 2 Right image). The role of sugars such as trehalose and polysaccharides may be to maintain cellular physiological activity, support cell structure, and increase cytoplasmic viscosity to reduce ice crystal growth. The role of antifreeze proteins (AFPs) is to reduce ice crystal formation and growth. Currently, trehalose, polysaccharides, and antifreeze proteins cannot freely penetrate into cells. Therefore, injecting these substances into cells through microinjection can increase protection during the low-temperature freezing process.

[0073] In Comparative Example 1, sodium alginate hydrogel was used, and no sugar or antifreeze protein microinjection was performed. The remaining steps were the same as those in the example, and the proportion of eggs with morphological integrity and water absorption capacity reached 60.5%.

[0074] In contrast, in Comparative Example 2, which was stored using conventional cryopreservation methods (no sodium alginate hydrogel, no sugar microinjection, 10% DMSO cryoprotectant solution, liquid nitrogen cooling, and water bath heating), the integrity of the eggs was approximately 9, 23 were dehydrated, and the morphological integrity of the eggs was 28.1% ( Figure 2 left).

[0075] In comparative example 3, the oocyte morphological integrity was 36.8% when the sugar injection was performed without using sodium alginate hydrogel and the conventional low-temperature preservation method was used in other steps.

[0076] In comparative example 4, the antifreeze protein injection was performed without using sodium alginate hydrogel, and the conventional low-temperature preservation method was used in other steps, and the morphological integrity of the eggs was 47.8%.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A cryoprotectant, characterized in that The invention comprises a cryoprotectant, trehalose and an ovarian protective solution; the cryoprotectant comprises one or more of DMSO, ethylene glycol or isopropanol; and the ovarian protective solution comprises BSA, Na2HPO4, NaCl, KCl, MgCl2·6H2O, CaCl2, tyrosine and glycine.

2. The cryoprotectant according to claim 1, wherein The volume fraction of the cryoprotectant is 10% to 40%.

3. A composition for cryopreservation of fish eggs or embryos, characterized in that: Comprising a biocompatible antifreeze substance, a sodium alginate solution and the cryoprotectant according to claim 1 or 2; The biocompatible antifreeze material includes one or more of trehalose, polysaccharide or antifreeze protein AFP.

4. Use of any of the following in cryopreservation of fish eggs or embryos: (I) The cryoprotectant according to claim 1 or 2; and / or (II) The composition according to claim 3.

5. A method for cryopreservation of fish eggs or embryos, characterized in that: The following steps are involved: Step 1: introducing the biocompatible antifreeze substance in the composition of claim 3 into fish eggs or embryos to obtain fish eggs or embryos introduced with the biocompatible antifreeze substance, and placing the fish eggs or embryos in the ovarian protective solution in the cryoprotectant of claim 1 to prepare an egg or embryo suspension; Step 2: mixing the egg or embryo suspension described in step 1 with the sodium alginate solution in the composition according to claim 3, and then dripping the mixture into a calcium chloride solution to form cell microspheres; Step 3, equilibrating the cell microspheres described in step 2 in the cryoprotectant according to claim 1 or 2 to obtain equilibrated cell microspheres; Step 4: Storing the balanced cell microspheres in liquid nitrogen to obtain frozen vitreous fish eggs or embryos.

6. The cryopreservation method according to claim 5, wherein: Step 3 specifically comprises: equilibrating the cell microspheres in step 2 in the cryoprotectant containing 10%, 20% or 40% of the cryoprotectant by volume.

7. The cryopreservation method according to claim 5 or 6, wherein: The volume of the storage unit for the frozen vitreous fish eggs or embryos in step 4 is less than 10 mL.

8. The cryopreservation method according to any one of claims 5 to 7, characterized in that: Also includes: Step 4: After obtaining the frozen glassy fish eggs or embryos, thawing is performed by heating at 20° C. to 30° C.

9. The cryopreservation method according to claim 8, wherein: Also includes: The frozen glassy fish eggs or embryos are The cryopreservation method according to claim 8, wherein after thawing, sodium citrate solution is used to remove sodium alginate on the surface of the cell microspheres to release the fish eggs or embryos.

10. The cryopreservation method according to claim 9, wherein: The concentration of the sodium citrate solution in step 2 ranges from 0.1 mol / L to 0.5 mol / L.

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