A method for cryopreservation of mongolian bream embryos

Abstract

The application relates to the field of biotechnology, and particularly discloses a method for freezing and preserving embryos of Pseudobrama mongolicus, (1) the embryos of Pseudobrama mongolicus are balanced in balance liquid one and balance liquid two for 8-12 minutes respectively; the balance liquid one is prepared by mixing a cryoprotectant and a diluent at a volume ratio of 1:1.8-2.4; the balance liquid two is prepared by mixing the cryoprotectant and the diluent at a volume ratio of 1.8-2.4:1; (2) the embryos of Pseudobrama mongolicus are transferred into the cryoprotectant, single embryos of Pseudobrama mongolicus are collected, and are placed on a metal block cooled by liquid nitrogen for 8-12 seconds, are placed in a frozen storage container and are sealed, and are stored in liquid nitrogen. The method reduces cell damage, improves the freezing and preserving effect, and has a high embryo hatching rate; the solid surface vitrification method is used to preserve single embryos in the form of droplets, the process is more refined, the temperature change of the temperature rising and falling process of each embryo can be ensured to be uniform, and the embryo preserving effect and the repeatability of the preserving scheme are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the field of biological material / tissue cryopreservation, and more particularly to the field of fish embryo cryopreservation. BACKGROUND

[0002] Due to the large volume of fish embryos (diameter ≥ 1 mm), double membrane structure, poor villus membrane permeability, and high water content of yolk sac, the cryopreservation of fish embryos is much more difficult than that of mammalian embryos. So far, only a few species of fish embryos have been successfully cryopreserved. For large-volume biological samples such as fish embryos, the method of cryopreservation can usually only use vitrification method, which is a method that uses high concentration of antifreeze and extremely fast cooling rate to make the solution reach a glassy state without ice crystals, thereby reducing the mechanical damage to biological samples. This method has been widely used because of its simplicity, speed, low cost, and better protection of embryos from cell damage caused by freezing. Zhang et al. used a cryoprotectant containing propylene glycol, methanol, glycerol, and sucrose to conduct a preliminary study on the cryopreservation of grouper embryos, and obtained a survival rate of 6.32%, and 39.85% of the surviving embryos eventually hatched successfully. Ding et al. used a programmed cooling method and a vitrification method to cryopreserve pagrus major embryos, and made a breakthrough in the cryopreservation and revival of embryos in liquid nitrogen. Then by detecting the survival rate of embryos, it was found that propylene glycol had the lowest toxicity to pagrus major embryos, and methanol had the highest toxicity.

[0003] Chen Songlin and Tian Yongsheng, professors of Huanghai Institute of Oceanography, Chinese Academy of Fishery Sciences, have published a method for vitrification cryopreservation of marine fish embryos. The scheme is as follows: fish embryo collection: select pre-blastula embryos; vitrification solution preparation: a mixture of base solution and antifreeze, the base solution (BS2) is: NaCl 2.472%, KCl 0.086%, CaCl2·2H2O 0.146%, MgCl2·6H2O 0.486%, NaHCO3 0.019%; antifreeze: 1,2-propanediol (PG), methanol (MeOH); vitrification solution formula: BS2: 50-67%, PG: 20-30%, MeOH: 13-20%; embryo cryopreservation before balancing treatment: the embryos are sequentially balanced in vitrification solutions with concentrations of 25%, 33%, 50%, 67% and 100% in five steps, and the treatment time is 45-55 min, and the treatment time of each step is 9-11 min; liquid nitrogen rapid freezing preservation: the balanced embryos 9-13 are taken into a 3mm diameter and 10cm long straw with 250ul vitrification solution, and after heat sealing, it is put into-196℃ liquid nitrogen for rapid freezing preservation; thawing and cultivation: the straw is removed from the liquid nitrogen and quickly inserted into a 39-40℃ water bath within 1-2 seconds to rapidly thaw, when the embryo is milky white and about to disappear, the straw is cut open, and the embryo is washed into a container with 2ml 0.125M sucrose eluent for 10min, and then sterilized seawater at 14-16℃ is added for cultivation.

[0004] Since the prior art lacks a freezing preservation technology for freshwater economic fish embryos, and the prior art uses 0.25mL straws to preserve fish embryos, the preservation method is rough, the cooling and heating cannot be uniform and stable, and the preservation effect is not good. In addition, the prior art cannot completely avoid the recrystallization phenomenon in the rewarming process of the vitrification solution, and the experimental results are accidental and have poor repeatability. SUMMARY

[0005] In view of the problem that the prior art lacks a freezing preservation technology for freshwater economic fish embryos, the present application provides a Mongolian perch embryo cryopreservation solution kit and a freezing preservation method to overcome the shortcomings of the prior art.

[0006] A method for freezing preservation of Mongolian perch embryos, comprising the following steps:

[0007] (1) The Mongolian perch embryos are balanced in ES1 and ES2 respectively for 8-12min;

[0008] The cryoprotectant and the diluent are mixed at a volume ratio of 1:1.8-2.4 to prepare the first balancing solution; the cryoprotectant and the diluent are mixed at a volume ratio of 1.8-2.4:1 to prepare the second balancing solution;

[0009] The diluent contains 6-10 g / L of sodium chloride, 0.3-0.7 g / L of potassium chloride and 12-18 g / L of glucose with water as solvent.

[0010] The cryoprotectant contains 3.5-5.0 g / L of sodium chloride, 0.2-0.4 g / L of potassium chloride, 7-11 g / L of glucose, 0.5-1.5 mmol / L of melatonin and 0.5-1.5 mg / L of Fe3O4 magnetic nanospheres with water as solvent, 8%-15% of methanol, 10%-20% of propylene glycol and 10%-20% of dimethyl sulfoxide by volume fraction.

[0011] (2) The embryos of P. mongolicus are transferred into the cryoprotectant CPA, and single embryos of P. mongolicus are collected, placed on a metal block cooled by liquid nitrogen for 8-12 s, placed in a cryopreservation container and sealed, and stored in liquid nitrogen.

[0012] Preferably, the embryos of P. mongolicus are in the tailbud stage.

[0013] The Fe3O4 magnetic nanospheres have a particle size of 100-800 nm, and preferably, the Fe3O4 magnetic nanospheres have a particle size of 500-600 nm.

[0014] Preferably, the cryoprotectant can be prepared by adding methanol, propylene glycol, dimethyl sulfoxide, melatonin and Fe3O4 magnetic nanospheres into the diluent, wherein the diluent has a volume fraction of 50%-70%.

[0015] Preferably, the diluent contains 7-9 g / L of sodium chloride, 0.4-0.6 g / L of potassium chloride and 14-16 g / L of glucose, and the cryoprotectant contains 3.5-5.0 g / L of sodium chloride, 0.2-0.4 g / L of potassium chloride, 8-10 g / L of glucose, 0.8-1.2 mmol / L of melatonin and 0.8-1.2 mg / L of Fe3O4 magnetic nanospheres with water as solvent, 9%-12% of methanol, 12%-16% of propylene glycol and 12%-16% of dimethyl sulfoxide by volume fraction.

[0016] Preferably, in step (1), the first equilibration solution is prepared by mixing the cryoprotectant and the diluent at a volume ratio of 1:2, and the second equilibration solution is prepared by mixing the cryoprotectant and the diluent at a volume ratio of 2:1.

[0017] In a preferred mode of the present application, the diluent contains 8 g / L of sodium chloride, 0.5 g / L of potassium chloride and 15 g / L of glucose with water as solvent.

[0018] The freezing protective agent contains 15% dimethyl sulfoxide by volume, 10% methanol by volume, 15% propylene glycol by volume, 1 mmol / L melatonin and 1 mg / L Fe3O4 magnetic nanometer microspheres; and wherein the sodium chloride content is 4.8 g / L, the potassium chloride content is 3 g / L and the glucose content is 9 g / L. The freezing protective agent can be prepared by using the diluent with 60% by volume as a solvent and adding methanol, propylene glycol, dimethyl sulfoxide, melatonin and Fe3O4 magnetic nanometer microspheres.

[0019] In step (2), preferably, the single Procypris precaudalis embryos are collected by using a freezing hook; the freezing container is preferably a freezing ring. The collected Procypris precaudalis embryos are placed on a metal block placed in liquid nitrogen, cooled for 10 s, inserted into a freezing ring placed in liquid nitrogen for freezing, sealed by a heat engine, and placed in a liquid nitrogen tank for quick freezing and preservation; the single embryos are preserved in the form of droplets. The metal block can be an aluminum alloy metal block.

[0020] The application provides a Procypris precaudalis embryo cryopreservation reagent, which comprises at least one of a diluent and a freezing protective agent.

[0021] The diluent contains 6-10 g / L sodium chloride, 0.3-0.7 g / L potassium chloride and 12-18 g / L glucose with water as a solvent.

[0022] The freezing protective agent contains 8%-15% methanol by volume, 10%-20% propylene glycol by volume, 10%-20% dimethyl sulfoxide, 3.5-5.0 g / L sodium chloride, 0.2-0.4 g / L potassium chloride, 7-11 g / L glucose, 0.5-1.5 mmol / L melatonin and 0.5-1.5 mg / L Fe3O4 magnetic nanometer microspheres. The freezing protective agent can be prepared by using the diluent described above with a volume fraction of 50%-70%. The Fe3O4 magnetic nanometer microspheres have a particle size of 100-800 nm, and preferably, the Fe3O4 magnetic nanometer microspheres have a particle size of 500-600 nm.

[0023] Preferably, the diluent contains 7-9 g / L sodium chloride, 0.4-0.6 g / L potassium chloride and 14-16 g / L glucose. The freezing protective agent contains 9%-12% methanol by volume, 12%-16% propylene glycol by volume, 12%-16% dimethyl sulfoxide, 3.5-5.0 g / L sodium chloride, 0.2-0.4 g / L potassium chloride, 8-10 g / L glucose, 0.8-1.2 mmol / L melatonin and 0.8-1.2 mg / L Fe3O4 magnetic nanometer microspheres. The freezing protective agent can be prepared by using the diluent with a volume fraction of 56%-65%.

[0024] As preferred, the diluent takes water as solvent, contains 8g / L sodium chloride, 0.5g / L potassium chloride and 15g / L glucose. The cryoprotective solution is prepared by adding methanol, propylene glycol, dimethyl sulfoxide, melatonin and Fe3O4 magnetic nanospheres into the above diluent as solvent with a volume fraction of 60%, and contains a volume fraction of 15% dimethyl sulfoxide, a volume fraction of 10% methanol, a volume fraction of 10% propylene glycol, 1mmol / L melatonin and 0.001mg / mL Fe3O4 magnetic nanospheres; wherein the sodium chloride content is 4.8g / L, the potassium chloride content is 3g / L and the glucose content is 9g / L.

[0025] The diluent and the cryoprotective agent can be mixed when used, and the equilibrium solution I and the equilibrium solution II are configured respectively.

[0026] The diluent and the cryoprotective agent CPA are mixed in a volume ratio of 1.8-2.4:1 to configure the equilibrium solution I,

[0027] Preferably, the diluent and the cryoprotective agent CPA are mixed in a volume ratio of 2:1 to configure the equilibrium solution I.

[0028] The diluent and the cryoprotective agent CPA are mixed in a volume ratio of 1:2 to configure the equilibrium solution II.

[0029] Preferably, the diluent and the cryoprotective agent CPA are mixed in a volume ratio of 1:2 to configure the equilibrium solution II.

[0030] After the Mongolian gudgeon embryos are frozen by the method, the thawing method is as follows: the frozen Mongolian gudgeon embryos are sequentially placed in 0.4-0.6mol / L and 0.15-0.35mol / L sucrose solutions, balanced at room temperature for 8-12min, and finally transferred to the above diluent.

[0031] Preferably, the frozen Mongolian gudgeon embryos are sequentially placed in 0.45-0.55mol / L and 0.2-0.3mol / L sucrose solutions, balanced at room temperature for 9-11min.

[0032] In a preferred mode of the present application, the frozen Mongolian gudgeon embryos are sequentially placed in 0.5mol / L and 0.25mol / L sucrose solutions, and balanced at room temperature for 10min.

[0033] The present application has the following beneficial effects:

[0034] The present application provides a Mongolian gudgeon embryo cryopreservation method, which adopts a cryopreservation solution formula mixed with multiple solvents and the antioxidant melatonin, reduces the toxicity of a single organic solvent, reduces the toxic damage and oxidative damage of cells in the cryopreservation process, improves the cryopreservation effect, and the embryo hatching rate reaches 19.57%.

[0035] The application adds Fe3O4 nano material in the cryoprotectant, improves the heat conduction performance of the solution, increases the temperature rising and falling rate, and improves the recrystallization phenomenon in the rewarming process.

[0036] The application adopts the solid surface vitrification method, and single embryos are preserved in the form of droplets, so that the process is more refined, the temperature change of each embryo in the temperature rising and falling process is ensured to be uniform, and the preservation effect and the repeatability of the preservation scheme are improved. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 To pick up single Mongolian catfish tail bud stage embryos using a freezing ring;

[0038] Figure 2 To place the freezing ring in a metal block for cooling;

[0039] Figure 3 To insert the freezing ring into the freezing ring sleeve;

[0040] Figure 4 To seal the freezing ring sleeve by using a heat sealer;

[0041] Figure 5 To hatch the Mongolian catfish fry successfully after thawing and recovery. DETAILED DESCRIPTION

[0042] The embodiments and comparative examples of the application will be described in detail below with reference to the accompanying drawings:

[0043] Example 1: The Mongolian catfish embryo cryopreservation technology of the application is used to preserve embryos and detect the survival condition, and the specific process is as follows:

[0044] Step 1): Preparation of diluent, and the order of adding solutes has no special requirements, and the formula is shown in Table 1:

[0045] Table 1: Composition and amount of diluent

[0046]

[0047] Step 2): Preparation of cryoprotectant (CPA), the solvent is the diluent prepared in step 1), and the order of adding solutes has no special requirements, and the formula is shown in Table 2:

[0048] Table 2: Formula of cryoprotectant (CPA)

[0049]

[0050] Note: The particle size of Fe3O4 magnetic nano microspheres is 500-600 nm

[0051] Step 3): Preparation of equilibrium solution ES1 and ES2, mix the CPA prepared in step 2 and the diluent prepared in step 1 in a ratio of 1:2 to obtain the equilibrium solution ES1 with one-third CPA concentration; mix the CPA prepared in step 2 and the diluent prepared in step 1 in a ratio of 2:1 to obtain the equilibrium solution ES2 with two-thirds CPA concentration.

[0052] Step 4): Collect the embryos of P. mongolicus, observe the development process of the embryos, and select the tail-bud stage embryos for cryopreservation.

[0053] Step 5): Place the aluminum alloy cooling metal block in liquid nitrogen and insert the freezing ring into the liquid nitrogen.

[0054] Step 6): Transfer the tail-bud stage embryos of P. mongolicus into the equilibrium solution ES1, balance for 10 min, then transfer into the equilibrium solution ES2 for 10 min, and finally transfer into the cryoprotectant CPA.

[0055] Step 7): Pick up a single tail-bud stage embryo of P. mongolicus with a freezing ring, quickly place it on the metal block for vitrification for 10 s, then insert it into the freezing ring, and the whole process is carried out on the liquid nitrogen, as shown in Figure 1 ; quickly place it on the metal block for vitrification for 10 s, then insert it into the freezing ring, and the whole process is carried out on the liquid nitrogen, as shown in Figure 2 ; then insert it into the freezing ring, and the whole process is carried out on the liquid nitrogen, as shown in Figure 3 . The single embryo is stored in the form of a droplet.

[0056] Step 8): Seal the freezing ring with a heat sealer, as shown in Figure 4 , then transfer it to a liquid nitrogen tank for storage.

[0057] After the embryos are frozen in liquid nitrogen for 10-30 s, they are taken out and thawed according to the following method and continue to hatch.

[0058] A) Take out the freezing ring containing the embryos of P. mongolicus from the liquid nitrogen, cut open the sealing place with scissors, take out the freezing ring, and quickly insert it into a 0.5 mol / L sucrose solution at 28℃ for 10 min.

[0059] B) Transfer the embryos of P. mongolicus into a 0.25 mol / L sucrose solution, balance at room temperature for 10 min, and finally transfer into the diluent.

[0060] The thawed embryos of P. mongolicus continue to hatch in the breeding water. The survival rate of P. mongolicus embryos after cryopreservation of different batches is shown in Table 3.

[0061] Table 3 Survival rate of P. mongolicus embryos after cryopreservation

[0062]

[0063] Pictures of hatched P. mongolicus fry after cryopreservation are shown in Figure 5 .

[0064] In the process of embryo cryopreservation of Procyprinus, the solid surface vitrification method is used to preserve single embryo in the form of droplet, the process is more refined, and the temperature change of each embryo during temperature rise and fall is ensured to be uniform, thereby improving the embryo preservation effect and the repeatability of the preservation scheme.

Claims

1. A method for cryopreservation of embryos of Pseudobagrus vachelli, characterized in that, Includes the following steps: (1) The embryos of Culter mongolica were equilibrated in equilibration solution one and equilibration solution two for 8-12 minutes respectively; Prepare equilibrium solution one by mixing the cryoprotectant and diluent at a volume ratio of 1:1.8-2.4; prepare equilibrium solution two by mixing the cryoprotectant and diluent at a volume ratio of 1.8-2.4:

1. The diluent uses water as a solvent and contains 6-10 g / L sodium chloride, 0.3-0.7 g / L potassium chloride, and 12-18 g / L glucose. The cryoprotectant uses water as a solvent and contains 8%-15% methanol, 10%-20% propylene glycol, and 10%-20% dimethyl sulfoxide by volume, as well as 3.5-5.0 g / L sodium chloride, 0.2-0.4 g / L potassium chloride, 7-11 g / L glucose, 0.5-1.5 mmol / L melatonin, and 0.5-1.5 mg / L Fe3O4 magnetic nanospheres with a particle size of 100-800 nm. (2) Transfer the Mongolian bream embryos to cryoprotectant, collect individual Mongolian bream embryos, place them on a metal block cooled by liquid nitrogen for 8-12 seconds, place them in a cryopreservation container and seal it, and preserve them in liquid nitrogen.

2. The method for cryopreservation of embryos of Procydiurn modestum according to claim 1, characterized in that, The first equilibration solution is prepared by mixing the cryoprotectant and the diluent at a volume ratio of 1:2; the second equilibration solution is prepared by mixing the cryoprotectant and the diluent at a volume ratio of 2:

1.

3. The method for freezing the embryos of Procyprinus major according to claim 1, wherein, In step (1), the oocytes are equilibrated in equilibration solution one and equilibration solution two for 10 minutes each.

4. The method for freezing the embryos of Procyonus mantschuricus according to claim 1, characterized in that, The diluent contains 7-9 g / L sodium chloride, 0.4-0.6 g / L potassium chloride, and 14-16 g / L glucose; The cryoprotectant contains 9%-12% methanol, 12%-16% propylene glycol, and 12%-16% dimethyl sulfoxide by volume, and also contains 3.5-5.0 g / L sodium chloride, 0.2-0.4 g / L potassium chloride, 8-10 g / L glucose, 0.8-1.2 mmol / L melatonin, and 0.8-1.2 mg / L Fe3O4 magnetic nanospheres.

5. The method for freezing the embryos of Procyonus mantschuricus according to claim 1, characterized in that, In step (2), a single Culter alburnus embryo is collected using a cryo-hook; the cryopreservation container is a cryo-ring.

6. The method for freezing the embryos of Procyonus mantschuricus according to claim 1, characterized in that, The embryo of the Mongolian bream described is in the tail bud stage.

7. A reagent for cryopreservation of embryos of Pseudobagrus fulvidraco, characterized in that, It includes at least one of a diluent and a cryoprotectant, wherein the diluent uses water as a solvent and contains 6-10 g / L sodium chloride, 0.3-0.7 g / L potassium chloride, and 12-18 g / L glucose; The cryoprotectant uses water as a solvent and contains 8%-15% methanol, 10%-20% propylene glycol, and 10%-20% dimethyl sulfoxide by volume, as well as 3.5-5.0 g / L sodium chloride, 0.2-0.4 g / L potassium chloride, 7-11 g / L glucose, 0.5-1.5 mmol / L melatonin, and 0.5-1.5 mg / L Fe3O4 magnetic nanospheres.

8. The reagent for cryopreservation of embryos of Pseudobagrus kena according to claim 7, wherein, The diluent contains 7-9 g / L sodium chloride, 0.4-0.6 g / L potassium chloride, and 14-16 g / L glucose; The cryoprotectant contains 9-12% (volume fraction) methanol, 12-16% (volume fraction) propylene glycol, 12-16% (volume fraction) dimethyl sulfoxide, and contains 3.5-5.0 g / L sodium chloride, 0.2-0.4 g / L potassium chloride, 8-10 g / L glucose, 0.8-1.2 mmol / L melatonin and 0.8-1.2 mg / L Fe3O4 magnetic nanoballs.

9. The reagent for cryopreservation of embryos of Pseudobagrus kena according to claim 7, wherein, The diluent contains 8 g / L sodium chloride, 0.5 g / L potassium chloride and 15 g / L glucose; the cryoprotectant contains 4.8 g / L sodium chloride, 3 g / L potassium chloride and 9 g / L glucose, 15% (volume fraction) dimethyl sulfoxide, 10% (volume fraction) methanol, 10% (volume fraction) propylene glycol, 1 mmol / L melatonin and 1 mg / L Fe3O4 magnetic nanoballs.

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