CN110521721B - Method for freezing and storing grouper embryos by utilizing non-permeable antifreeze agent and open carrier - Google Patents
Method for freezing and storing grouper embryos by utilizing non-permeable antifreeze agent and open carrier Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Abstract
The invention relates to a method for freezing and preserving grouper embryos by utilizing a non-permeable antifreeze agent and an open carrier, belonging to the technical field of ultralow-temperature preservation of fish embryos in low-temperature biology, wherein 1mol/L of non-permeable sugar solution is used as an antifreeze agent to obtain embryos which develop to a sarcomere stage, a tail bud stage or an embryo rotation stage for freezing and preserving; the open carrier is a plastic pipe with two open ends, and one end is sealed by a bolting silk net; placing the embryo into a refrigerant to be treated for 4-10 minutes; then pouring the processed embryo into an open carrier, and vertically immersing the processed embryo into liquid nitrogen with an upward bottom opening for freezing and storing; the method for freezing and storing the embryos has large quantity, does not cause solution damage to the embryonic cells of the fishes, and has high survival rate and hatching rate of the frozen embryos, the survival rate of the embryos of the zebra carps frozen and stored by the method can reach 31.30 percent, and the hatching rate can reach 41.28 percent.
Description
The technical field is as follows:
the invention belongs to the technical field of ultralow-temperature preservation of fish embryos in low-temperature biology, and particularly relates to a method for freezing and preserving grouper embryos by using a non-permeable antifreeze agent and an open carrier.
Technical background:
the fish embryo cryopreservation research has only been about 60 years old so far. Because the fish embryo has large volume (1-7mm), low cell permeability, double-layer membrane and high yolk content, internal ice crystals are easily formed during freezing, the freezing damage sensitivity is high, the freezing preservation of the fish embryo is more difficult than that of fish sperms and mammal embryos, the fish embryo is still in an exploration and research stage, and the fish embryo freezing preservation method cannot be applied to actual production and establishment of germplasm resource libraries.
At present, a 0.25ml thin tube is generally used for freezing and storing the embryo of the fish as a main bearing tool for freezing the embryo, a vitrification solution containing a high-concentration mixed antifreeze is used as a freezing protective agent, and the infiltration treatment is generally carried out by gradually adding the vitrification solution. However, as the thin tube is used as a carrier, each tube can contain 20-25 embryos at most, the freezing quantity of the embryos in unit time is limited, large-batch freezing experiments cannot be carried out, and the survival quantity of the embryos preserved by freezing the fish embryos is limited; furthermore, the concentration of the cryoprotectant to be vitrified is at least 40-60% (Chilobrachys huazhao, Chilobrachys jezoensis, Low-temperature biomedical technology, Beijing, science publishers, 1994), when the embryo is exposed to the high-concentration antifreeze for too long time, the structure and the function of the embryo are obviously damaged, the survival rate and the hatching rate of the embryo are reduced, and even the mature embryo after vitrification freezing can not be continuously developed and cultured due to malformation.
The invention content is as follows:
in order to solve the technical problems, the invention provides a method for rapidly freezing and preserving the zebra fish by using a non-permeable antifreeze agent and an open carrier, which can improve the freezing and preserving effect of embryos, greatly shorten the time required by the freezing process and improve the freezing quantity of the embryos in unit time.
The invention is realized by the following technical scheme:
a method for cryopreservation of grouper embryos using a non-penetrating antifreeze and an open vehicle, the method comprising the steps of:
(1) preparing an impermeable antifreeze agent: preparing 1mol/L non-permeable sugar solution by taking filtered seawater sterilized at high temperature as a basic solution; the sugar is one of sucrose, trehalose or glucose;
(2) collecting and culturing grouper embryos: obtaining fertilized eggs of the epinephelus coioides, culturing the fertilized eggs in seawater at the temperature of 22-24 ℃, and freezing and storing the fertilized eggs when the embryos are developed to the sarcomere stage, the tail bud stage or the embryo body rotation stage;
(3) preparing an open carrier, namely preparing about 40-50ml of plastic tube and a bolting silk net, wherein the height of the plastic tube is 116mm, the diameter of the plastic tube is 28-30mm, two ends of the plastic tube are opened, and one end of the plastic tube is sealed by the bolting silk net;
furthermore, the mesh diameter of the bolting silk net is 0.180mm, and the specification is 60 multiplied by 60 mm.
(4) Embryo freezing: adding the prepared non-permeable sugar solution into a culture dish, placing a certain amount of the Mylopharyngodon Piceus embryos, and treating for 4-10 minutes; pouring the processed embryo into the open carrier in the step (3), and vertically immersing the processed embryo into liquid nitrogen for freezing and storing with an opening at the bottom upwards; if the food is stored for a long time, after the food is frozen for 20 minutes, the opening at the other end is blocked, and the food is placed in a liquid nitrogen tank for long-term storage;
(5) embryo thawing and culturing: placing the filtered seawater sterilized at high temperature into a glass beaker, and controlling the temperature of the filtered seawater at 25-35 ℃; placing the open carrier containing the frozen embryos in the step (4) in filtered seawater, gently stirring and unfreezing; then, the beaker was placed in a biochemical incubator at 26 ℃ and half an hour later, the seawater was replaced and the cultivation was continued.
Compared with the prior art, the invention has the following beneficial effects:
(1) the antifreeze used in the invention is only non-permeable sucrose, trehalose or glucose, has simple components and no toxicity, and can not cause solution damage to fish embryonic cells. Other fish embryo cryopreservation liquids contain micromolecule antifreezes (dimethyl sulfoxide, methanol and the like) with strong permeability, and because of strong toxicity, the embryo cryopreservation liquids inevitably cause solution damage, thereby reducing the survival rate of the frozen embryo.
(2) The number of the frozen embryos is large, the number of the frozen embryos is limited, only 20 embryos can be frozen and preserved at one time, the freezing efficiency is low, the method can freeze and preserve more than 1000 embryos, and the freezing preservation efficiency is greatly improved.
(3) The temperature reduction and thawing rate is high, and the embryo open carrier which is communicated up and down can enable the surface of the embryo to be directly contacted with liquid nitrogen, so that the effect of rapid temperature reduction is achieved; when in unfreezing, the embryo can be directly contacted with seawater at the temperature of 25-35 ℃, and the defect of low temperature conduction efficiency of freezing carriers such as plastic straws and the like is overcome.
(4) The survival rate and the hatching rate of the frozen embryos are high, the survival rate of the embryos of the zebra carps frozen and stored by the method can reach 31.30%, and the hatching rate can reach 41.28%; the survival rate reported to date is 5.15% (Tian et al, 2018, Theriogenology).
Description of the drawings:
FIG. 1 is a schematic view of an open carrier fabrication;
FIG. 2 is a schematic view of an open carrier frozen embryo handling.
The specific implementation mode is as follows:
the following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1: survival rate of embryo treated by non-permeable antifreeze
Respectively preparing 1mol/L lactose, fructose, glucose, mannitol, astragalus polysaccharide, sucrose and trehalose by using filtered and sterilized seawater, respectively treating embryos of the zebra fish at 4-6 sarcomere stage, 16 sarcomere stage, tail bud stage and early hatching stage for 60min, treating 300 embryos in each sample, and repeating for 3 times. The treated embryos were cultured in an incubator at 26 ℃. Embryo culture conditions: the temperature of the seawater is 26.71 +/-1.26 ℃, the dissolved oxygen is 6-10mg/L, the pH value is 7.45-8.49, and the salinity of the seawater is 33.03 +/-0.75 per mill. And 5 hours after the embryo is cultured, counting the survival rate of the embryo.
The results show that the embryo treated by sucrose, trehalose and glucose has higher survival rate and hatching rate, and the specific results are shown in table 1.
TABLE 1 survival rate and hatching rate of Mylopharyngodon piceus embryos at different periods after treatment with seven kinds of non-permeable cryoprotectants
Example 2: non-penetrating antifreeze concentration and treatment time
Sucrose was selected as an impermeable antifreeze according to the results in table 1 in example 1. Sterilizing and filtering seawater to obtain basic solution, and mixing with 0.5-2M sucrose solution. Selecting Myxocyprinus striatus embryos at the muscle section stage and tail bud stage development stage under a stereo microscope for processing. Embryos are equilibrated at room temperature for 4-9 minutes in petri dishes containing different molarity sucrose solutions. The morphology of the treated embryos was observed by using a microscope, and the specific results are shown in Table 2, wherein 1M sucrose was used for treating the sarcomere embryos for 6-7min, the tail bud embryos for 5-7min, 70-80% of the embryos appeared in a dehydrated state, and the embryos appeared as spheroids, which is the most suitable treatment condition.
TABLE 2 statistics of the embryo morphology of Myxocyprinus variegatus after different time treatment with sucrose solutions of different concentrations
"+", 70-80% of embryos under microscope showed dehydrated state, a half of a sphere of the volume of round embryos, where molarity and treatment time were optimal;
"-", not optimal.
Example 3: cryopreservation of Mylopharyngodon mackerel sarcomere embryos
The specific method comprises the following steps:
(1) preparing an impermeable antifreeze agent: preparing 1mol/L sucrose solution by taking filtered seawater sterilized at high temperature as a basic solution;
(2) collecting and culturing grouper embryos: artificially propagating fertilized eggs of the epinephelus coioides, culturing the fertilized eggs in seawater at the temperature of 22-24 ℃, observing the development of embryos by using an inverted biomicroscope, and freezing and storing the fertilized eggs after the development reaches the sarcomere stage;
(3) preparing an open carrier: a50 ml conical bottom centrifuge tube and a 0.180mm silk screen were prepared. First, the conical section of the bottom end of the centrifuge tube was cut off, approximately 3cm high. Cutting a circular opening with a diameter of 25mm from the middle of the centrifuge tube cap, and placing a 60 × 60mm 0.180mm bolting silk net between the bottle body and the cap, and screwing tightly, as shown in fig. 1;
(4) embryo freezing: adding the prepared sucrose solution into a culture dish, placing a certain amount of the Epinephelus coioides embryos, and treating for 4-10 minutes. Then, the embryos in the culture dish are poured into the embryo freezing open carrier prepared in the step (3), and are vertically immersed into liquid nitrogen (-196 ℃) with the opening facing upwards for freezing and storing for 24 hours, as shown in figure 2. If the product is stored for a long time, after 20 minutes, the cut conical part in the step (3) is inversely inserted into the bottom opening of the open carrier, and the product is sealed and placed in a liquid nitrogen tank for long-term storage;
(5) and (3) unfreezing the embryo: the filtered seawater sterilized at high temperature was placed in a glass beaker, and the temperature of the water in the beaker was controlled at 26 ℃ with a water bath. Placing the open carrier containing the frozen embryos in step (4) in a beaker, and gently stirring the water until no lumps are evident in the water. Then, the beaker was taken out of the water bath, placed in a biochemical incubator set at 26 ℃, and the seawater was changed after half an hour.
And transferring the embryos in the beaker to an 8L transparent incubator, and adopting micro-aeration and micro-fluid water culture, wherein the water temperature is controlled to be 26 +/-1 ℃, the pH value is 7.45-8.4, the salinity is 30 per thousand, and the dissolved oxygen is 6-10 mg/L. The result of cryopreservation of the Mylopharyngodon Piceus myotomentosa embryos is shown in Table 3.
TABLE 3 frozen survival statistics of Myxocyprinus striatus myotomentosa embryos
Example 4: cryopreservation of embryos at tail bud stage of epinephelus coioides
(6) And selecting the embryos at the tail bud stage of the epinephelus coioides under a stereo microscope for freezing and storing. Filtering seawater sterilized at high temperature to obtain basic solution, and mixing with 1M sucrose solution. Placing the embryos in culture dishes containing 1M sucrose solution at room temperature, balancing for 5 min, 6 min and 7min respectively, placing the embryos in a self-made open carrier, directly inserting the embryos into liquid nitrogen (-196 ℃), freezing for 20 min, inversely inserting the cut conical parts into an opening at the bottom of the open carrier, sealing, and placing in a liquid nitrogen tank for long-term storage; the other steps are the same as in example 3.
Placing the filtered seawater sterilized at high temperature into a glass beaker, setting the temperature of a water bath kettle to be 26 ℃, placing an open carrier containing frozen embryos into the beaker when the temperature of the water inside and outside the beaker is consistent, and slightly stirring the water until no obvious blocks exist in the water. The beaker was then removed from the water bath and placed in a biochemical incubator set at 26 ℃. And after half an hour, transferring the embryos in the beaker to an 8L transparent incubator, and culturing by adopting micro-aeration and micro-fluid water, wherein the water temperature is controlled to be 26 +/-1 ℃, the pH value is 7.45-8.4, the salinity is 30 per thousand, and the dissolved oxygen is 6-10 mg/L. The cryopreservation results of the embryos at the tail bud stage of the epinephelus coioides are shown in table 4.
TABLE 4 frozen survival statistics of embryos at tail bud stage of Epinephelus coioides
Example 5: cryopreservation of embryos in tail bud stage of epinephelus coioides by using wheat pipes
And selecting the embryos at the tail bud stage of the epinephelus coioides under a stereo microscope for freezing and storing. Using filtered seawater sterilized at high temperature as a base solution, a 35% PMG3S vitrification solution was prepared. Placing the embryo in a culture dish containing 35% PMG3S solution at room temperature, balancing for 5, 6 and 7min by five steps, respectively, sucking the embryo into 0.25mL wheat tube, sealing both ends with sealer, and directly inserting into liquid nitrogen (-196 deg.C) for freezing and storing for 24 hr.
Placing the filtered seawater sterilized at high temperature in a glass beaker, setting the temperature of a water bath kettle at 26 ℃, placing the straw containing the frozen embryo in the beaker when the water temperature inside and outside the beaker is consistent, slightly stirring until thawing, and cutting the straw to release the embryo. The beaker was then removed from the water bath and placed in a biochemical incubator set at 26 ℃. And after half an hour, transferring the embryos in the beaker to an 8L transparent incubator, and culturing by adopting micro-aeration and micro-fluid water, wherein the water temperature is controlled to be 26 +/-1 ℃, the pH value is 7.45-8.4, the salinity is 30 per thousand, and the dissolved oxygen is 6-10 mg/L. The results of cryopreservation of the embryos at the tail bud stage of Epinephelus coioides by using a wheat straw are shown in Table 5.
TABLE 5 statistics of embryo survival rate in tail bud stage of Myxocyprinus striatus preserved by freezing in straw
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (2)
1. A method for cryopreservation of grouper embryos using a non-penetrating antifreeze and an open vehicle, the method comprising the steps of:
(1) preparing an impermeable antifreeze agent: preparing 1mol/L non-permeable sugar solution by taking filtered seawater sterilized at high temperature as a basic solution; the sugar is one of sucrose, trehalose or glucose;
(2) collecting and culturing grouper embryos: obtaining fertilized eggs of the epinephelus coioides, culturing the fertilized eggs in seawater at the temperature of 22-24 ℃, and freezing and storing the fertilized eggs when the embryos are developed to the sarcomere stage, the tail bud stage or the embryo body rotation stage;
(3) preparing an open embryo freezing carrier by preparing a plastic tube of 40-50ml and a bolting silk net, wherein the plastic tube has a height of 116mm and a diameter of 28-30mm, two ends of the plastic tube are open, and one end of the plastic tube is sealed by the bolting silk net;
(4) embryo freezing: adding the prepared non-permeable sugar solution into a culture dish, placing a certain amount of the Mylopharyngodon Piceus embryos, and treating for 4-10 minutes; pouring the processed embryo into the open embryo freezing carrier in the step (3), and vertically immersing the processed embryo into liquid nitrogen with an upward opening for freezing and storing;
embryo thawing and culturing: placing the filtered seawater sterilized at high temperature into a glass beaker, and controlling the temperature of the filtered seawater at 25-35 ℃; immersing the open embryo freezing carrier containing the frozen embryo in the step (4) into filtered seawater, and slightly stirring and unfreezing; then, the beaker is placed in a biochemical incubator at 26 ℃, the seawater is replaced after half an hour, and the cultivation is continued.
2. The method of claim 1, wherein the bolting silk mesh has a mesh diameter of 0.180mm and a gauge of 60 x 60 mm.
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| CN106417251A (en) * | 2016-09-21 | 2017-02-22 | 中国水产科学研究院黄海水产研究所 | High-concentration vitrification solution and ultra-low temperature cryopreservation method for grouper embryos |
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| CN106417251A (en) * | 2016-09-21 | 2017-02-22 | 中国水产科学研究院黄海水产研究所 | High-concentration vitrification solution and ultra-low temperature cryopreservation method for grouper embryos |
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| "High survival of mouse oocytes/embryos after vitrification without permeating cryoprotectants followed by ultra-rapid warming with an IR laser pulse";Bo jin et al.;《Scientific Reports》;20150319;摘要，第2页左栏2-3段，右栏1-4段，第5页左栏第4-6段、右栏第1-7段，表2 * |
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