CN110521721B - Method for freezing and storing grouper embryos by utilizing non-permeable antifreeze agent and open carrier - Google Patents

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

Method for cryopreservation of grouper embryos using impermeable antifreeze and open carriers

Technical field:

The invention belongs to the technical field of ultra-low temperature preservation of fish embryos in cryobiology, and particularly relates to a method for cryopreserving grouper embryos by using a non-penetrating antifreeze agent and an open carrier.

technical background:

Cryopreservation of fish embryos is only about 60 years old. Due to the large size (1-7mm) of fish embryos, low cell permeability, double membrane, and high yolk content, internal ice crystals are easily formed during freezing, and the sensitivity to freezing damage is high. Embryos are difficult, still in the exploratory research stage, and have not yet been applied to actual production and establishment of germplasm resources.

At present, fish embryo cryopreservation generally uses a 0.25ml thin tube as the main carrier for embryo freezing, and vitrification solution containing a high concentration of mixed antifreeze is a cryoprotectant. Generally, the method of gradually adding vitrification solution is used for infiltration treatment. However, because the thin tube is used as a carrier, each tube can hold up to 20-25 embryos. The number of embryos frozen per unit time is limited, and large-scale freezing experiments cannot be carried out, which limits the number of surviving fish embryo cryopreservation embryos; The concentration of vitrification should be at least 40%-60% (Hua Zezhao, Ren Hesheng. Low temperature biomedical technology. Beijing: Science Press, 1994). When embryos are exposed to high concentrations of antifreeze for too long, It will cause obvious damage to the structure and function of the embryo, resulting in a decrease in the survival rate and hatching rate of the embryo, and even lead to the abnormality of the surviving embryo after vitrification and the inability to continue to develop and cultivate.

Invention content:

In order to solve the above-mentioned technical problems, the present invention provides a method for rapidly cryopreserving moire grouper by utilizing a non-penetrating antifreeze agent and an open carrier, which can improve the cryopreservation effect of embryos and can greatly shorten the freezing time. The time required for the process increases the number of embryos frozen per unit time.

The present invention is achieved through the following technical solutions:

A method for cryopreserving grouper embryos utilizing a non-penetrating antifreeze and an open carrier, the method comprising the following steps:

(1) Impermeable antifreeze preparation: take the filtered seawater sterilized by high temperature as the base solution to prepare 1mol/L impermeable sugar solution; Described sugar is a kind of in sucrose, trehalose or glucose;

(2) Collection and culture of grouper embryos: Obtain fertilized eggs of moire grouper, culture the fertilized eggs in seawater at 22-24 °C, and freeze the embryos until they develop to the sarcomere stage, tail bud stage or embryo body rotation stage. save;

(3) open carrier preparation: prepare about 40-50ml plastic tube and sieve mesh, the plastic tube height is 100-116mm, and the diameter is 28-30mm, and both ends of the plastic tube are open, and one end is sealed with a mesh mesh;

Further, the mesh diameter of the screen mesh is 0.180mm, and the specification is 60×60mm.

(4) Embryo freezing: add the prepared non-permeable sugar solution to the petri dish, place a certain number of moire grouper embryos, and process for 4-10 minutes; then pour the processed embryos into the Open carrier, the bottom opening is vertically immersed in liquid nitrogen for cryopreservation; for long-term storage, after 20 minutes of freezing, the other end of the opening is blocked and placed in a liquid nitrogen tank for long-term storage;

(5) Embryos thawing and culture: place the high-temperature sterilized filtered seawater in a glass beaker, and control the temperature of the filtered seawater at 25-35°C; put the open carrier containing the frozen embryos in the step (4) Placed in filtered seawater, gently stirred and thawed; then, the beaker was placed in a biochemical incubator at 26°C, and the seawater was replaced after half an hour to continue cultivation.

The beneficial effects of the present invention compared with the prior art are as follows:

(1) The antifreeze used in the present invention has only impermeable sucrose, trehalose or glucose, and the composition is simple and non-toxic, and will not cause "solution damage" to fish embryo cells. Other fish embryo cryopreservation solutions contain small molecule antifreezes (dimethyl sulfoxide, methanol, etc.) with strong permeability. Due to their strong toxicity, they will inevitably cause "solution damage" to the embryos, thereby reducing freezing. Embryo survival.

(2) The number of cryopreserved embryos is large. Currently, the commonly used open carrier for cryopreservation of fish embryos is generally a 0.25ml straw. The number of cryopreserved embryos is limited, and only 20 embryos can be cryopreserved at a time. The freezing efficiency is low. The method can cryopreserve more than 1000 embryos, which greatly improves the cryopreservation efficiency.

(3) The rate of cooling and thawing is fast, and the open carrier for the embryos that is connected up and down can directly contact the surface of the embryos with liquid nitrogen to achieve the effect of rapid cooling; It solves the disadvantage of low temperature conduction efficiency of freezing vehicles such as plastic straws.

(4) The survival rate and hatching rate of frozen embryos are high, the survival rate of moire grouper embryos can reach 31.30% and the hatching rate can reach 41.28% by using the method of the present invention; the currently reported survival rate is 5.15% (Tian et al. ., 2018, Theriogenology).

Description of drawings:

Figure 1 is a schematic diagram of the production of an open vehicle;

Figure 2 Schematic diagram of the operation of freezing embryos in an open carrier.

Detailed ways:

The specific embodiments of the present invention will be further described in detail below with reference to the examples. The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

Example 1: The survival rate of embryos treated with impermeable antifreeze

1 mol/L lactose, fructose, glucose, mannitol, astragalus polysaccharide, sucrose, trehalose were prepared with filtered sterilized seawater, respectively. The pre-embryos were incubated for 60 min, and 300 embryos were processed for each sample, which was repeated 3 times. The treated embryos were cultured in a 26°C incubator. Embryo culture conditions: seawater temperature 26.71±1.26℃, dissolved oxygen 6-10mg/L, pH=7.45～8.49, seawater salinity 33.03±0.75‰. The survival rate of embryos was counted after 5 hours of embryo culture.

The results showed that the survival rate and hatching rate of embryos treated with sucrose, trehalose and glucose were higher. The specific results are shown in Table 1.

Table 1. Survival rate and hatching rate of moire grouper embryos at different stages after treatment with seven non-permeable cryoprotectants

Example 2: Impermeable Antifreeze Concentration and Treatment Time

According to the results in Table 1 of Example 1, sucrose was selected as the impermeable antifreeze. Use sterile filtered seawater as the base solution with 0.5-2M sucrose solution. The moire grouper embryos at the sarcomeric and tail bud developmental stages were selected for processing under a stereo microscope. Embryos were equilibrated in petri dishes containing various molar concentrations of sucrose solutions for 4-9 minutes at room temperature. Use a microscope to observe the morphology of the treated embryos. The specific results are shown in Table 2. Using 1M sucrose to treat the sarcomere stage embryos for 6-7min and the tail bud stage embryos for 5-7min, 70-80% of the embryos showed a dehydration state, and the embryos appeared Concave sphere, this time is the optimal processing condition.

Table 2 Morphological statistics of moire grouper embryos treated with different concentrations of sucrose solution for different times

"+", 70-80% of the embryos are dehydrated under the microscope, half of the volume of the round embryo is a concave sphere, and the molar concentration and treatment time are the optimal conditions at this time;

"-", non-optimal condition.

Example 3: Cryopreservation of sarcomeric stage embryos of moire grouper

The specific method is as follows:

(1) Preparation of impermeable antifreeze agent: prepare 1 mol/L sucrose solution with the filtered seawater sterilized at high temperature as the base solution;

(2) Collection and culture of grouper embryos: artificially propagate the fertilized eggs of moire grouper, culture the fertilized eggs in seawater at 22-24 °C, observe the development of embryos with an inverted biological microscope, and cryopreservation when they develop to the sarcomere stage ;

(3) Open carrier preparation: prepare a 50ml conical bottom centrifuge tube and a 0.180mm sieve mesh. First, cut off the tapered part of the bottom end of the centrifuge tube, about 3 cm high. Then cut a circular opening with a diameter of 25mm in the middle of the centrifuge tube cap, and place a 60×60mm 0.180mm sieve mesh between the bottle body and the cap and tighten it, as shown in Figure 1;

(4) Embryos freezing: add the prepared sucrose solution to a petri dish, place a certain number of moire grouper embryos, and process for 4-10 minutes. Then, the embryos in the petri dish were poured into the embryo freezing open carrier prepared in the step (3), and the opening was vertically immersed in liquid nitrogen (-196°C) for 24 hours, as shown in Figure 2. For long-term storage, the tapered part cut out in the step (3) can be inserted upside down into the bottom opening of the open carrier after 20 minutes, sealed and placed in a liquid nitrogen tank for long-term storage;

(5) Embryos thawing: The high-temperature sterilized filtered seawater was placed in a glass beaker, and the water temperature in the beaker was controlled at 26° C. with a water bath. The open carrier containing the frozen embryos in the step (4) is placed in a beaker, and the water body is gently stirred until there are no obvious lumps in the water. Then, the beaker was taken out of the water bath and placed in a biochemical incubator with a temperature set to 26°C, and the seawater was replaced after half an hour.

The embryos in the beaker were transferred to an 8L transparent incubator, cultured in micro-aerated, microfluidic water, the water temperature was controlled at 26±1°C, the pH was 7.45-8.4, the salinity was 30‰, and the dissolved oxygen was 6-10mg/L . The results of cryopreservation of sarcomere stage embryos of moire grouper are shown in Table 3.

Table 3 Frozen survival statistics of moire grouper sarcomere stage embryos

Example 4: Cryopreservation of embryos at the tail bud stage of moire grouper

(6) The embryos at the tail bud stage of the moire grouper were selected for cryopreservation under a stereo microscope. Use autoclaved filtered seawater as the base solution with 1 M sucrose solution. After equilibrating the embryos in a petri dish containing 1 M sucrose solution for 5, 6 and 7 min at room temperature, the embryos were placed in a self-made open carrier, directly inserted into liquid nitrogen (-196 °C), and frozen for 20 min. Afterwards, the cut conical part was inserted upside down into the bottom opening of the open carrier, sealed and placed in a liquid nitrogen tank for long-term storage; other steps were the same as in Example 3.

Put the high-temperature sterilized filtered seawater in a glass beaker, set the temperature of the water bath to 26°C, and when the water temperature inside and outside the beaker is the same, place the open carrier containing the frozen embryos in the beaker, and gently stir the water until there is no more water in the water. Obvious lumps. Then, the beaker was removed from the water bath and placed in a biochemical incubator with a temperature set to 26°C. Half an hour later, the embryos in the beaker were transferred to an 8L transparent incubator, cultured in micro-aerated, microfluidic water, the water temperature was controlled at 26±1°C, the pH was 7.45-8.4, the salinity was 30‰, and the dissolved oxygen was 6 -10mg/L. The results of cryopreservation of embryos at the tail bud stage of moire grouper are shown in Table 4.

Table 4 Frozen survival statistics of embryos at the tail bud stage of moire grouper

Example 5: Cryopreservation of moire grouper tail bud stage embryos using straws

Embryos at the tail bud stage of moire grouper were selected for cryopreservation under a stereo microscope. The 35% PMG3S vitrification solution was prepared with high temperature sterilized filtered seawater as the base solution. The embryos were placed in a petri dish containing 35% PMG3S solution at room temperature. After each step was equilibrated for 5, 6 and 7 min in a five-step method, the embryos were sucked into a 0.25 mL straw, and the two ends were sealed with a sealer. Directly inserted into liquid nitrogen (-196°C) and stored frozen for 24 hours.

Place the high-temperature sterilized filtered seawater in a glass beaker, set the temperature of the water bath to 26°C, and when the water temperature inside and outside the beaker is the same, place the straw containing frozen embryos in the beaker, stir gently until thawed, and cut the wheat The tube will release the embryo to the embryo. Then, the beaker was removed from the water bath and placed in a biochemical incubator with a temperature set to 26°C. Half an hour later, the embryos in the beaker were transferred to an 8L transparent incubator, cultured in micro-aerated, microfluidic water, the water temperature was controlled at 26±1°C, the pH was 7.45-8.4, the salinity was 30‰, and the dissolved oxygen was 6 -10mg/L. The results of cryopreservation of embryos at the tail bud stage of moire grouper using straws are shown in Table 5.

Table 5. Statistics on the survival rate of embryos at the tail bud stage of moire grouper using straw cryopreservation

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the technical principles of the present invention, several improvements and modifications can also be made. These improvements and modifications It 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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