Epinephelus lanceolatus frozen sperm activation liquid

Abstract

The invention discloses an epinephelus lanceolatus frozen sperm activating solution which is characterized in that the activating solution is a mixed aqueous solution of calcium chloride and sodium chloride and / or potassium chloride, and the osmotic pressure is 1000mOsmol / kg. The activation liquid can improve the activity of the frozen sperms of the epinephelus lanceolatus after activation.

Description

Technical Field

[0001] The invention belongs to the technical field of fish genetic resource protection, and particularly relates to an activation liquid for frozen sperm of grouper. Background Art

[0002] The saddleback grouper (Epinephelus lanceolatus), commonly known as dragon grouper or dragon gall, is widely distributed in the Indo-Pacific region and is the world's largest coral reef fish, reaching up to 2.7 meters in length and weighing up to 400 kg. Saddleback groupers are an important commercial fish for marine fishing and aquaculture. Due to the easy availability of large quantities of sperm, they are often used as sires in the grouper industry, resulting in hybrids such as the tiger-dragon hybrid grouper and the cloud-dragon grouper. Therefore, the cryopreservation technology and cryopreserved sperm of saddleback groupers have extremely high economic and application value.

[0003] However, after ultra-low temperature cryopreservation, saddle grouper sperm may suffer from freezing damage such as cell membrane damage, DNA damage, oxidative damage, acrosome and flagella damage, and its physical and chemical properties will change, resulting in decreased sperm motility after activation. After successful cryopreservation of saddle grouper sperm, existing technology usually uses filtered seawater for activation, allowing sperm movement and fertilization with the egg to complete the fertilization process; however, after activation, sperm motility is only 75-82% (see Yang S, et al. Optimization of a sperm cryopreservation protocol for giant grouper (Epinephelus lanceolatus). Aquaculture, 2022, 555: 738-186). This shows that using filtered seawater to activate sperm does not achieve the optimal activation effect, and there is room for improvement. Currently, there are no relevant literature or patents on sperm activation of grouper. Therefore, the development of saddle grouper frozen sperm activation solution will help improve the sperm motility of saddle grouper frozen sperm after activation. Summary of the Invention

[0004] The invention aims to provide a saddle grouper frozen sperm activation liquid to improve the activity of the saddle grouper frozen sperm after activation.

[0005] To achieve the above objectives, the present invention adopts the following technical solutions.

[0006] The invention discloses an activation solution for frozen sperm of grouper, which is characterized by comprising a mixed aqueous solution of calcium chloride, sodium chloride and / or potassium chloride.

[0007] The osmotic pressure of the saddle grouper frozen sperm activation solution is 1000mOsmol / kg.

[0008] The present invention's frozen saddle grouper sperm activation solution comprises calcium chloride and sodium chloride and / or potassium chloride as solutes, maintaining an osmotic pressure of 1000 mOsmol / kg and achieving an activation effect superior to filtered seawater. Sodium chloride, a monovalent inorganic salt, regulates the influx of sodium ions and the efflux of hydrogen ions in sperm cells, maintaining osmotic pressure balance inside and outside the sperm cells and stabilizing the pH value. Potassium chloride, a monovalent inorganic salt, maintains osmotic pressure balance inside and outside the sperm cells and promotes sperm membrane hyperpolarization. Calcium chloride, a divalent inorganic salt, provides a large calcium source for sperm cells, helping to activate calcium-dependent signaling pathways, releasing energy, and improving sperm motility.

[0009] As a preferred embodiment of the present invention, the activating solution for frozen sperm of Epinephelus saddleback is a mixed aqueous solution of 200 mM calcium chloride and 200 mM sodium chloride.

[0010] As a preferred embodiment of the present invention, the activating solution for frozen sperm of Epinephelus saddleback is a mixed aqueous solution of 200 mM calcium chloride and 200 mM potassium chloride.

[0011] As a preferred embodiment of the present invention, the saddle grouper frozen sperm activation solution is a mixed aqueous solution of 143mM calcium chloride, 143mM sodium chloride, and 143mM potassium chloride.

[0012] As a preferred embodiment of the present invention, the saddle grouper frozen sperm activation solution is a mixed aqueous solution of 91mM calcium chloride, 91mM sodium chloride, and 273mM potassium chloride.

[0013] As a preferred embodiment of the present invention, the activation solution for frozen sperm of grouper is a mixed aqueous solution of 231mM calcium chloride, 77mM sodium chloride and 77mM potassium chloride; this embodiment has the best activation effect.

[0014] The method for using the saddle grouper frozen sperm activation liquid of the present invention comprises the following steps: (1) rapidly transferring the saddle grouper sperm stored at ultra-low temperature in liquid nitrogen to a 40°C water bath for thawing; (2) after the frozen sperm is thawed, fully mixing the thawed sperm with fresh eggs; and (3) adding the saddle grouper frozen sperm activation liquid to activate the thawed sperm and complete the fertilization process.

[0015] The amount of the frozen sperm activation solution of grouper is 10% of the sperm volume. 3 times.

[0016] The ratio of thawed sperm to fresh eggs is 10 6 :1.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention develops for the first time a saddle grouper frozen sperm activation liquid, which effectively promotes sperm activation and improves the motility of saddle grouper frozen sperm.

[0019] Furthermore, the activating liquid for frozen sperm of grouper of the present invention has simple ingredients, using only 2 to 3 kinds of inorganic salts and no other complex ingredients; therefore, it is simple to prepare and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Results of experiments comparing the activation effects of artificial sea salt and sucrose under different osmotic pressures are shown.

[0021] Figure 2 Shows the experimental results comparing the activation effects of sodium chloride, potassium chloride, and calcium chloride under different osmotic pressures. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below through specific implementation cases so that those skilled in the art can better understand and implement the technical solutions of the present invention.

[0023] Experimental Example 1:

[0024] In order to explore the effects of multi-ion activation solution (artificial sea salt) and ion-free activation solution (sucrose) on the activation of frozen sperm of saddle grouper, Experimental Example 1 of this application configured artificial sea salt activation solution and sucrose activation solution.

[0025] The following concentrations of artificial sea salt activation solutions were prepared respectively: 13.5 g / L, 22.5 g / L, 31.5 g / L, 40.5 g / L, 45 g / L, 49.5 g / L, 58.5 g / L, 67.5 g / L, and 76.5 g / L; the corresponding preparation methods were as follows: artificial sea salt was added to deionized water and dissolved to obtain the artificial sea salt activation solution; the corresponding osmotic pressures were: 300 mOsmol / kg, 500 mOsmol / kg, 700 mOsmol / kg, 900 mOsmol / kg, 1000 mOsmol / kg, 1100 mOsmol / kg, 1300 mOsmol / kg, 1500 mOsmol / kg, and 1700 mOsmol / kg (see Table 1).

[0026] The following concentrations of sucrose activation solutions were prepared respectively: 300 mM, 500 mM, 700 mM, 900 mM, 1000 mM, 1100 mM, 1300 mM, 1500 mM, and 1700 mM; the corresponding preparation method was: adding sucrose to deionized water and dissolving it to obtain the sucrose activation solution; the corresponding osmotic pressures were: 300 mOsmol / kg, 500 mOsmol / kg, 700 mOsmol / kg, 900 mOsmol / kg, 1000 mOsmol / kg, 1100 mOsmol / kg, 1300 mOsmol / kg, 1500 mOsmol / kg, and 1700 mOsmol / kg (see Table 1).

[0027] Table 1

[0028]

[0029] The saddle grouper sperm cryopreserved in liquid nitrogen was quickly transferred to a 40°C water bath to be thawed for 7 seconds. Then, artificial sea salt activation solution and sucrose activation solution were used to activate the saddle grouper frozen sperm in the breeding pond. The saddle grouper frozen sperm was activated by contact with the saddle grouper frozen sperm. The activation time was 20 seconds. The results of activating the saddle grouper frozen sperm are shown in the figure. Figure 1 shown.

[0030] Figure 1 The results showed that: (1) when artificial sea salt and sucrose solution were used to activate sperm, the activation effect was best at 900-1100 mOsmol / kg; (2) when sperm was activated at an osmotic pressure of 700-1300 mOsmol / kg, artificial sea salt had a better activation effect than sucrose, which indicated that the ions in artificial sea salt were beneficial to the activation of frozen sperm of grouper.

[0031] Example 2

[0032] In order to explore the effects of different ions on the activation of frozen sperm of grouper, this Example 2 prepared sodium chloride activation solution, potassium chloride activation solution, and calcium chloride activation solution for experiment.

[0033] The following concentrations of sodium chloride activation solution were prepared respectively: 150 mM, 250 mM, 350 mM, 450 mM, 500 mM, 550 mM, 650 mM, 750 mM, and 850 mM; the corresponding preparation method was: sodium chloride was added to deionized water and dissolved to obtain the sodium chloride activation solution; the corresponding osmotic pressures were: 300 mOsmol / kg, 500 mOsmol / kg, 700 mOsmol / kg, 900 mOsmol / kg, 1000 mOsmol / kg, 1100 mOsmol / kg, 1300 mOsmol / kg, 1500 mOsmol / kg, and 1700 mOsmol / kg (see Table 2).

[0034] The following concentrations of potassium chloride activation solution were prepared respectively: 150mM, 250mM, 350mM, 450mM, 500mM, 550mM, 650mM, 750mM, and 850mM; the corresponding preparation method was: adding potassium chloride to deionized water and dissolving it to obtain the potassium chloride activation solution; the corresponding osmotic pressures were: 300mOsmol / kg, 500mOsmol / kg, 700mOsmol / kg, 900mOsmol / kg, 1000mOsmol / kg, 1100mOsmol / kg, 1300mOsmol / kg, 1500mOsmol / kg, and 1700mOsmol / kg (see Table 2).

[0035] The following concentrations of calcium chloride activation solution were prepared respectively: 100 mM, 167 mM, 233 mM, 300 mM, 333 mM, 367 mM, 433 mM, 500 mM, and 567 mM; the corresponding preparation method was: adding calcium chloride to deionized water and dissolving it to obtain the calcium chloride activation solution; the corresponding osmotic pressures were: 300 mOsmol / kg, 500 mOsmol / kg, 700 mOsmol / kg, 900 mOsmol / kg, 1000 mOsmol / kg, 1100 mOsmol / kg, 1300 mOsmol / kg, 1500 mOsmol / kg, and 1700 mOsmol / kg (see Table 2).

[0036] Table 2

[0037]

[0038] The saddle grouper sperm cryopreserved in liquid nitrogen was quickly transferred to a 40°C water bath to be thawed for 7 seconds. Then, sodium chloride activation solution, potassium chloride activation solution, and potassium chloride activation solution were used to activate the saddle grouper frozen sperm in the breeding pond. The saddle grouper frozen sperm was activated by contact with the saddle grouper frozen sperm. The activation time was 20 seconds. The results of activating the saddle grouper frozen sperm are shown in the figure. Figure 2 shown.

[0039] Figure 2 The results showed that: (1) Sodium chloride and calcium chloride solutions had the best activation effect at 900-1100 mOsmol / kg; (2) Potassium chloride solution had the best activation effect at 1000-1100 mOsmol / kg; (3) All three activation solutions had the best activation effect at an osmotic pressure of 1000 mOsmol / kg.

[0040] Example 3

[0041] To explore the effects of different combinations of ions on the activation of frozen sperm of grouper, this Example 3 prepared a combination activation solution of sodium chloride, potassium chloride, and calcium chloride based on the results of Example 2 (the best activation effect was achieved under an osmotic pressure of 1000 mOsmol / kg).

[0042] Prepare the activation solution using the following sodium chloride / potassium chloride / calcium chloride concentrations:

[0043] Combined activation solution 1: 250mM sodium chloride + 250mM potassium chloride;

[0044] Combined activation solution 2: 200 mM sodium chloride + 200 mM calcium chloride;

[0045] Combined activation solution 3: 200 mM potassium chloride + 200 mM calcium chloride;

[0046] Combined activation solution 4: 143mM sodium chloride + 143mM potassium chloride + 143mM calcium chloride;

[0047] Combined activation solution 5: 273mM sodium chloride + 91mM potassium chloride + 91mM calcium chloride;

[0048] Combined activation solution 6: 91mM sodium chloride + 273mM potassium chloride + 91mM calcium chloride;

[0049] Combined activation solution 7: 77mM sodium chloride + 77mM potassium chloride + 231mM calcium chloride;

[0050] The above-mentioned combined activation solution was prepared by adding different combinations of sodium chloride / potassium chloride / calcium chloride to deionized water for dissolution to obtain a sodium chloride / potassium chloride / calcium chloride combined activation solution for frozen saddle grouper sperm; the corresponding osmotic pressures were all 1000 mOsmol / kg and pH 8.0 (see Table 3).

[0051] The saddle grouper sperm ultra-low temperature cryopreserved in liquid nitrogen was quickly transferred to a 40°C water bath for thawing for 7 seconds. The above-mentioned combined activation liquid was used to activate the saddle grouper frozen sperm in the breeding pond. The saddle grouper frozen sperm was activated by contact with the saddle grouper frozen sperm. The activation time was 20 seconds. The results of activating the saddle grouper frozen sperm are shown in Table 3.

[0052] Table 3

[0053]

[0054] Note: In Table 3, different letters indicate significant differences, while the same letters indicate no significant differences.

[0055] The results in Table 3 show that the activation effects of combination activation solutions 2, 3, 4, 6, and 7 on the frozen sperm of Epinephelus saddleback are better than those of the artificial sea salt activation solution in the control group, that is, better than the seawater used in the prior art. In other words, the above-mentioned combination activation solutions can improve the motility of the frozen sperm of Epinephelus saddleback; among them, combination activation solution 7 (77 mM sodium chloride + 77 mM potassium chloride + 231 mM calcium chloride) has the best activation effect.

Claims

1. A frozen sperm activation solution for grouper, characterized in that: A mixed aqueous solution of calcium chloride and sodium chloride and / or potassium chloride with an osmotic pressure of 1000mOsmol / kg.

2. The activating solution for frozen sperm of grouper according to claim 1, characterized in that: A mixed aqueous solution of 200 mM calcium chloride and 200 mM sodium chloride.

3. The activating solution for frozen sperm of grouper according to claim 1, characterized in that: A mixed aqueous solution of 200 mM calcium chloride and 200 mM potassium chloride.

4. The activating solution for frozen sperm of grouper according to claim 1, characterized in that: A mixed aqueous solution of 143mM calcium chloride, 143mM sodium chloride, and 143mM potassium chloride.

5. The activating solution for frozen sperm of grouper according to claim 1, characterized in that: A mixed aqueous solution of 91mM calcium chloride, 91mM sodium chloride, and 273mM potassium chloride.

6. The activating solution for frozen sperm of grouper according to claim 1, characterized in that: A mixed aqueous solution of 231mM calcium chloride, 77mM sodium chloride, and 77mM potassium chloride.

7. The activating solution for frozen sperm of grouper according to claim 1, characterized in that: The method for using the saddle grouper frozen sperm activation liquid comprises the following steps: (1) rapidly transferring the saddle grouper sperm that has been ultra-low temperature frozen in liquid nitrogen to a 40° C. water bath for thawing; (2) after the frozen sperm is thawed, fully mixing the thawed sperm with fresh eggs; and (3) adding the saddle grouper frozen sperm activation liquid to activate the thawed sperm and complete the fertilization process.

8. The activating solution for frozen sperm of grouper according to claim 7, characterized in that: The amount of the frozen sperm activation solution of grouper is 10% of the sperm volume. 3 times.

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