Formula for maintaining high motility of epinephelus lanceolatus sperm and method of use

Abstract

The application discloses a formula for maintaining high activity of fresh sperm of Epinephelus lanceolatus, and is characterized by a water solution composed of any two of sodium chloride, potassium chloride and calcium chloride, and the osmotic pressure is 1000 mOsmol / kg. The formula can guarantee high activity of sperm after short-time activation and maintain activity of sperm after long-time movement, and can make the activity of sperm after 300 seconds of activation reach 78.53+4.03%.

Description

Technical Field

[0001] This invention belongs to the technical field of fish genetic resource protection, specifically relating to a formula and method for maintaining high sperm motility in saddleback grouper. Background Technology

[0002] The Queensland reef fish (Epinephelus lanceolatus), also known as the Queensland reef fish, spotted reef fish, or saddleback reef fish, is the largest reef fish distributed throughout the Indo-Pacific region. This species is extremely large, reaching a maximum length of 2.7 meters and a weight of up to 400 kilograms. However, due to its high economic value in the live reef fish trade, coupled with its long lifespan, late sexual maturity, and spawning colony characteristics, this species has suffered severe overfishing in recent decades, leading to a sharp decline in wild populations. Therefore, the Queensland reef fish is currently listed as a protected species in several countries. Nevertheless, despite the increasing depletion of wild resources, the Queensland reef fish remains an important species in grouper aquaculture due to its superior growth performance.

[0003] However, the insufficient supply of grouper fry severely restricts the sustainable development of its commercial aquaculture. A major bottleneck in artificial breeding is the insufficient number of mature male broodstock, highlighting the importance and urgency of improving sperm utilization efficiency in aquaculture practice. Currently, existing technologies use filtered and disinfected seawater to activate fresh grouper sperm, enabling sperm to fertilize the egg. However, experiments revealed that while seawater activation of fresh sperm for 20 seconds can achieve 90% sperm motility, after 300 seconds, sperm motility drops to only about 50%. Since artificial insemination typically takes about 5 minutes, the decline in sperm motility after 300 seconds of seawater activation affects the fertilization rate and reduces sperm utilization efficiency. Furthermore, there are currently no reports on how to maintain high sperm motility in grouper. Summary of the Invention

[0004] The purpose of this invention is to provide a formula and method for maintaining the high motility of fresh sperm from grouper, thereby improving the motility of fresh sperm after prolonged motility.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A formula for maintaining the high motility of fresh sperm in grouper, characterized by an aqueous solution composed of any two of sodium chloride, potassium chloride, and calcium chloride, with an osmotic pressure of 1000 mOsmol / kg.

[0007] When sodium chloride and potassium chloride are used in combination, the preferred molar concentration ratio between the two is 1:1 to 1:3.

[0008] When sodium chloride and calcium chloride are used in combination, the preferred molar concentration ratio is 3:1.

[0009] When potassium chloride and calcium chloride are used in combination, the preferred molar concentration ratio is 7:1.

[0010] As a preferred embodiment of the present invention, the formula for maintaining the high motility of fresh sperm in saddleback grouper is an aqueous solution composed of sodium chloride and potassium chloride in a molar concentration ratio of 1:1.

[0011] The formula for maintaining high sperm motility in saddleback grouper described in this invention is used as a fresh sperm activating solution. The specific usage method is as follows: (1) Apply at a sperm-to-egg ratio of 10... 6 :1. After thoroughly mixing fresh sperm and fresh eggs; (2) Add 10 ml of sperm volume 3 This formula solution, which doubles the concentration of fresh sperm motility, fully activates fresh sperm and maintains high motility for an extended period, enabling fertilization to complete.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] This invention is the first to develop a formula for maintaining high sperm motility in fresh grouper. This formula enhances sperm motility after activation, ensuring high motility both after short-term activation and after prolonged motility, achieving a motility of 78.53±4.03% after 300 seconds of motility. This invention effectively improves fertilization rates by maintaining high sperm motility in fresh grouper, thereby promoting the production of purebred or hybrid grouper. Furthermore, this formula uses only any two of the three inorganic salts—sodium chloride, potassium chloride, and calcium chloride—to provide excellent activation and high motility maintenance effects, without using other complex ingredients, making it simple to prepare and low in cost. Attached Figure Description

[0014] Figure 1 A bar chart showing the effect of artificial sea salt solutions with different osmotic pressures on sperm motility;

[0015] Figure 2 Bar chart showing the effect of sodium chloride, potassium chloride, and calcium chloride solutions with different osmotic pressures on sperm motility.

[0016] Figure 3 This is a bar chart showing the effect of a combined solution of sodium chloride, potassium chloride, and calcium chloride on sperm motility.

[0017] Above the bar chart, different letters indicate significant differences, while the same letters indicate no significant differences.

[0018] At the top of the bar chart, ns indicates no significant difference (P > 0.05), * indicates a significant difference (P < 0.05), ** indicates a significant difference (P < 0.01), and *** indicates a significant difference (P < 0.001). Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the reagents or materials used in the embodiments are all from commercial sources.

[0020] In the following examples, the sperm motility test method is as follows: timing begins from the addition of sperm activation solution (specifically: artificial sea salt solution; sodium chloride, potassium chloride, and calcium chloride solution; and a combination of sodium chloride, potassium chloride, and calcium chloride solution), and the proportion of sperm with motility reaching 10 μm / s is calculated after 20 seconds and 300 seconds of activation.

[0021] Example 1

[0022] To investigate the effect of (artificial) seawater on the sperm motility of the saddle-banded grouper, an artificial sea salt solution was prepared in this embodiment.

[0023] The concentrations of the artificial sea salt solutions are: 22.5 g / L, 27.0 g / L, 31.5 g / L, 36.0 g / L, 40.5 g / L, 45.0 g / L, 49.5 g / L, 54.0 g / L, 58.5 g / L, 63.0 g / L, and 67.5 g / L. The corresponding preparation methods are: dissolving artificial sea salt in deionized water to obtain the artificial sea salt solution. The corresponding osmotic pressures are: 500 mOsmol / kg, 600 mOsmol / kg, 700 mOsmol / kg, 800 mOsmol / kg, 900 mOsmol / kg, 1000 mOsmol / kg, 1100 mOsmol / kg, 1200 mOsmol / kg, 1300 mOsmol / kg, 1400 mOsmol / kg, and 1500 mOsmol / kg.

[0024] The sperm of the grouper were activated in the culture pond using artificial sea salt solutions of different concentrations. The sperm were activated through contact with the grouper sperm. The sperm motility results after 20 seconds and 300 seconds of activation are as follows: Figure 1 As shown. The results showed that (1) the activation effect of fresh sperm was best when artificial sea salt solution was used with an osmotic pressure of 900~1000 mOsmol / kg; (2) the sperm motility could reach 90% after activating with artificial sea salt solution for 20 seconds, but dropped sharply to about 50% after 300 seconds.

[0025] Example 2

[0026] To investigate the effects of different ions on the sperm motility of grouper saddleback, this example prepared solutions of sodium chloride, potassium chloride, and calcium chloride.

[0027] The concentrations of the prepared sodium chloride solutions are: 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, 700mM, and 750mM; the corresponding preparation methods are: dissolving sodium chloride in deionized water to obtain sodium chloride solutions; the corresponding osmotic pressures are: 500mOsmol / kg, 600mOsmol / kg, 700mOsmol / kg, 800mOsmol / kg, 900mOsmol / kg, 1000mOsmol / kg, 1100mOsmol / kg, 1200mOsmol / kg, 1300mOsmol / kg, 1400mOsmol / kg, and 1500mOsmol / kg.

[0028] The concentrations of the prepared potassium chloride solutions are: 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, 700mM, and 750mM. The corresponding preparation method is to dissolve potassium chloride in deionized water to obtain the potassium chloride solution. The corresponding osmotic pressures are: 500mOsmol / kg, 600mOsmol / kg, 700mOsmol / kg, 800mOsmol / kg, 900mOsmol / kg, 1000mOsmol / kg, 1100mOsmol / kg, 1200mOsmol / kg, 1300mOsmol / kg, 1400mOsmol / kg, and 1500mOsmol / kg.

[0029] The concentrations of the prepared calcium chloride solutions are: 167mM, 200mM, 233mM, 267mM, 300mM, 333mM, 367mM, 400mM, 433mM, 467mM, and 500mM. The corresponding preparation methods are: dissolving calcium chloride in deionized water to obtain calcium chloride solutions. The corresponding osmotic pressures are: 500mOsmol / kg, 600mOsmol / kg, 700mOsmol / kg, 800mOsmol / kg, 900mOsmol / kg, 1000mOsmol / kg, 1100mOsmol / kg, 1200mOsmol / kg, 1300mOsmol / kg, 1400mOsmol / kg, and 1500mOsmol / kg.

[0030] The sperm of the grouper was activated in the aquaculture pond using sodium chloride, potassium chloride, and potassium chloride solutions. The sperm was activated through contact with the grouper sperm. The sperm motility results were as follows after 20 seconds and 300 seconds of activation: Figure 2 As shown in the figure. The results showed that all three component solutions had the best activation effect at an osmotic pressure of 1000 mOsmol / kg. However, after 300 seconds of activation, sperm motility also dropped significantly to about 60% or below. Specifically, after activation for 300 seconds with sodium chloride, potassium chloride, and potassium chloride solutions at an osmotic pressure of 1000 mOsmol / kg, the sperm motility was 58.02±3.17%, 60.22±1.40%, and 58.09±3.19%, respectively.

[0031] Therefore, in order to obtain a better activation effect, the osmotic pressure of the formula solution for maintaining high sperm motility in grouper should be 1000 mOsmol / kg.

[0032] Example 3

[0033] To investigate the effects of different ion combinations on sperm activation in grouper saddleback, Example 3 prepared a combined solution of sodium chloride, potassium chloride, and calcium chloride.

[0034] The prepared sodium chloride / potassium chloride / calcium chloride combined formulation solutions are: sodium chloride + potassium chloride, sodium chloride + calcium chloride, and potassium chloride + calcium chloride, combined in molar ratios of 7:1, 5:1, 3:1, 1:1, 1:3, 1:5, and 1:7, respectively, to prepare combined formulation solutions with an osmotic pressure of 1000 mOsmol / kg. The corresponding preparation method is as follows: different combinations of sodium chloride / potassium chloride / calcium chloride are dissolved in deionized water to obtain sodium chloride / potassium chloride / calcium chloride combined formulation solutions of grouper sperm. The osmotic pressure of the combined formulation solution is 1000 mOsmol / kg, and the pH is 8.0.

[0035] The combined formulation solutions prepared above were used to activate the sperm of *Gymnocypris saddleback* in the culture pond. The sperm were activated through contact with the sperm. The sperm motility results after 20 seconds and 300 seconds of activation are as follows: Figure 2As shown. The results showed that (1) under an osmotic pressure of 1000 mOsmol / kg, the formulation solutions of sodium chloride, potassium chloride and calcium chloride in pairs could improve sperm motility after 300 seconds of activation; (2) under an osmotic pressure of 1000 mOsmol / kg, the following combination formulation solutions could achieve sperm motility of over 90% after 20 seconds of activation and over 75% after 300 seconds of activation, which is better than the existing seawater activation effect: 250 mM sodium chloride + 250 mM potassium chloride (1 :1), 125mM sodium chloride + 375mM potassium chloride (1:3), 333mM sodium chloride + 111mM calcium chloride (3:1), 412mM potassium chloride + 59mM calcium chloride (7:1); (3) The optimal combination solution is 250mM sodium chloride + 250mM potassium chloride, with an osmotic pressure of 1000mOsmol / kg. After activation for 20 seconds, the sperm motility is 96.18±1.24%, and after activation for 300 seconds, the sperm motility is as high as 78.53±4.03%.

Claims

1. A formula for maintaining the high motility of fresh sperm in grouper, characterized in that, An aqueous solution composed of any two of sodium chloride, potassium chloride, and calcium chloride, with an osmotic pressure of 1000 mOsmol / kg.

2. The formula for maintaining high sperm motility in saddleback grouper according to claim 1, characterized in that, When sodium chloride and potassium chloride are used in combination, the molar concentration ratio of the two is 1:1 to 1:

3.

3. The formula for maintaining high sperm motility in saddleback grouper according to claim 1, characterized in that, When sodium chloride and calcium chloride are used in combination, the molar concentration ratio of the two is 3:

1.

4. The formula for maintaining high sperm motility in saddleback grouper according to claim 1, characterized in that, When potassium chloride and calcium chloride are used in combination, the molar concentration ratio of the two is 7:

1.

5. The formula for maintaining high sperm motility in saddleback grouper according to claim 1, characterized in that, An aqueous solution composed of sodium chloride and potassium chloride in a 1:1 molar concentration ratio, with an osmotic pressure of 1000 mOsmol / kg.

6. The formula for maintaining high sperm motility in saddleback grouper according to claim 1, characterized in that, Used as a fresh sperm activator.

Images