Leiocassis longirostris artificial insemination buffer solution and use method thereof

Abstract

The invention discloses a leiocassis longirostris artificial insemination buffer solution and a use method thereof.The method mainly comprises the steps that seminal fluid is protected and diluted through a mixed solution of sodium chloride, sodium bicarbonate, potassium chloride, calcium carbonate, glucose and the like, then a trypsin solution is used for reducing the viscidity of leiocassis longirostris eggs, the eggs are prevented from being rapidly bonded and clustered when encountering water, the artificial insemination time is prolonged, and the leiocassis longirostris artificial insemination effect is improved. Therefore, the egg fertilization rate and hatching rate are improved. Before artificial propagation, 20 g of sodium chloride, 0.1 g of sodium bicarbonate, 0.2 g of potassium chloride, 0.1 g of anhydrous calcium chloride and 20 g of glucose are weighed and dissolved in water at the normal temperature, the volume is fixed to 1 L, and a buffer base solution is prepared and stored at the temperature of 4 DEG C. During artificial insemination, a basic solution of a buffer solution is diluted by 10 times with water, trypsin with the concentration of 0.001% is added, the mixture is mixed to prepare the buffer solution, the buffer solution needs to be prepared and used immediately, the buffer solution with the volume 2 times that of eggs is added after seminal fluid and the eggs of the leiocassis longirostris are rapidly and uniformly stirred, stirring is performed, 10% fetal calf serum is added after 1 min, and the mixture enters a hatching link after being uniformly stirred.

Description

Technical Field

[0001] The invention belongs to the field of animal genetic breeding, and particularly relates to a long-snouted catfish artificial insemination buffer solution and a use method thereof. Background Art

[0002] The long-snake catfish (Leiocassis longirostris) belongs to the order Siluriformes, family Bagridae, genus Catfish. Because of its long snout, it is often called the long-snake catfish, also known as catfish, river catfish, fat catfish, and Huaiwang fish. It is a demersal fish that often inhabits deep, rocky areas with slow currents. Its lack of scales and intramuscular spines, coupled with its delicious and nutritious meat, have made it a popular choice. There's a popular saying, "If you haven't eaten river catfish, you don't know the taste of fish." Although artificial breeding research for the long-snake catfish was successful in Sichuan in the 1980s and subsequently promoted and industrialized nationwide, some regions have struggled to master key technologies for artificial breeding, cultivation, transportation, and disease prevention, particularly in the crucial stages of artificial breeding. The high breeding cost has become a bottleneck for the large-scale breeding of long-snouted catfish. Therefore, improving the reproductive capacity of long-snouted catfish is of great value for its artificial breeding.

[0003] Current artificial insemination techniques for longnose catfish artificial breeding present the following challenges: 1. Synchronization between broodstock rearing and gonadal development. 2. Technical bottlenecks in inducing spawning and insemination: Male longnose catfish require laparotomy to extract semen, which is typically small and viscous. Furthermore, longnose catfish eggs are viscous, causing them to quickly clump upon contact with water during artificial insemination, significantly impacting fertilization and hatchability. Furthermore, the timing and operational requirements for artificial insemination are critical. These include the extreme sensitivity of sperm-egg contact to water quality (pH, temperature, and dissolved oxygen). Fluctuations in water quality (such as impurities or sudden temperature changes) or uneven mixing of sperm and eggs can significantly reduce fertilization rates (normally around 60% to 80%, but can fall below 50% if the technique is unstable). 3. Challenges in incubation and environmental control of fertilized eggs. The stickiness of the fertilized eggs makes incubation management difficult and requires stringent environmental parameters, all of which impact hatching success rates. 4. The conflict between disease prevention and control and production efficiency, including the spread of disease caused by parent fish stress, which can be transmitted to offspring through sperm and eggs, leading to reduced fry survival rates. 5. The limitations of sperm preservation technology. Longnose catfish sperm maintains a short motility at room temperature (usually less than an hour). Currently, commonly used cryopreservation (4°C) or cryopreservation (liquid nitrogen) technologies are not yet mature, resulting in low sperm recovery rates and impracticality for long-distance transportation or long-term storage. This necessitates on-site sperm collection for artificial insemination, limiting the flexibility of large-scale production.

[0004] In response to the above problems, the present invention has precisely developed a long-snout catfish artificial insemination buffer solution and a method of use thereof to improve the stability and production efficiency of artificial insemination. Summary of the Invention

[0005] To address the low fertilization and hatching rates of existing long-snouted catfish eggs, the present invention provides a long-snouted catfish artificial insemination buffer and its use method. Using the buffer during the long-snouted catfish artificial insemination process effectively protects and dilutes semen, improving semen activity while also preventing eggs from rapidly clumping and sticking to water. This extends the artificial insemination time and improves egg fertilization and hatching rates. The present invention is achieved through the following technical means:

[0006] The present invention first discloses a method for preparing a long-snout catfish artificial insemination buffer solution, comprising:

[0007] (1) Prepare the buffer base solution: 20 g of sodium chloride, 0.1 g of sodium bicarbonate, 0.2 g of potassium chloride, 0.1 g of anhydrous calcium chloride, and 20 g of glucose. Dissolve them in water at room temperature and adjust the volume to 1 L to prepare the buffer base solution. Store at 4°C until use.

[0008] (2) During artificial insemination, water is added to dilute the buffer solution 10 times, and then 0.001% trypsin is added to obtain a long-snout catfish artificial insemination buffer solution.

[0009] The invention also discloses a long-snout catfish artificial insemination buffer solution prepared according to the preparation method.

[0010] The present invention also discloses a method for using the above-mentioned long-snout catfish artificial insemination buffer solution, comprising:

[0011] The buffer solution was prepared and used immediately. After the semen and eggs of the long-snout catfish were quickly stirred evenly, a buffer solution twice the volume of the eggs was added and stirred. After 1 minute, 10% fetal bovine serum was added and stirred evenly before entering the incubation stage.

[0012] The beneficial effects of the present invention are:

[0013] After using the buffer solution, the agglomeration rate of the eggs during artificial insemination was greatly reduced, the average fertilization rate of the long-snouted catfish eggs increased by about 12.7%, and the average hatching rate increased by about 13.2%, which greatly improved the efficiency of artificial breeding and increased economic benefits. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. That is, the embodiments described are only part of the embodiments of the present invention, rather than all embodiments.

[0015] Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the invention as claimed, but is merely intended to represent selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0016] Example 1

[0017] A preparation method and use method of a long-snout catfish artificial insemination buffer solution, comprising:

[0018] (1) Prepare the buffer base solution: 20 g of sodium chloride, 0.1 g of sodium bicarbonate, 0.2 g of potassium chloride, 0.1 g of anhydrous calcium chloride, and 20 g of glucose. Dissolve them in water at room temperature and adjust the volume to 1 L to prepare the buffer base solution. Store at 4°C until use.

[0019] (2) During artificial insemination, water is added to dilute the buffer solution 10 times, and then 0.001% trypsin is added to obtain a long-snout catfish artificial insemination buffer solution;

[0020] (3) The buffer solution was prepared and used immediately. After the semen and eggs of the long-snout catfish were quickly stirred evenly, a buffer solution twice the volume of the eggs was added and stirred. After 1 minute, 10% fetal bovine serum was added and stirred evenly before entering the incubation stage.

[0021] Comparative Example 1

[0022] The artificial insemination buffer solution for long-snout catfish was not used, and the remaining operations were the same as in Example 1.

[0023] Test example

[0024] On May 28, 2023 and June 2, 2024, two consecutive years of artificial breeding trials of long-snout catfish were carried out, with a male-female ratio of 6: 1, 24 female parents for each induced spawning, and the same method was used for induced spawning for all parents. Among them, the eggs obtained from 12 fish were artificially inseminated using water as a buffer solution during the artificial insemination link (as a control group), and the remaining 12 fish were artificially inseminated using the buffer solution described in Example 1. After the artificial insemination, a sticky plate was used for hatching, and the fertilization rate was counted during the hatching process, and the hatching rate was counted after the hatching was completed. The results showed that the fish eggs using the buffer solution were significantly less sticky and clumping than the fish eggs in the control group. After 24 hours of incubation, 6 sticky egg plates (3 each in the buffer and non-buffer groups) were randomly selected to count the fertilization rates. The average fertilization rate of the eggs in the buffer group was increased by about 12.7%. Because long-snout catfish eggs tend to stick to water, fertilized eggs tend to clump together, significantly increasing the risk of fish outbreaks and Saprolegnia infection, which in turn reduces hatching rates. Using a buffer solution reduced stickiness in fertilized eggs, and Saprolegnia infection improved during the hatching process. Six randomly selected egg-holding plates (three each in the buffer-treated and untreated groups) were incubated individually. The hatching rates were calculated after the fry emerged from the hatching fry. The average hatching rate in the buffer-treated group was approximately 13.2% higher. The specific results are shown in Table 1.

[0025] Table 1 Results of artificial breeding of longnose catfish in 2023 and 2024

[0026]

[0027] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for preparing a buffer solution for artificial insemination of long-snouted catfish, comprising: (1) Prepare the buffer base solution: 20 g of sodium chloride, 0.1 g of sodium bicarbonate, 0.2 g of potassium chloride, 0.1 g of anhydrous calcium chloride, and 20 g of glucose. Dissolve them in water at room temperature and adjust the volume to 1 L to prepare the buffer base solution. Store at 4°C until use. (2) During artificial insemination, water is added to dilute the buffer solution 10 times, and then 0.001% trypsin is added to obtain a long-snout catfish artificial insemination buffer solution.

2. An artificial insemination buffer solution for long-snout catfish prepared according to the preparation method according to claim 1.

3. A method for using the artificial insemination buffer solution for long-snouted catfish according to claim 2, comprising: The buffer solution was prepared and used immediately. After the semen and eggs of the long-snout catfish were quickly stirred evenly, a buffer solution twice the volume of the eggs was added and stirred. After 1 minute, 10% fetal bovine serum was added and stirred evenly before entering the incubation stage.

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