阅读说明：本技术 一种现配现用的提高黄河鲶鱼受精率的精液稀释液及其制备方法 (Semen diluent for improving fertilization rate of yellow river catfish in-situ preparation and preparation method thereof ) 是由 冯志云 高祥云 康鹏天 邵淑娟 王全意 贾旭龙 靳玉楼 高大珍 柴成举 徐丽 于 2021-05-28 设计创作，主要内容包括：本发明提供的现配现用的提高黄河鲶鱼受精率的精液稀释液及其制备方法,属于鱼类精液稀释液领域,主要解决大规模人工繁育黄河鲶时往往因为精液不够而影响人工繁育规模的问题,该现配现用的提高黄河鲶鱼受精率的精液稀释液,包括鲶雄鱼精液、水,所述鲶雄鱼精液与水以1∶1的比例混合激活,激活后的精液与0.6％～0.8％浓度的葡萄糖溶液以1∶10的比例稀释,葡萄糖溶液为0.8％浓度的葡萄糖溶液。该种精液稀释液的制备方法,包括如下步骤：步骤1：配置0.8％浓度的葡萄糖作为等渗液；步骤2：采集黄河鲶雄鱼的精液；步骤3：步骤2采集的精液与黄河水比例为1∶1进行混合激活；步骤4：用步骤3激活后的精液与0.8％浓度的葡萄糖溶液以1∶10的比例进行稀释保存。(The invention provides a sperm diluent for improving the fertilization rate of a yellow river catfish and a preparation method thereof, belonging to the field of fish sperm diluent and mainly solving the problem that the artificial breeding scale is influenced because the sperm is insufficient when the yellow river catfish is artificially bred in a large scale. The preparation method of the semen diluent comprises the following steps: step 1: preparing 0.8% glucose as isotonic solution; step 2: collecting semen of male catfish in yellow river; and step 3: mixing and activating the semen collected in the step 2 and the yellow river water according to the proportion of 1: 1; and 4, step 4: diluting and storing the semen activated in the step 3 and a glucose solution with the concentration of 0.8% in a ratio of 1: 10.)

1. The utility model provides a semen diluent of improvement yellow river catfish fertility rate that just matches usefulness which characterized in that: comprises the sperm of the male catfish and water, the sperm of the male catfish and the water are mixed and activated according to the proportion of 1: 1, and the activated sperm is diluted with the glucose solution with the concentration of 0.6 percent to 0.8 percent according to the proportion of 1: 10.

2. The semen diluent for improving the fertilization rate of the yellow river catfish as claimed in claim 1, wherein the semen diluent comprises: the glucose solution is a 0.8% glucose solution.

3. The semen diluent for improving the fertilization rate of the yellow river catfish as claimed in claim 2, wherein the semen diluent comprises: the water is yellow river water of Lanzhou section at the upstream of the yellow river after filtering, aerating and oxygenating.

4. A preparation method of semen diluent for improving the fertilization rate of yellow river catfish in-situ preparation is characterized by comprising the following steps: the method comprises the following steps:

step 1: preparing 0.8% glucose as isotonic solution;

step 2: collecting semen of male catfish in yellow river;

and step 3: mixing and activating the semen collected in the step 2 and the yellow river water according to the proportion of 1: 1;

and 4, step 4: diluting and storing the semen activated in the step 3 and a glucose solution with the concentration of 0.8% in a ratio of 1: 10.

5. The method for preparing the semen diluent for improving the fertilization rate of the yellow river catfish as claimed in claim 4, which comprises the following steps: the method for collecting the semen of the male catfish in the step 1 comprises the following steps: before semen collection, stopping feeding the feed, wiping the belly of the male catfish during semen collection, slightly squeezing the male catfish from front to back along the belly by using a thumb and a forefinger, discarding the semen and urine at the front end, and then putting the semen into a dry culture dish to shield light for later use.

Technical Field

The invention belongs to the field of fish semen diluent, and particularly relates to a ready-to-use semen diluent for improving the fertilization rate of a yellow river catfish and a preparation method thereof.

Background

The yellow river catfish (silurus Lanzhouensis) is also called Lanzhou catfish, namely the catfish and the Clarias fuscus, belongs to the order of Parasilurus and the family of Parasilurus, and is an important native famous economic fish in the middle and upstream of the yellow river. The yellow river Gansu section is the origin and main habitat of the yellow river catfish. The catfish in the yellow river has the advantages of fast growth, strong adaptability, low temperature resistance, low oxygen resistance and other good ecological habits, tender meat quality, rich nutrient components, fine and smooth mouthfeel, less thorns, more meat, high nutrient value and excellent quality, enjoys the reputation of 'live ginseng in the yellow river', is widely favored by people, and presents good utilization prospect. The catfish is a rare local characteristic fish germplasm resource with great development and utilization potential in a yellow river basin with relatively deficient fish germplasm resources.

The growth speed of male catfish of the yellow river catfish is obviously slower than that of female catfish, and the semen is less after sexual maturity, so that the artificial breeding scale is affected due to insufficient semen when the artificial breeding is carried out on a large scale in production. In recent years, due to the continuous expansion of the culture scale of the yellow river catfish and the requirement of artificial propagation, the semen of the yellow river catfish is preserved by a low-temperature freezing preservation technology, which has important significance for optimizing the artificial propagation and seedling production technology and improving the artificial propagation scale, but the cost of the semen diluent for low-temperature freezing preservation is higher, and the fertilization rate is higher when the semen diluent is prepared in practice in practical application. The invention takes the yellow river catfish as a research object, aims to improve the fertilization rate of the catfish, adopts glucose solutions with different concentrations to carry out experimental observation on the sperm motility of the yellow river catfish, finds the concentration of the sperm diluent most suitable for fertilization, ensures the sperm to have the maximum motility, improves the fertilization rate, provides a foundation for large-scale artificial propagation of the yellow river catfish, and enriches the sperm biological content of the catfish in the family silurus.

Disclosure of Invention

The invention aims to provide a semen diluent for improving the fertilization rate of clarias fuscus in the existing preparation and a preparation method thereof, so as to solve the problem that the artificial breeding scale is influenced by insufficient semen when the clarias fuscus is artificially bred in a large scale.

In order to solve the problems, the invention adopts the technical scheme that:

a semen diluent for improving the fertilization rate of the yellow river catfish which is prepared for use at present comprises a catfish semen and water, wherein the catfish semen and the water are mixed and activated according to the proportion of 1: 1, and the activated semen and a glucose solution with the concentration of 0.6-0.8% are diluted according to the proportion of 1: 10.

Further, the glucose solution is a 0.8% concentration glucose solution.

Further, the water is yellow river water in Lanzhou section at the upstream of the yellow river after filtering, aerating and oxygenating.

Further, the preparation method of the semen diluent for improving the fertilization rate of the yellow river catfish in the prior use comprises the following steps:

step 1: preparing 0.8% glucose as isotonic solution;

step 2: collecting semen of male catfish in yellow river;

and step 3: mixing and activating the semen collected in the step 2 and the yellow river water according to the proportion of 1: 1;

and 4, step 4: diluting and storing the semen activated in the step 3 and a glucose solution with the concentration of 0.8% in a ratio of 1: 10.

Further, the method for collecting the semen of the male catfish in the step 1 comprises the following steps: before semen collection, stopping feeding the feed, wiping the belly of the male catfish during semen collection, slightly squeezing the male catfish from front to back along the belly by using a thumb and a forefinger, discarding the semen and urine at the front end, and then putting the semen into a dry culture dish to shield light for later use.

The invention has the following beneficial effects:

the semen diluent for improving the fertilization rate of the yellow river catfish, which is prepared in the prior art, takes a glucose solution with the concentration of 0.6-0.8% as an isotonic solution, so that the sperm vigorous movement time is long, and the sperm life is prolonged.

When a 0.8% glucose solution is used as an isotonic solution, the duration of vigorous sperm motility is longest and the lifetime of sperm is also longest.

The catfish sperm is activated by yellow river water in a ratio of 1: 1, and then treated by using 0.8% of glucose as isotonic solution, the strong movement time of the sperm is obviously longer than that of the sperm treated by the isotonic solution, because the osmotic pressure of the external environment is reduced after the yellow river water is added, the strong movement time of the sperm is prolonged, the osmotic pressure of the isotonic solution is between the highest concentration and the death concentration of the sperm, the catfish sperm is an osmotic pressure solution close to a sperm preserving solution, and in the treatment of adding the equal osmotic solution after the yellow river water is added, the sperm activity is higher than that of adding the equal osmotic solution and then adding the yellow river water, the rapid movement time of the sperm is prolonged by adopting the method, the chance of meeting sperm and eggs is increased, the fertility rate can be improved, the fertility can reach 85%, the hatching rate can reach 83%, and is higher than that of the seminal fluid.

Detailed Description

The present invention is further illustrated by the following specific experiments.

Materials and methods

1.1 test materials

4-year-old mature male fishes of the yellow river catfish cultured in the yellow river catfish breeding field.

1.2 test methods

1.2.1 semen Collection

Selecting healthy yellow river catfish to temporarily culture in a water pool, stopping feeding the feed during the period, wiping off the abdomen of the catfish during sperm collection, slightly squeezing the abdomen of the catfish from front to back by using a thumb and a forefinger, discarding semen and urine at the front end, and then putting the semen into a dry culture dish to shade the light for later use.

1.2.2 sperm motility Observation

The slide was placed under a 16X 40-fold microscope, and the following solution 1 drop was aspirated into each slide with a pipette: (1) yellow river water (yellow river water in Lanzhou section at the upstream of the yellow river after filtering aeration and oxygenation treatment); (2) 0.2% glucose solution; (3) 0.4% glucose solution; (4) 0.6% glucose solution; (5) 0.8% glucose solution; (6) 1.0% glucose solution; (7) 1.2% glucose solution; (8) physiological saline; (9) purified water; (10) distilled water;

then, semen is picked up by a dissecting needle and is sequentially mixed with the solution on the glass slide, and the movement of the semen is observed by a microscope. Timing was initiated at the time of mixing, and observations were based on a visual field, sperm motility grade referenced to the criteria of Wujing Gui: the violent movement time is that only a violent turning black spot is seen in the visual field from the mixing of the semen and the solution, and the shape and the movement route of the sperms are not clear; the slow movement time is the movement state from the state that the individual shape and the movement route of the sperms can be distinguished to the state that the movement ability of the sperms is lost; the tail-shaking movement time is that the sperms basically do weak tail-shaking movement in situ until the swinging is stopped.

The end of the tail-shaking movement is determined as the end of the previous movement stage and the beginning of the next movement stage of the batch of sperms, and the end of the tail-shaking movement is determined as the stopping of 95% of sperms in the visual field, wherein 70% of sperms in the visual field are converted from the previous movement stage to the other movement stage.

1.3 data statistics and analysis

All collected data are sorted by Excel software, and then SPSS 17.0 is used for carrying out One way ANOVA method variance analysis on the data. When the difference is significant, Duncan's method is used for multiple comparison, the significance level is (P < 0.05), and the result is expressed by (mean. + -. standard deviation).

Results and analysis

2.1 comparison of the time periods of vigorous movements of Clarias fuscus sperm in different concentrations of glucose solutions and different waters

Note: 1: the same column number indicates that the difference is obvious when different letters are annotated on the shoulder (P is less than 0.05), and the difference is not obvious when the same letters are annotated on the shoulder (P is more than 0.05); 2: the background mineralization degree of the culture water is 386.5 mg/L.

As can be seen from Table 1, in the yellow river water, the duration of the vigorous sperm movement is (19.85 + -4.81) s, the duration of the vigorous sperm movement is correspondingly prolonged with the increase of the glucose concentration in the range of 0.2% -0.8%, the duration of the vigorous sperm movement is (36.20 + -2.79) s in the glucose concentration of 0.8%, the duration of the vigorous sperm movement is shortened with the continuous increase of the concentration, the difference between the 0.8% glucose and the 0.6% glucose is not significant, and the difference between the 0.8% glucose and the 1.0% glucose is very significant, which indicates that the duration of the vigorous sperm movement is longest when the glucose concentration is 0.8%, and the duration of the vigorous sperm movement is gradually reduced with the continuous increase of the glucose concentration.

The glucose concentration is in the range of 0.2% -0.8%, the sperm lifetime is prolonged as the glucose concentration increases, reaching a maximum of (91.32 ± 6.27) s at 0.8%, and then as the glucose concentration continues to increase, the sperm lifetime is significantly shortened.

This can result in: when the glucose solution with the concentration of 0.6-0.8% is used as the isotonic solution, the vigorous movement time of the sperms is long, and the service life of the sperms is also prolonged, particularly when the glucose solution with the concentration of 0.8% is used as the isotonic solution, the vigorous movement time of the sperms is longest, and the service life of the sperms is also longest.

The following experiments were performed with different treatment methods to obtain experimental data:

2.2 comparison of Furce movement time of Clarias fuscus sperm by different treatment methods

Table 2: comparison of Furce movement time of Clarias fuscus sperm by different treatment methods

Note: 1: the same column number indicates that the difference is obvious when different letters are annotated on the shoulder (P is less than 0.05), and the difference is not obvious when the same letters are annotated on the shoulder (P is more than 0.05); 2: the background mineralization degree of the yellow river water is 386.5 mg/L;

as is clear from Table 2, the third treatment method showed a longer vigorous sperm motility and a longer sperm cell life than the fourth treatment method, and the sperm cell life of the third treatment method was (96.16. + -. 4.62) s.

Comparison of seminal fluid stock and dilutions

As can be seen from Table 3, the sperm dilution obtained by the third method in Table 2 has fertilization rate and hatching rate higher than those of the sperm stock solution treatment group, and the fertilization rate and hatching rate can reach 85% and 83%.

Discussion of the related Art

3.1 comparison of the time for vigorous movement of Clarias fuscus sperm in different concentrations of glucose solution and different water

In a glucose solution with the concentration of 0.2-0.8%, the violent movement time of the sperms of the catfish fry is gradually prolonged along with the rise of the concentration of the glucose, reaches the maximum at the concentration of 0.8%, and then is gradually reduced along with the continuous rise of the concentration of the glucose; the sperm lifetime of the catfish is gradually prolonged along with the increase of the concentration of the glucose, reaches the maximum at the concentration of 0.8 percent, and then gradually decreases along with the continuous increase of the concentration of the glucose. It can thus be derived: in 0.8% glucose, the duration of the vigorous sperm motility and the duration of the sperm longevity were the longest.

Comparison of time periods of intense movement of Clarias fuscus sperm in isotonic solutions of different concentrations

From the research, the influence of glucose solutions with different concentrations on the vitality and the life span of the yellow river catfish sperms is different and has very remarkable effect. In the low concentration glucose solution, the activity of the yellow river catfish sperm is gradually strengthened, and in the 0.8 percent glucose solution, the sperm activity reaches the highest. In solutions above 0.8% concentration, sperm motility is inhibited. It can be concluded that the sperm reaches the highest at 0.8%, indicating that the glucose concentration of 0.8% is close to the osmotic pressure of the seminal plasma of the catfish in the yellow river.

In the artificial insemination process, the isotonic solution is used for cleaning yolk substances flowing out from broken eggs in the eggs, and the yolk substances can reduce the combination chance of sperms and the eggs in the fertilization process and reduce the fertilization rate. An isotonic solution is a solution with an osmotic pressure higher than that of the hatching water. The Clarias fuscus sperm is activated by treating with yellow river water, and then treated with 0.8% glucose as isotonic solution, and the violent movement time of the sperm is obviously longer than that of the sperm treated with the isotonic solution. The osmotic pressure of the isotonic solution is between the highest concentration and the death concentration of the sperms, the osmotic pressure solution is close to the sperm preservation solution, and the sperm activity is higher than that of the treatment of adding the isotonic solution after adding the isotonic solution in the treatment of adding the yellow river water after adding the yellow river water dropwise.

Conclusion

The activity of the yellow river catfish sperm is gradually prolonged along with the increase of the concentration in the glucose solution with the concentration of 0.2 to 0.8 percent, the violent movement time of the sperm is gradually shortened if the concentration of the glucose solution is continuously increased, and the corresponding sperm life is also gradually shortened; in the artificial insemination process of the yellow river catfish, glucose with the concentration of 0.8% is used as isotonic solution, the specific method is that the semen and the yellow river water are mixed and activated at the ratio of 1: 1, the activated semen and the glucose solution with the concentration of 0.8% are diluted at the ratio of 1: 10, and then the artificial insemination is carried out. The method prolongs the rapid movement time of the sperms, increases the chance of meeting the sperms and the eggs, is beneficial to improving the fertilization rate, and the fertilization rate can reach 85 percent, the hatching rate can reach 83 percent and is higher than that of the seminal fluid stock solution.

A preparation method of a semen diluent for improving the fertilization rate of yellow river catfish in-situ preparation comprises the following specific operation steps:

step 1: preparing 0.8% glucose as isotonic solution;

step 2: collecting the semen of the male catfish: selecting healthy yellow river catfish to temporarily culture in a water pool, stopping feeding the feed during the period, wiping off the abdomen of the catfish during sperm collection, slightly squeezing the abdomen of the catfish from front to back by using a thumb and a forefinger, discarding semen and urine at the front end, and then putting the semen into a dry culture dish to shade for later use;

and step 3: mixing and activating the semen collected in the step 2 and the yellow river water according to the proportion of 1: 1;

and 4, step 4: diluting and storing the semen activated in the step 3 and a glucose solution with the concentration of 0.8% in a ratio of 1: 10.