CN117147548A - Novel method for sperm survival rate - Google Patents

Abstract

The invention relates to the technical field of sperm detection, and discloses a new method for sperm survival rate, which includes the following detection steps: S1: reagent preparation, configuring eosin Y dyeing solution and neutral red dyeing solution according to the dosage, and then adding eosin Y dyeing solution and neutral red dyeing solution. After the Y dye solution and neutral red dye solution are prepared, prepare the buffer solution for later use; S2: Semen collection and processing, collect semen from farmed animals, and then place the collected semen in a constant temperature water bath for insulation treatment. The semen after heat preservation is then highly diluted and estimated to obtain an estimated score. By combining the estimated scores during semen collection and processing and the detection data of various sperm survival rates, the sperm survival rate of farmed animals is detected. The detection accuracy is high and the detection is relatively convenient. It is convenient for animal husbandry and breeding institutions to measure the sperm survival rate of farmed animals. detection, promotion and use value is high.

Description

A new approach to sperm survival rates

Technical field

The invention relates to the technical field of sperm detection, specifically a new method of sperm survival rate.

Background technique

Sperm is divided into animal sperm and plant sperm. It is the male germ cell in the sexual reproduction process of animals, the germ cell of male animals, the male gamete in anisogametic reproduction, and the haploid germ cell produced by the sperm organ. In life, sperm are more Most of them refer to mature male reproductive cells, formed in the testes. Semen is an organic matter. Semen contains fructose and protein, as well as some enzymes, inorganic salts and organic salts.

In the process of livestock breeding, the reproductive efficiency of farmed animals is an important factor affecting the profitability of livestock breeding institutions. Therefore, it is necessary to regularly detect the sperm survival rate of farmed animals. The sperm survival rate is an important indicator for evaluating semen quality. Although the measurement under an optical microscope is simple and convenient, the detection accuracy is poor. Therefore, those skilled in the art have proposed a new method of sperm survival rate to solve the above-mentioned technical problems.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides a new method of sperm survival rate, which solves the existing problem of poor detection accuracy when using an optical microscope for detection.

In order to achieve the above objectives, the present invention is realized through the following technical solutions: a new method of sperm survival rate, including the following detection steps:

S1: Reagent preparation

Prepare the eosin Y dye solution and neutral red dye solution according to the dosage, and then prepare the buffer solution after the eosin Y dye solution and neutral red dye solution are prepared and set aside;

S2: Semen collection and processing

Collect semen from farmed animals, then place the collected semen in a constant-temperature water bath for insulation treatment. Then, the semen after insulation treatment is highly diluted and estimated to obtain an estimation score;

S3: Survival rate detection

Use a microsampler to take 10l of semen and drop it on the glass slide. Then use the eosin Y stain and neutral red stain prepared in step S1 to detect the sperm survival rate on the glass slide. After the detection is completed, use SPSS The software performs statistical analysis and obtains test results;

S4: Conclusion analysis

Combining the estimated score in step S2 and the detection result in step S3, the sperm survival rate of the tested animal is obtained.

Preferably, in step S1, both the eosin Y dyeing solution and the neutral red dyeing solution are purchased from Shanghai Reagent Factory No. 3, and the eosin Y dyeing solution is prepared with distilled water.

Preferably, in step S1, the concentration of the eosin Y dyeing solution is 0.15%, the pH value of the neutral red dyeing solution is 7.4, and the concentration of the buffer solution is 0.01%.

Preferably, in the step S2, the temperature of the constant-temperature water bath is 37 degrees Celsius, the semen is highly diluted, and the sperm outline and sperm spacing are determined using a light microscope.

Preferably, in step S2, when estimating the sperm under the field of view, the motility of 10% of the sperm moving in a straight line is 0.1, and the motility of 20% of the sperm moving in a straight line is 0.2, and so on, 1 is a perfect score.

Preferably, in step S3, count 200 sperm per sample and calculate the sperm survival rate. The sperm survival rate calculation formula is: survival rate = (total sperm number - non-linear motion sperm number)/total sperm number * 100%.

Preferably, in step S3, when using eosin Y staining solution to detect the sperm survival rate on the glass slide, add 10tA 0.15% eosin Y staining solution to the glass slide and mix it evenly, and then Then add 2011% aniline black or aniline blue as the background color, and then use another slide to make a smear. After natural drying, it can be seen under a microscope that the live sperm are not colored, the cytoplasm is transparent, and the dead sperm are red.

Preferably, in step S3, when neutral red dye is used to detect the sperm survival rate on the slide, neutral red dye and buffer are added dropwise to the slide, mixed evenly, and then covered after staining for 2 minutes. The film was observed under a bright field 400x microscope within half an hour. Dead sperm were stained red, live sperm were not stained, and the cytoplasm was transparent.

The present invention provides a new method of sperm survival rate. It has the following beneficial effects:

1. The present invention detects the sperm survival rate of farmed animals by combining the estimated scores during semen collection and processing and the detection data of various sperm survival rates. The detection accuracy is high, the detection is relatively convenient, and it is convenient for animal husbandry and breeding institutions to carry out breeding. The detection of animal sperm survival rate has high promotion and use value.

2. The present invention regularly detects the sperm survival rate of the breeding animals in the breeding institution, and uses the data of the sperm survival rate to screen out the optimal breeding animals among the breeding animals, thereby improving the breeding success rate of the breeding institution, and at the same time It can also improve the economic benefits of breeding institutions.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Example 1:

The embodiment of the present invention provides a new method for sperm survival rate, which includes the following detection steps:

S1: Reagent preparation

Prepare the eosin Y dye solution and neutral red dye solution according to the dosage, and then prepare the buffer solution after the eosin Y dye solution and neutral red dye solution are prepared and set aside;

S2: Semen collection and processing

Collect semen from farmed animals, then place the collected semen in a constant-temperature water bath for insulation treatment. Then, the semen after insulation treatment is highly diluted and estimated to obtain an estimation score;

S3: Survival rate detection

Use a microsampler to take 10l of semen and drop it on the glass slide. Then use the eosin Y stain and neutral red stain prepared in step S1 to detect the sperm survival rate on the glass slide. After the detection is completed, use SPSS The software performs statistical analysis and obtains test results;

S4: Conclusion analysis

Combining the estimated score in step S2 and the detection result in step S3, the sperm survival rate of the tested animal is obtained.

In step S1, both the eosin Y dyeing solution and the neutral red dyeing solution were purchased from Shanghai Reagent Factory No. 3, and the eosin Y dyeing solution was prepared with distilled water.

In step S1, the concentration of the eosin Y dyeing solution is 0.15%, the pH value of the neutral red dyeing solution is 7.4, and the concentration of the buffer solution is 0.01%.

In step S2, the temperature of the constant-temperature water bath is 37 degrees Celsius. The semen is highly diluted and the sperm outline and sperm spacing are determined using a light microscope.

In step S2, when estimating the sperm in the field of view, 10% of the sperm moving in a straight line have a motility of 0.1, 20% of the sperm moving in a straight line have a motility of 0.2, and so on, with 1 being a perfect score.

In step S3, count 200 sperm per sample and calculate the sperm survival rate. The sperm survival rate calculation formula is: survival rate = (total sperm number - non-linear sperm number)/total sperm number * 100%.

In step S3, when using eosin Y dye to detect the sperm survival rate on the slide, add 10tA 0.15% eosin Y dye to the slide and mix it evenly, and then add 2011% aniline Black or aniline blue is used as the background color, and then another slide is used to make a smear. After natural drying, observation under a microscope shows that the living sperm are not colored, the cytoplasm is transparent, and the dead sperm are red.

The detection of sperm motility is an important part of semen testing. Its level directly reflects the sperm quality and sperm viability. The reduction of sperm motility will significantly affect the fertilization ability of sperm. Eosin-aniline black staining method and neutral red The dyeing method is used to calculate sperm viability. The difference is that eosin Y is an acidic dye with anions and can combine with substances with cations. The cell membrane of dead sperm is damaged and can penetrate into the sperm and combine with cations to turn red.

Since the cell membrane of live sperm is intact, eosin Y cannot penetrate into the cell without coloring. Neutral red is a water-soluble dye that can be stored in the cell through the cell membrane of live sperm to color it; when the sperm cell membrane and lysosome membrane are destroyed Finally, the dye cannot be taken up, so the dead sperm are not stained.

In step S3, when using neutral red dye to detect the sperm survival rate on the slide, add neutral red dye and buffer on the slide and mix them evenly. After staining for 2 minutes, add a cover slip and place a cover slip in the bright field. Under a 400x microscope, within half an hour, dead sperm were stained red, live sperm were not stained, and the cytoplasm was transparent.

This step is based on the comparison of two methods for measuring sperm motility. The difference between the two methods is not obvious. However, during the test process, it was found that the determination of sperm motility by eosin dye is affected by its concentration. A too high concentration will affect the sperm. Causes loss or even kills sperm. Neutral red staining can describe and identify the morphology of live sperm under an optical microscope. This is better than eosin Y staining and heating. It has promotion and application value in the detection of semen quality. .

In step S3, the drip pressure method can also be used for detection. The operation steps of the drip pressure method are: take the same amount of semen, eosin aqueous solution and aniline black solution as mentioned above, and first drop them into a small petri dish, penicillin vial or small centrifuge tube ( Generally less than 1.5ml), after staining for 3 minutes, take a drop on the slide, then cover it with a cover slip, and conduct direct microscopic examination. The dead sperm are stained red, the live sperm are not stained, and the cytoplasm is transparent. Count 200 for each specimen. sperm and calculate the sperm motility rate.

Example 2:

The embodiments of the present invention provide experimental comparative analysis examples based on the above embodiments.

Three cows were randomly selected from a dairy farm in Longde County, Ningxia, and their semen was collected. After the semen was collected, it was placed in a constant-temperature water bath for constant-temperature storage according to the treatment method of the above embodiment, and then used an optical microscope. The semen is highly diluted and then smeared on a glass slide for detection. The detection methods are three detection methods: evaluation score, eosin Y staining solution and neutral red staining solution. The detection information data are shown in Table 1:

Table 1

It can be seen from the information data in Table 1 that when three different detection methods were used to detect the semen of three cows respectively, the three detection methods of evaluation score, eosin Y dye, and neutral red dye were effective for the same cow. The discrepancies in the detection data are all less than 0.1. Therefore, the above-mentioned detection method of the present application has high detection accuracy and can be promoted and used in livestock breeding institutions.

Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art will understand that various changes, modifications, and substitutions can be made to these embodiments without departing from the principles and spirit of the invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (8)

1. A new method for sperm survival rate, which is characterized by including the following detection steps: S1: Reagent preparation Prepare the eosin Y dye solution and neutral red dye solution according to the dosage, and then prepare the buffer solution after the eosin Y dye solution and neutral red dye solution are prepared and set aside; S2: Semen collection and processing Collect semen from farmed animals, then place the collected semen in a constant-temperature water bath for insulation treatment. Then, the semen after insulation treatment is highly diluted and estimated to obtain an estimation score; S3: Survival rate detection Use a microsampler to take 10l of semen and drop it on the glass slide. Then use the eosin Y stain and neutral red stain prepared in step S1 to detect the sperm survival rate on the glass slide. After the detection is completed, use SPSS The software performs statistical analysis and obtains test results; S4: Conclusion analysis Combining the estimated score in step S2 and the detection result in step S3, the sperm survival rate of the tested animal is obtained. 2. A new method of sperm survival rate according to claim 1, characterized in that, in the step S1, both the eosin Y dyeing solution and the neutral red dyeing solution are purchased from Shanghai No. 3 Reagent Factory, and the eosin Y dyeing solution and the neutral red dyeing solution are purchased from Shanghai Reagent Factory No. Y dye liquor is prepared with distilled water. 3. A new method of sperm survival rate according to claim 1, characterized in that, in the step S1, the concentration of the eosin Y dyeing solution is 0.15%, and the pH value of the neutral red dyeing solution is 7.4, The concentration of buffer is 0.01%. 4. A new method of sperm survival rate according to claim 1, characterized in that, in the step S2, the temperature of the constant temperature water bath is 37 degrees Celsius, and the semen is highly diluted to determine the sperm outline and Sperm spacing. 5. A new method of sperm survival rate according to claim 1, characterized in that, in the step S2, when estimating the sperm under the field of view, the motility of 10% of the sperm moving in a straight line is 0.1, 20% goes straight forward as 0.2, and so on, 1 is a perfect score. 6. A new method of sperm survival rate according to claim 1, characterized in that in the step S3, 200 sperm are counted for each sample to calculate the sperm survival rate. The sperm survival rate calculation formula is: survival rate = (Total sperm count – Non-linear sperm count)/Total sperm count*100%. 7. A new method of sperm survival rate according to claim 1, characterized in that, in the step S3, when using eosin Y staining solution to detect the sperm survival rate on the glass slide, Add 10tA 0.15% eosin Y stain and mix it evenly, then add 2011% aniline black or aniline blue as the background color, then use another slide to make a smear, dry it naturally, and place it under a microscope Observation shows that live sperm are not colored, the cytoplasm is transparent, and dead sperm are red. 8. A new method of sperm survival rate according to claim 1, characterized in that, in the step S3, when using neutral red dye to detect the sperm survival rate on the glass slide, Add neutral red dye and buffer and mix evenly. After staining for 2 minutes, add a cover slip and observe under a bright field 400x microscope within half an hour. Dead sperm are stained red, live sperm are not stained, and the cytoplasm is transparent.