WO2017156844A1 - Kit for evaluating sperm quality after in vitro capacitation and method of use thereof - Google Patents

Abstract

Provided are a kit for evaluating the sperm quality after in vitro capacitation and a method of use thereof. The kit comprises a detection buffer 1, a BWW culture solution, a solution for in vitro capacitation, a fluorescent reagent, a sialidase standard stock solution and a detection buffer 2. The detection buffer 1 is a phosphate buffer or a BWW culture solution. The detection buffer 2 is a phosphate buffer containing 0.05 mmol/l of sodium acetate and 0.25 μg/μl of bovine serum albumin.

Description

Kit for evaluating sperm quality after in vitro capacitation and using method thereof Technical field

The invention belongs to the technical field of medical detection, and particularly relates to a kit for evaluating sperm quality after in vitro capacitation and a using method thereof.

Background technique

With the continuous development of the social industry, the total incidence of infertile couples has increased significantly. Statistics of the European Reproductive Society in 2004: 25% of women of childbearing age who are unable to become pregnant within 1 year, 15% of whom seek treatment, and 50% of male factors cause infertility. The causes of male infertility are sexual dysfunction (1.7%), varicocele (12.3%), reproductive tract infection (6.6%), congenital dysplasia (2.1%) acquired acquired disease (2.6%), endocrine disorders (0.6%), immune factors (3.1%), other abnormalities (3%), but up to 60% to 75% of patients can not find the cause, called idiopathic male infertility, they only show less sperm, Abnormal sperm quality such as weak sperm or abnormal sperm disease. Unexplained male infertility may be caused by a variety of factors, such as long-term stress environmental factors causing endocrine disorders, reactive oxygen species and genetic defects.

For many years, traditional semen routine analysis has been the most basic clinical indicator for judging male fertility. The true cause of clinical infertility in the majority of male infertility patients remains unclear. In particular, about one-third of infertile patients in clinical practice, the results of routine semen analysis in men are normal and near normal. These patients are clinically classified as unexplained infertility. Therefore, for a long time, clinical diagnosis of male infertility has great difficulties. The main reason is that conventional semen analysis only measures sperm count, viability, activity rate and morphology. These indicators can only reflect the most basic semen quality, and do not reflect all sperm functions, such as sperm nuclei maturation and DNA damage, sperm and human zona pellucida binding reaction and penetration, acrosome status and response and binding to yolk membrane Ability, etc. Therefore, special sperm function tests need to be established to determine these functions. Sperm capacitation is a necessary condition for the combination of sperm and egg to form fertilized eggs. Although there have been some advances in the study of sperm capacitation, there are still many unexplained problems.

In clinical work, some of the primary or secondary infertile male patients use existing conventional semen quality tests to indicate that there is no abnormality in sperm and semen quality. Currently used sperm detection methods are CASA (Computer Aided Semen Analysis), and anti-sperm antibodies. Today's most widely used CASA technology and some morphological related tests can mainly evaluate sperm activity The ratio of ability to live sperm, and thus the ability to fertilize into the female reproductive tract, and rely on the current sperm function evaluation system, there are certain defects in the evaluation of sperm quality and function, and it is difficult to provide patients with appropriate clinical consultation. In fact, this is just one aspect of evaluating sperm function, and we should consider more factors that may not be revealed that may affect sperm motility.

In the prior patent 201310431847.0, a method for detecting sialidase affecting sperm function is proposed. The first step is to visualize the expression of sialidase in sperm by single sperm fluorescence intensity analysis, or to visually observe the expression of sialidase in sperm by microscopic fluorescence imaging; if sialidase is highly expressed in sperm, In the second step, the sialidase assay for sperm motility is determined by the in vitro capacitation solution, and the sialidase activity is detected by fluorescence. This patent discloses the expression of sialidase protein molecules in sperm and the detection of essential activities. However, the method includes the expression detection of sperm sialidase NEU1/3, which requires fluorescence microscopy or flow cytometry, and has the following disadvantages: 1) complicated technical process; 2) high demand for experimental equipment and operator skills and experience; Not suitable for use in clinical laboratories and routine technicians; 4) The use of abcam kits is expensive. In addition, due to the different sperm counts of different samples, the expressed protein values are different, and the corresponding enzyme activity values are also different. Therefore, it is not scientific to simply compare the enzyme activities of different samples.

Summary of the invention

In view of the above technical problems, the present invention provides a kit for assessing sperm quality after in vitro capacitation and a method of using the same. Through the detection of sialidase activity in the energy-receiving solution after sperm capacitation in vitro, the sperm quality after capacitation is evaluated, and the diagnosis basis and clinical consultation are provided for male infertility patients with unknown clinical causes.

In order to achieve the above object, the present invention adopts the following technical solutions:

A kit for assessing sperm quality after in vitro capacitation, characterized in that the kit comprises a detection buffer 1, a BWW culture solution, an in vitro energy-receiving solution, a fluorescent reagent, a sialidase standard stock solution and a detection buffer 2 .

The detection buffer 1 is PBS (phosphate buffer) or BWW culture solution.

The detection buffer 2 was PBS containing 0.05 mmoL/L sodium acetate and 0.25 μg/μl bovine serum albumin. The buffer system for assay buffer 2 was specifically formulated for detection of sialidase activity.

Preferably, the sodium acetate has a pH of 5-6 and is the best working environment for salivary enzyme activity.

The in vitro capacitation solution is HTF (human oviduct fluid) containing HSA (human serum albumin) 5 mg/ml.

The standard stock solution of sialidase is Clostridium perfringens sialidase (AUS) at a concentration of 5 U/ml, which can be conveniently diluted to a concentration gradient of 5/2.5/1.25/0.625/0.312/0 mU/ml. The method for detecting a small amount of sperm of the present invention is applied to Clostridium perfringens sialidase.

The fluorescent reagent is 2'-4-methylumbelliferyl-α-D-N-acetylneuraminic acid, which has high sensitivity.

The specific structure of the 2'-4-methylumbelliferyl-α-D-N-acetylneuraminic acid sodium is as follows:

Preferably, the concentration of the fluorescent reagent is 10 mM, providing a concentrated solution of the fluorescent reagent, which can ensure stability, is not easy to be decomposed and quenched, and can reduce volume and facilitate boxing.

The method for using the kit for assessing sperm quality after in vitro capacitation according to the present invention: the specific steps are as follows:

A, sperm washing

Collect fresh liquefied semen samples, test buffer 1 to wash the sperm, fully remove the residual seminal plasma components, and then centrifuge to separate the sperm.

In the present invention, the detection buffer 1 is first added to the semen sample for washing, and then centrifuged to obtain a sperm precipitate. This is because the seminal plasma itself is sticky, and it can not completely precipitate all the sperm in the semen by centrifuging it directly. After adding the detection buffer 1 to dilute it, the centrifugal operation is better.

Preferably, the semen is centrifuged 3 times to ensure that the number of spermatozoa is reduced as much as possible to ensure sperm motility without detecting interference from the seminal plasma.

B, in vitro capacitation of sperm

BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO ₂ cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO ₂ cells. The sperm was incubated for 3 to 4 hours in the chamber; the supernatant was centrifuged again, and the supernatant was collected as the sample to be tested. After getting the sperm with better vigor in the upstream method, it will be processed immediately, and the experimental results are more scientific and objective.

Preferably, the number of upstream sperm is controlled to be 1× ^{10 7} or more, which is set according to the need for detection sensitivity.

C. Determination of sialidase activity in sperm capacitation solution

(1) Dilute the sialidase standard stock solution to a sialidase standard solution at a concentration gradient of 5/2.5/1.25/0.625/0.312/0 mU/ml using the detection buffer 2, and sequentially add each sialic acid to the test plate. Enzyme standard solution and sample to be tested.

The gradient of the standard solution is set based on the amount of sperm sialidase released into the capacitation liquid after capacitation and the sensitivity detectable by the method of the present invention.

Preferably, the detection plate is a 96-well blackboard. The invention adopts the fluorescence method to determine the activity of sialidase, and the black detection plate is optimal for sensitivity and specificity, and can protect the fluorescent dye from being quenched by light, thereby greatly improving the sensitivity of detection.

(2) Dilute the fluorescent reagent with the detection buffer 2, add it to each of the above sialidase standard solutions and the wells of the sample to be tested, and then place the assay plate at 37 ° C, incubate in the dark for 1-2 hours, and then use the enzyme. The spectrometer reads the fluorescence intensity of each well, calculates the sialidase activity of the sample to be tested according to the standard curve of sialidase activity, and finally obtains the value of sialidase activity/sperm number.

Preferably, the concentration of the fluorescent reagent diluted to 0.05 mM with the detection buffer 2 is the optimum working concentration.

Preferably, the microplate reader reads the fluorescence intensity of each well at a wavelength of excitation wavelength and emission wavelength of 365 nm and 450 nm, respectively. 365 nm is the best excitation wavelength and 450 nm is the optimal emission wavelength.

Preferably, the sample in the well for detection has a sialidase activity in the range of 0.3 to 5 mU/ml, and if it is exceeded, the sialidase activity value of the sample to be tested in the well should be adjusted within this linear range.

The beneficial effects of the invention are:

1. The reagents in the kit of the invention all select the conventional test reagent, and combine the speciality of the sperm detection to reduce the experiment cost, and the required instruments and equipments and the consumables are few, only the centrifuge, the microplate reader, the EP tube. , the gun head and the test board are all routine laboratory configurations; and the operation method is simple, only for centrifugation, sample loading, etc. There is no need to practice multiple times to obtain the operation skills, and can be widely used in clinical laboratories and routines. Used by technicians.

2. The kit of the present invention is used for assessing the quality of sperm after capacitation, according to the number of sperm in the unit. The activity value of liquefaction enzyme is a critical value for expressing sialidase activity in the human population, and the sperm function can be understood deeper through the unit enzyme activity value in the clinic, and can be applied to reproductive assist such as test tube baby.

3. Because the number of sperm in different samples is different, the protein values expressed are different, and the corresponding enzyme activities are different. Therefore, it is not scientific to simply compare the enzyme activities of different samples, because if the number of sperm in one sample is small, However, the quality of sperm is very high, while the sperm count of another sample is very large, but the sperm quality is low. If the enzyme activity value is simply compared, the former is likely to lose to the latter, but the actual situation is that the former is higher in quality than the latter. Therefore, the detection of the sialidase activity / sperm number per unit enzyme activity value, the detection results are more objective, accurate and reliable.

4. The invention fully cleans the sperm, completely removes the residual seminal plasma component, so that the residual protein and sialidase in the seminal plasma does not affect the accuracy of the detection result; the standard set when the sperm protein concentration is optimized is optimized. Protein concentration gradient and standard enzyme activity gradient set when the enzyme is active. If the gradient setting is unreasonable, it will affect the sensitivity and accuracy of the result. Using the black detection plate to measure the enzyme activity can significantly reduce the quenching of the fluorescent reagent. The negative effects, thereby greatly improving the stability, sensitivity and accuracy of the experimental results; the final test results are designed as the value of the number of sialidase/sperm, which can reduce the difference between different samples and make them more comparable Sexuality guarantees the stability and reliability of test results.

DRAWINGS

Figure 1 shows the sialidase activity of uncapacity sperm and capacitated sperm (A is a mouse and B is a human) in the kit of the present invention.

detailed description

The substantial content of the present invention will be further described in detail below in conjunction with the specific embodiments.

Example 1

A kit for assessing sperm quality after in vitro capacitation, the kit comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2; Detection buffer 1 was a phosphate buffer; the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 μg/μl bovine serum albumin.

The kit of the present invention measures the sialidase activity of the uncaptured sperm and the capacitated sperm (A is a mouse, B is a human), and the results show that the sperm can release the sialidase into the capacitate after capacitation.

Example 2

A kit for assessing sperm quality after in vitro capacitation, the kit comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2; The detection buffer 1 was a BWW culture solution; the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 μg/μl bovine serum albumin.

The in vitro capacitation solution is HTF containing HSA 5 mg/ml.

Example 3

A kit for assessing sperm quality after in vitro capacitation, the kit comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2; Detection buffer 1 was a phosphate buffer; the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 μg/μl bovine serum albumin.

The in vitro capacitation solution is HTF containing HSA 5 mg/ml.

The standard stock solution of sialidase is Clostridium perfringens sialidase at a concentration of 5 U/ml.

Example 4

A kit for assessing sperm quality after in vitro capacitation, the kit comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2; Detection buffer 1 was a phosphate buffer; the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 μg/μl bovine serum albumin.

The in vitro capacitation solution is HTF containing HSA 5 mg/ml.

The standard stock solution of sialidase is Clostridium perfringens sialidase at a concentration of 5 U/ml.

The fluorescent reagent is sodium 2'-4-methylumbelliferyl-α-D-N-acetylneuraminic acid.

The sodium acetate has a pH of 5.5.

Example 5

A kit for assessing sperm quality after in vitro capacitation, the kit comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2; Detection buffer 1 was a phosphate buffer; the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 μg/μl bovine serum albumin.

The in vitro capacitation solution is HTF containing HSA 5 mg/ml.

The standard stock solution of sialidase is Clostridium perfringens sialidase at a concentration of 5 U/ml.

The fluorescent reagent is sodium 2'-4-methylumbelliferyl-α-D-N-acetylneuraminic acid.

The concentration of the fluorescent reagent was 10 mM.

The sodium acetate has a pH of 5.

Example 6

A kit for assessing sperm quality after in vitro capacitation, the kit comprising a detection buffer 1, a BWW culture solution, an in vitro capacitation solution, a fluorescent reagent, a sialidase standard stock solution, and a detection buffer 2; Detection buffer 1 was a phosphate buffer; the detection buffer 2 was a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 μg/μl bovine serum albumin.

The in vitro capacitation solution is HTF containing HSA 5 mg/ml.

The standard stock solution of sialidase is Clostridium perfringens sialidase at a concentration of 5 U/ml.

The fluorescent reagent is sodium 2'-4-methylumbelliferyl-α-D-N-acetylneuraminic acid.

The concentration of the fluorescent reagent was 10 mM.

The sodium acetate has a pH of 6.

Example 7

The method for using the kit for assessing sperm quality after in vitro capacitation according to the present invention has the following specific steps:

A, sperm washing

Collecting fresh liquefied semen specimens, detecting buffer 1 to wash the sperm, fully removing the residual seminal plasma components, and then separating the sperm by centrifugation;

B, in vitro capacitation of sperm

BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO ₂ cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO ₂ cells. Incubate the sperm in the box for 3 to 4 hours; centrifuge, the collected supernatant is centrifuged again, and the supernatant is collected as the sample to be tested;

C. Determination of sialidase activity in sperm capacitation solution

(1) Dilute the sialidase standard stock solution to a sialidase standard solution at a concentration gradient of 5/2.5/1.25/0.625/0.312/0 mU/ml using the detection buffer 2, and sequentially add each sialic acid to the test plate. Enzyme standard solution and sample to be tested.

(2) Dilute the fluorescent reagent with the detection buffer 2, add it to each of the above sialidase standard solutions and the wells of the sample to be tested, and then place the assay plate at 37 ° C, incubate in the dark for 1-2 hours, and then use the enzyme. Reader The fluorescence intensity of each well was calculated from the sialidase activity standard curve to calculate the sialidase activity of the sample to be tested, and finally the value of sialidase activity/sperm number was obtained.

Example 8

The method for using the kit for assessing sperm quality after in vitro capacitation according to the present invention has the following specific steps:

A, sperm washing

Collecting fresh liquefied semen specimens, detecting buffer 1 to wash the sperm, fully removing the residual seminal plasma components, and then separating the sperm by centrifugation;

B, in vitro capacitation of sperm

BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO ₂ cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO ₂ cells. Incubate the sperm in the box for 3 to 4 hours; centrifuge, the collected supernatant is centrifuged again, and the supernatant is collected as the sample to be tested;

C. Determination of sialidase activity in sperm capacitation solution

(1) Dilute the sialidase standard stock solution to a sialidase standard solution at a concentration gradient of 5/2.5/1.25/0.625/0.312/0 mU/ml using the detection buffer 2, and sequentially add each sialic acid to the test plate. Enzyme standard solution and sample to be tested.

(2) Dilute the fluorescent reagent with the detection buffer 2, add it to each of the above sialidase standard solutions and the wells of the sample to be tested, and then place the detection plate at 37 ° C, incubate for 1 hour in the dark, and then use a microplate reader. The fluorescence intensity of each well was read, and the sialidase activity of the sample to be tested was calculated based on the standard curve of sialidase activity, and finally the value of sialidase activity/number of sperm was obtained.

The detection board is a 96-hole blackboard.

Example 9

The method for using the kit for assessing sperm quality after in vitro capacitation according to the present invention has the following specific steps:

A, sperm washing

Collecting fresh liquefied semen specimens, detecting buffer 1 to wash the sperm, fully removing the residual seminal plasma components, and then separating the sperm by centrifugation;

B, in vitro capacitation of sperm

BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO ₂ cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO ₂ cells. Incubate the sperm in the box for 3 to 4 hours; centrifuge, the collected supernatant is centrifuged again, and the supernatant is collected as the sample to be tested;

C. Determination of sialidase activity in sperm capacitation solution

(1) Dilute the sialidase standard stock solution to a sialidase standard solution at a concentration gradient of 5/2.5/1.25/0.625/0.312/0 mU/ml using the detection buffer 2, and sequentially add each sialic acid to the test plate. Enzyme standard solution and sample to be tested.

(2) Dilute to the fluorescent reagent at a concentration of 0.05 mM with the detection buffer 2. , adding it to each of the above sialidase standard solutions and the wells of the sample to be tested, and then placing the test plate at 37 ° C, and incubating in the dark for 2 hours, the excitation plate has an excitation wavelength and an emission wavelength of 365 nm and 450 nm, respectively. The fluorescence intensity of each well was read at the wavelength. The sialidase activity of the sample to be tested was calculated from the standard curve of sialidase activity, and finally the value of sialidase activity/number of sperm was obtained.

The detection board is a 96-hole blackboard.

Example 10

The method for using the kit for assessing sperm quality after in vitro capacitation according to the present invention has the following specific steps:

A, sperm washing

Collecting fresh liquefied semen specimens, detecting buffer 1 to wash the sperm, fully removing the residual seminal plasma components, and then separating the sperm by centrifugation;

B, in vitro capacitation of sperm

BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO ₂ cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO ₂ cells. Incubate the sperm in the box for 3 to 4 hours; centrifuge, the collected supernatant is centrifuged again, and the supernatant is collected as the sample to be tested;

C. Determination of sialidase activity in sperm capacitation solution

(1) Dilute the sialidase standard stock solution to a sialidase standard solution at a concentration gradient of 5/2.5/1.25/0.625/0.312/0 mU/ml using the detection buffer 2, and sequentially add each sialic acid to the test plate. Enzyme standard solution and sample to be tested.

(2) Dilute to the concentration of the fluorescent reagent with the detection buffer 2 to 0.05 mM, add it to each of the above sialidase standard solutions and the wells of the sample to be tested, and then place the test plate at 37 ° C, and incubate for 1.5 hours in the dark. After that, the microplate reader reads each at a wavelength of excitation wavelength and emission wavelength of 365 nm and 450 nm, respectively. Pore fluorescence intensity. The sialidase activity of the sample to be tested was calculated from the standard curve of sialidase activity, and finally the value of sialidase activity/number of sperm was obtained.

The detection board is a 96-hole blackboard.

Example 11

Consumables (all sterilized): 15ml EP tube, 1.5ml EP tube, 3 kinds of specifications (1ml/200μl/10μl), 96-well transparent plate, 96-hole blackboard

Instrument: high speed cryogenic centrifuge, microplate reader

The method for using the kit for assessing sperm quality after in vitro capacitation according to the present invention has the following specific steps:

A, sperm washing

1. Collect freshly liquefied semen specimens 4-5ml (according to the patient's sperm count, preservation and transport on ice).

2. Add an equal volume of PBS and blow evenly.

3. Centrifuge at low speed (1500g/5min) and discard the supernatant.

4. Add PBS to 3-4 ml and blow evenly.

5. Centrifuge at low speed (1500g5min), discard the supernatant, leave the supernatant to about 300μl, and blow evenly.

6. Add PBS to 1 ml, centrifuge at high speed (4 degrees, 12000 rpm/5 min), discard the supernatant as much as possible.

B, in vitro capacitation of sperm

1. BWW was added to the sperm pellet, and the upstream method was cultured in a 37 ° C 5% CO 2 cell incubator for 30 min, the upstream sperm was harvested, the sperm count was counted (1× ^{10 7} or more), and the sperm was precipitated by low speed centrifugation (1500 g/5 min).

2. Add the in vitro capacitation solution and incubate for 4 hours in a 37 ° C 5% CO 2 cell incubator.

3. Centrifuge at low speed (500g/5min) and collect the supernatant.

4. Centrifuge at low speed (5000g/15min) and collect the supernatant.

C. Determination of sialidase activity in sperm capacitation solution

1. Dilute the sialidase standard stock solution to a sialidase standard solution at a concentration gradient of 5/2.5/1.25/0.625/0.312/0mU/ml using the detection buffer 2, and then in a 96-well assay plate (black). 50 μl of each sialidase standard solution was added.

2. Dilute to a concentration of 0.05 mM with a detection reagent 2, and add it to each of the above Inside the well, 50 μl/well.

3. Place the 96-well assay plate (black) at 37 ° C, incubate for 1-2 hours in the dark, and then read the fluorescence intensity of each well with a microplate reader at an excitation/emission wavelength of 365/450 nm, according to sialic acid. The enzyme activity standard curve calculates the sialidase activity of the sample to be tested.

4. Finally, the sialidase activity/sperm number obtained will be calculated and the unit enzyme activity value (U AUS/10 ⁴ sperm) will be calculated.

The sialidase activity of the sample used for the assay should be in the range of 0.3-5 mU/ml. If it is exceeded, the sialidase activity value of the sample to be tested in the well should be adjusted within this linear range.

Claims (10)

1. A kit for assessing sperm quality after in vitro capacitation, characterized in that the kit comprises a detection buffer 1, a BWW culture solution, an in vitro energy-receiving solution, a fluorescent reagent, a sialidase standard stock solution and a detection buffer 2 The detection buffer 1 is a phosphate buffer or a BWW culture solution; the detection buffer 2 is a phosphate buffer containing 0.05 mmol/l sodium acetate and 0.25 μg/μl bovine serum albumin.
2. The kit for assessing sperm quality after in vitro capacitation according to claim 1, wherein the in vitro capacitation solution is HTF containing HSA 5 mg/ml.
3. The kit for assessing sperm quality after in vitro capacitation according to claim 1, wherein the standard sialidase stock solution is Clostridium perfringens sialidase at a concentration of 5 U/ml.
4. The kit for assessing sperm mass after in vitro capacitation according to claim 1, wherein the fluorescent reagent is 2'-4-methylumbelliferyl-α-DN-acetylneuraminic acid sodium.
5. The kit for assessing sperm quality after in vitro capacitation according to claim 4, wherein the concentration of the fluorescent reagent is 10 mM.
6. The kit for assessing sperm quality after in vitro capacitation according to claim 1, wherein the sodium acetate has a pH of 5-6.
7. The method for using a kit for assessing sperm quality after in vitro capacitation according to claim 1, wherein the specific steps are as follows:

   A, sperm washing

   Collecting fresh liquefied semen specimens, detecting buffer 1 to wash the sperm, fully removing the residual seminal plasma components, and then separating the sperm by centrifugation;

   B, in vitro capacitation of sperm

   BWW culture solution was added to the sperm isolated in step A, cultured in a 5% CO ₂ cell incubator by the upstream method, the upstream sperm was harvested, the sperm count was counted, and the in vitro capacitation solution was cultured in 5% CO ₂ cells. Incubate the sperm in the box for 3 to 4 hours; centrifuge, the collected supernatant is centrifuged again, and the supernatant is collected as the sample to be tested;

   C. Determination of sialidase activity in sperm capacitation solution

   (1) Dilute the sialidase standard stock solution to a sialidase standard solution at a concentration gradient of 5/2.5/1.25/0.625/0.312/0 mU/ml using the detection buffer 2, and sequentially add each sialic acid to the test plate. Enzyme standard solution and sample to be tested;

   (2) Dilute the fluorescent reagent with the detection buffer 2, add it to each of the above sialidase standard solutions and the wells of the sample to be tested, and then place the assay plate at 37 ° C, incubate in the dark for 1-2 hours, and then use the enzyme. The spectrometer reads the fluorescence intensity of each well, calculates the sialidase activity of the sample to be tested according to the standard curve of sialidase activity, and finally obtains the value of sialidase activity/sperm number.
8. The method of using a kit for assessing sperm quality after in vitro capacitation according to claim 7, wherein the detecting plate is a 96-well blackboard.
9. The method of using a kit for assessing the quality of sperm in vitro after in vitro according to claim 7, characterized in that the concentration of the fluorescent reagent is diluted with the detection buffer 2 to 0.05 mM.
10. The method for using the kit for evaluating the quality of sperm in vitro after in vitro according to claim 7, characterized in that the microplate reader reads the fluorescence intensity of each well at a wavelength of excitation wavelength and emission wavelength of 365 nm and 450 nm, respectively. .

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