CN110763751A - Method for detecting fertilization capability of frozen bovine semen and application of method - Google Patents
Method for detecting fertilization capability of frozen bovine semen and application of method Download PDFInfo
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- CN110763751A CN110763751A CN201911022724.5A CN201911022724A CN110763751A CN 110763751 A CN110763751 A CN 110763751A CN 201911022724 A CN201911022724 A CN 201911022724A CN 110763751 A CN110763751 A CN 110763751A
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Abstract
The application discloses a method for detecting the fertilization capability of frozen bovine semen and application thereof. The method for detecting the fertilization capability of the frozen bovine semen comprises the steps of detecting the fertilization capability index of the frozen bovine semen on the semen in the semen, and judging the fertilization capability of the frozen bovine semen according to the fertilization capability index of the frozen bovine semen; the indexes of the fertilization capability of the frozen bovine semen comprise sperm motility rate, sperm motility, mitochondrial membrane channel pore activity and sperm DNA damage. The method for detecting the fertilization capability of the frozen bovine semen uses the sperm motility rate, the sperm motility, the mitochondrial membrane channel hole motility and the sperm DNA damage as indexes to detect the sperm, can quickly, accurately and effectively judge the fertilization capability of the frozen bovine semen through the detection of the indexes, provides a new objective and effective analysis method for the fertilization capability detection of the frozen bovine semen, and has important significance for customs quick detection and biological breeding.
Description
Technical Field
The application relates to the field of bovine semen activity detection, in particular to a method for detecting bovine frozen semen fertilization capability and application thereof.
Background
China is a big country of animal husbandry, imports the semen of high-quality livestock, particularly the frozen bovine semen, is used for crossbreeding, and is vital to improving the yield of dairy cows and the quality of beef cattle in China; the fertilization ability of the frozen bovine semen directly determines the efficiency of rapid propagation.
The traditional method for judging sperm fertilization capability mainly depends on observing the motility and linear motion state of the sperm under a common microscope by naked eyes, or detecting the motility rate, the acrosome integrity rate and the like of the sperm through dyeing. The problem of strong subjectivity exists in both microscope observation and dyeing detection, and the judgment of a detection result is greatly influenced by human factors; meanwhile, the detection result is also influenced by the environmental temperature, the sperm incubation time and the like.
In recent years, the detection of the activity of bovine semen has been receiving increasing attention, and with the development of molecular biotechnology, various methods for detecting the activity of bovine semen have emerged, such as weak sperm-related gene expression detection, which tries to determine the activity of bovine semen through gene detection, and also, for example, determine the activity of bovine semen through the linear movement speed of sperm or the activity of mitochondria; however, the bovine semen activity parameter indexes obtained by different detection methods are different from the evaluation result of the fertilization capability of the bovine frozen semen, so that the method is extremely disadvantageous to the detection of the fertilization capability of the bovine frozen semen, particularly to the customs quick detection of the fertilization capability of the imported bovine frozen semen. Therefore, an effective objective method for rapidly and accurately determining the fertilization capability of the frozen bovine semen is urgently needed.
Disclosure of Invention
The object of the present application is to provide a comprehensive improved method for detecting the fertilization capability of frozen bovine semen and the use thereof.
The following technical scheme is adopted in the application:
the application discloses a method for detecting the fertilization capability of frozen bovine semen, which comprises the steps of detecting the fertilization capability index of the frozen bovine semen on the semen in the semen, and judging the fertilization capability of the frozen bovine semen according to the fertilization capability index of the frozen bovine semen; the indexes of the fertilization capability of the frozen bovine semen comprise sperm motility rate, sperm motility, mitochondrial membrane channel pore activity and sperm DNA damage. Wherein the sperm motility rate, the sperm motility and the activity of the mitochondrial membrane channel holes are in direct proportion to the fertilization capability of the frozen bovine semen; sperm DNA damage is inversely related to the fertility of frozen bovine semen.
Wherein, the sperm motility rate, namely the survival rate of the sperm, refers to the proportion of the viable sperm in unit semen; sperm motility refers to the percentage of sperm in unit semen which is moving forward; mitochondrial membrane transit pore (MPTP) is a nonspecific and calcium ion-dependent channel composed of both mitochondrial inner and outer membrane components; sperm DNA damage is a parameter that reflects sperm DNA integrity, with less sperm DNA damage, higher sperm quality, and correspondingly greater fertility.
The key point of the application lies in that the indexes of sperm motility rate, sperm motility, mitochondrial membrane channel pore activity and sperm DNA damage are creatively adopted as the basis for judging the fertilization capability of the frozen bovine semen. On one hand, the research of the application proves that the indexes are indeed directly related to the fertilization capability of the frozen bovine semen, so that the indexes can be used as judgment indexes; on the other hand, the indexes can be quantitatively detected through instruments or equipment, compared with a method of directly observing by naked eyes through a microscope, the method is less influenced by artificial subjective factors, and the detection result is more accurate.
The key point of the application is that the sperm motility rate, the sperm motility, the mitochondrial membrane channel pore activity or the sperm DNA damage are adopted as the judgment indexes of the bovine frozen semen fertilization capability; as to how to quantitatively detect each index, the prior art can be referred to; of course, in order to ensure that the detection result is more accurate and effective, the detailed detection of each index is defined in detail in the preferred technical scheme of the application, which is described in the following technical scheme. It is understood that the detection of each index provided in the following detailed technical solutions is only a preferred solution, and in the case of low detection requirements, it is not excluded that other solutions may also be adopted to detect each index, and the detection is not specifically limited herein.
Preferably, in the method for detecting the fertilization capability of the frozen bovine semen, the method for detecting the sperm motility rate comprises the steps of staining the sperm by trypan blue, and judging the sperm motility rate according to the staining condition.
The basic principle of trypan blue staining for identifying the sperm motility rate is that when the cell membrane structure is complete, trypan blue can be excluded so that the trypan blue cannot enter cells; while spermatids with lost activity or incomplete cell membranes have increased permeability of the cell membranes and can be stained blue by trypan blue. Loss of cell membrane integrity is generally considered, i.e., the cell is considered dead. Therefore, live sperm cells and dead sperm cells can be distinguished very simply and rapidly by means of trypan blue staining. It is understood that trypan blue is a preferred chemical dye specifically employed in one implementation of the present application, and that other chemical dyes, such as PI, FDA, AlamarBlue, Hoechst 33258, Hoechst 33342, DAPI, JC-1, AO, calcein, etc., may also be employed, without excluding that detection requirements are low.
Preferably, in the method for detecting the fertilization capability of the frozen bovine semen, the method for detecting the sperm motility comprises detecting the sperm motility by using a sperm analyzer.
The sperm analyzer is a conventional sperm activity performance analysis instrument, the sperm analyzer is used for detecting sperm motility (unit%), proctopathy (STR%), curve speed vcl (micrometer/s), linearity LIN (%), linearity speed vsl (micrometer/s), whip frequency BCF (HZ), swinging WOB (%), and other performance indexes, and the result shows that only sperm motility in the performance indexes is in relatively obvious positive correlation with the fertilization capability of the frozen bovine semen, and the correlation of other performance indexes is poor or even irrelevant; therefore, the sperm motility is used as an index for detecting the fertilization capability of the frozen bovine semen.
Preferably, in the method for detecting the fertilization capability of the frozen bovine semen, the method for detecting the activity of the channel hole of the mitochondrial membrane comprises the steps of staining the sperm by using calcein-AM as a fluorescence probe, and judging the activity of the channel hole of the mitochondrial membrane through the fluorescence change of calcein in the mitochondria.
It should be noted that the method for detecting the channel pore activity of the mitochondrial membrane by using calcein-AM is only a preferred method adopted in one implementation scheme of the present application, and other methods can also be adopted in the case of low detection requirements, which is not excluded.
Preferably, the GENMED living cell mitochondrial membrane channel pore fluorescence detection kit is used for detecting the mitochondrial membrane channel pore.
Preferably, in the method for detecting the fertilization capability of the frozen bovine semen, the method for detecting sperm DNA damage comprises the step of detecting sperm DNA damage by using a DNA damage comet assay kit.
The application also discloses application of the method in sperm fertilization ability detection.
It will be appreciated that although the method of the present application has been developed with particular reference to bovine frozen semen, the underlying principle is to detect and reflect the fertility of semen or sperm, and is not limited to bovine frozen semen, and other semen or sperm fertility detections may equally be used with the method of the present application.
The beneficial effect of this application lies in:
the method for detecting the fertilization capability of the frozen bovine semen detects the sperm by taking the sperm motility rate, the sperm motility, the mitochondrial membrane channel hole activity and the sperm DNA damage as indexes, and can quickly, accurately and effectively judge the fertilization capability of the frozen bovine semen through the detection of the indexes; provides a new objective and effective analysis method for the fertilization capability detection of the frozen bovine semen and has important significance for customs quick detection and biological breeding.
Drawings
FIG. 1 is a graph showing cleavage of semen A in the example of the present application;
FIG. 2 is a graph showing the cleavage of semen B in the example of the present application;
FIG. 3 is a graph showing cleavage of semen C in the example of the present application;
FIG. 4 is a diagram showing blastocyst development of semen A in the example of the present application;
FIG. 5 is a diagram showing blastocyst development of semen B in the example of the present application;
FIG. 6 is a diagram showing blastocyst development of semen C in the example of the present application;
FIG. 7 is a graph showing the result of trypan blue staining of semen A in the example of the present application;
FIG. 8 is a graph showing the result of trypan blue staining of semen B in the example of the present application;
FIG. 9 is a graph showing the result of trypan blue staining of C semen in the examples of the present application;
FIG. 10 is a graph showing the result of the detection of the acrosomal integrity rate of sperm A in the example of the present application;
FIG. 11 is a graph showing the result of the detection of the acrosome integrity rate of semen B in the example of the present application;
FIG. 12 is a graph showing the result of the acrosome integrity detection staining of semen C in the example of the present application;
FIG. 13 is a graph showing the results of the detection of mitochondrial membrane channel pores in triplicate semen in an example of the application;
FIG. 14 is a graph showing the results of the mitochondrial membrane potential detection of three semen samples in the example of the present application;
FIG. 15 is a graph showing the results of indirect measurement of mitochondrial activity of sperm by three semen MTT methods in the examples of the present application;
FIG. 16 is a graph showing the results of fluorescence measurement of mitochondrial oxidative stress reactive oxygen species in three sperm cells in the examples of the present application;
FIG. 17 is a graph showing the results of DNA damage detection on sperm cells in triplicate semen in an example of the present application;
FIG. 18 is a graph showing the results of the detection of the relative mRNA expression levels of the four genes SMCP, TEKT3, DNAH1 and TCTE in three semen samples of the present application.
Detailed Description
At present, methods for detecting semen activity are various, but not all detection methods or parameters can accurately and effectively reflect semen fertilization capability, particularly fertilization capability of frozen bovine semen. In view of the above, the present application provides a method for detecting the fertilization ability of frozen bovine semen, that is, the fertilization ability index of frozen bovine semen is detected for the semen in the semen, and the fertilization ability index of frozen bovine semen includes the sperm motility rate, the sperm motility, the mitochondrial membrane channel pore activity and the sperm DNA damage.
Although, parameters and detection methods for sperm motility, mitochondrial membrane channel pore activity and sperm DNA damage are known; however, the inventors of the present application have creatively discovered that, among the numerous semen activity parameters, the four parameters are closely related to the fertilization capability of the frozen bovine semen, and therefore propose the fertilization capability detection method of the frozen bovine semen of the present application.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application. In the following examples, unless otherwise specifically defined or indicated, reference is made to the prior art for other procedures or reagents not specifically indicated.
Examples
Materials and methods
1. Main instruments and reagents
The optical microscope was purchased from Olympus, Japan, the fluorescence microscope was purchased from Leica, Germany, the carbon dioxide incubator was purchased from Thermo Fisher Scientific, USA, the micropipettor and centrifuge were purchased from Eppendorf, Germany, the electronic balance was purchased from Shanghai Zhengguang medical instruments, the sperm analyzer was a Viagra sperm analyzer (WlJY-9000) purchased from Beijing Weilii New century technology, and the electric thermostatic water bath was purchased from JinghongKD-S24. Other disposable slides, coverslips, centrifuge tubes and 35mm petri dishes are conventional test equipment.
The kit required for detecting the relevant indexes comprises: GENMED universal DNA damage comet assay kit, GENMED-MTT colorimetric method cell propagation quantitative detection kit, GENMED living cell mitochondrial membrane channel hole (MPTP) fluorescence detection kit, GENMED living cell mitochondrial inner membrane function (membrane potential) fluorescence determination kit, GENMED sperm cell ATP bioluminescence quantitative detection kit and sperm trichrome (triple) staining kit.
The test is carried out by adopting the frozen bovine semen of three different batches of Holstein bull at different times, which is obtained from the cow center in Beijing City, wherein the frozen bovine semen is specifically the frozen bovine semen which is numbered 11101938 in 2006 and 10 months, and the frozen bovine semen is marked as semen A; frozen bovine frozen semen, numbered 11109006 at 10 months of 2016, in this case labeled as semen B; frozen bovine frozen semen, numbered 11101912 at month 4 of 2018, in this case labeled as C semen.
2. Detection of various indexes of frozen bovine semen
(1) Sperm analyzer detection
In the embodiment, a Vientine sperm analyzer is used for detecting various indexes of frozen bovine semen obtained from a cow center in Beijing, such as sperm motility, linear speed, curve speed, sperm head side swing amplitude, linear LIN, swing WOB, side swing amplitude ALH, average moving angle MAD (degree), whipping frequency BCF and the like of three different batches at different time. Thawing 3 frozen sperms in a water bath thermostat at 37 deg.C, smearing on glass slide, observing under microscope, and analyzing the statistical data with a Vientine sperm analyzer.
(2) Sperm acrosome integrity detection
In this example, sperm acrosome integrity was measured using a sperm trichrome staining kit, which specifically includes mixing thawed semen, adding 50 microliters of GENMED staining solution (Reagent A), incubating in a 37 ℃ constant temperature water bath for 15 minutes, immediately transferring 10 microliters to a clean slide, covering with a cover glass, immediately observing and counting under an optical microscope under 1000 times oil-scope, and counting 200 sperm per field. The sperm cells stained blue are dead cells, while the sperm cells not stained are live cells; the sperms with green heads are acrosome damaged sperms, and the sperms with red heads are dead sperms with complete acrosomes; the sperm stained partly green and partly red in the head were dead sperm with acrosome damage.
Blue stained sperm cells are dead cells and non-stained sperm cells are live cells, 1.5 ml of the centrifuge tube is placed in a mini-bench centrifuge and centrifuged for 10 minutes at a speed of 600g, or 2500RPM, such as EPPENDORF5415, the supernatant is carefully removed and 500 microliters of the GENMED clear solution is added, the mixture is placed in a mini-bench centrifuge and centrifuged for 10 minutes at a speed of 600g, the supernatant is carefully removed and 500 microliters of the GENMED fixative is carefully removed, the mixture is incubated for 60 minutes at room temperature, the mixture is placed in a mini-bench centrifuge and centrifuged for 10 minutes at a speed of 600g, the supernatant is carefully removed and 100 microliters of the GENMED clear solution is added, the mixture is immediately transferred to one end of a clean slide, dried in air with a cover slip or another slide push piece, 200 microliters of the GENMED stain is carefully added to the slide, the sample surface is covered, the mixture is placed in a 40 ℃ constant temperature incubator for 5 minutes, the GENMED stain is immediately removed, carefully add 200 microliters of the GENMED wash solution to the slide to cover the surface of the sample, immediately remove the GENMED wash solution, carefully add 200 microliters of the GENMED trisstain to the slide to cover the surface of the sample, incubate at room temperature for 30 minutes, immediately remove the GENMED trisstain, carefully add 200 microliters of the GENMED wash solution to the slide to cover the surface of the sample, and immediately remove the GENMED wash solution. Mounted, then immediately under the optical microscope 1000 times oil microscope observation and counting, each field count 200 sperm.
And (3) judging when counting sperms: pink acrosome and light brown acrosome back region, which is the intact acrosome of the living sperm; colorless acrosomes and light brown rear acrosomes, which are acrosomes or acrosomes lost after reaction, namely positive acrosomes react; blue spermatids, pink acrosomes and light brown acrosomes rear regions, which are dead sperm unreacted intact acrosomes; colorless acrosomes and the posterior region of colorless acrosomes, which are degenerated acrosomes or acrosomal deletions of sperm after death or cryopreservation and after injury.
Sperm viability was analyzed using trichromatic sperm staining, first using trypan blue (trypan blue) staining, which was detected based on the principle that trypan blue is repelled by live sperm cells, including motile and non-motile sperm, while dead sperm cell uptake dye shows red color. Second, intact or unreacted acrosomes are distinguished using either Monglar or tiger Red (Rose Bengal) dye in combination with acrosomal contents, and Bismarck Brown Y (Bismark Brown) dye identifies post-acrosomal region (post-acrosomal region) staining. The acrosome deletion state can be clearly observed by a common optical microscope without using a phase-contrast microscope.
(3) Mitochondrial membrane channel pore assay for sperm cells
Mitochondrial membrane channel pores (MPTP) are nonspecific and calcium ion-dependent channels composed of both mitochondrial inner and outer membrane components. During apoptosis or necrosis, mitochondrial contents are released into the cytosol through membrane access pores. The opening of membrane channel pores significantly alters mitochondrial permeability. Excessive calcium ion entry, oxidation of mitochondrial glutathione, and increased levels of reactive oxygen species, etc., lead to the sustained opening of membrane channel pores, resulting in the release of cytochrome C and the disappearance of the mitochondrial membrane potential. calcein-AM, also known as bismethyliminodiacetic acid sodium fluorescein-acetoxymethyl ester [ bis (carboxymethyethyl amino methyl fluorescein) -acetoxymethyl ester ], as a fluorescent probe: first, a suitable molecular weight is 622kd, less than 1.5 kd; second, there is little film binding; third, not a substrate for mitochondrial enzymes; fourth, it readily enters the cell and its mitochondria. After entering cells, calcein-AM is cleaved by intracellular esterase to produce calcein with polarity and strong fluorescence. Calcein enters mitochondria and is captured by mitochondria. While calcein in the cytoplasm is quenched by its quencher cobalt ion. Once the mitochondrial membrane channel pore was transiently opened, calcein was released and quenched by cobalt ions. Changes in the fluorescence of calcein within mitochondria indicate the open state of membrane channel pores.
The method specifically adopts a GENMED living cell mitochondrial membrane channel pore (MPTP) fluorescence detection kit to detect the mitochondrial membrane channel pore, and specifically comprises the following steps:
before the experiment, the GENMED staining solution (Reagent B) in the kit in a refrigerator with the temperature of-20 ℃ is placed in an ice tank to be melted, and the GENMED neutralizing solution (Reagent C) is placed in a constant-temperature water tank with the temperature of 37 ℃ to be preheated. Then 100. mu.l of GENMED staining solution (Reagent B) was removed to a new 1.5 ml centrifuge tube, 100. mu.l of GENMED neutralization solution (Reagent C) was added, mixed, labeled as GENMED staining solution, and placed in the dark. The following operations were then carried out:
will be 1 × 106The individual sperm cells were transferred into a 1.5 ml centrifuge tube, centrifuged in a mini-bench centrifuge for 5 minutes at a speed of 300g, or 2000RPM, e.g., eppendorf5415, the supernatant carefully removed, 200 microliters of 37 ℃ preheated GENMED clearance solution (Reagent A) were added, the cell particle population was homogenized, centrifuged in a mini-bench centrifuge for 5 minutes at a speed of 300g, or 2000RPM, e.g., eppendorf5415, the supernatant carefully removed, 100 microliters of GENMED staining solution containing GENMED staining solution (Reagent B) and GENMED neutralizing solution (Reagent C) were added, homogenized well, and incubated in a 37 ℃ cell culture chamber for 20 minutes without light.
After the incubation was complete, the cells were centrifuged in a mini-bench centrifuge for 5 minutes at 300g, or 2000RPM, e.g., eppendorf5415, the supernatant carefully removed, 100 microliters of 37 ℃ pre-heated GENMED clearance (Reagent A) was added, the cell particle population was mixed, and the cells were centrifuged in a mini-bench centrifuge for 5 minutes at 300g, or 2000RPM, e.g., eppendorf5415, and the supernatant carefully removed. Repeatedly washing with GENMED cleaning solution, adding 100 μ l of GENMED cleaning solution (Reagent A) preheated at 37 deg.C, mixing cell particle group, placing in fluorescence spectrophotometer, detecting every 5 min for 30 min; wherein, the excitation wavelength of the fluorescence spectrophotometer is 488nm, and the emission wavelength is 505 nm.
If the relative fluorescence unit value (RFU) decreases, this indicates an increase in mitochondrial membrane channel pore activity (MPTP).
(4) Mitochondrial Activity assay
In the embodiment, the mitochondrial activity is indirectly measured by adopting MTT, and the cell activity is specifically measured by adopting a GENMED-MTT colorimetric cell proliferation quantitative detection kit, so that the mitochondrial activity is indirectly reflected. The method specifically comprises the following steps:
resuscitating 3 different batches of frozen bovine semen, carefully adding GENMED staining solution (Reagent A) into each well of a well plate, placing the well plate into an incubator at 37 ℃, incubating for 4 hours, placing the well plate into a well plate centrifuge for centrifugation for 10 minutes at a speed of 300g, carefully removing the culture solution containing GENMED staining solution (Reagent A) from each well to ensure that no liquid remains in the culture well, then carefully adding 100 microliters of GENMED dissolving solution (Reagent B) into each well of a 96 well plate, placing the well plate into the incubator at 37 ℃, incubating for 1 hour, gently shaking the 96 well plate to mix the well plate, immediately placing the well plate into an enzyme reader for measurement, and generating an absorption peak at a wavelength of 570 nm: the light absorption value is in positive linear correlation with the cell amount and reflects the cell activity, thereby indirectly reflecting the mitochondrial activity.
(5) Mitochondrial membrane potential determination
JC-1 is an ideal fluorescent probe widely used for detecting mitochondrial membrane potential (mitogenic membrane potential) △ Ψ m, and the current example adopts a GENMED live cell mitochondrial inner membrane function (membrane potential) fluorescence measurement kit to detect the mitochondrial membrane potential, and specifically comprises the following steps:
3 different batches of frozen semen are recovered, the semen cells are moved into a 2 ml EP tube after recovery, GENMED cleaning solution (Reagent C) is added into the EP tube, the cleaning solution (Reagent C) is carefully moved out into a new 2 ml conical centrifugal tube, and the eluted cells are collected. Placing the cell culture medium into a 37 ℃ incubator for 1 minute, adding 1 ml of complete cell culture medium, transferring the cell culture medium into a 2 ml conical centrifuge tube, placing the centrifuge tube into a desktop centrifuge for 5 minutes at the speed of 300g, carefully removing supernatant, adding GENMED cleaning solution (Reagent C), gently sucking up and down by using a 200 microliter gun head, uniformly mixing cell particle groups, transferring the cell culture medium into a 1.5 ml centrifuge tube, adding GENMED staining working solution containing GENMED staining solution (Reagent A) and GENMED diluting solution (Reagent B), slightly whirling and shaking for 2 seconds, placing the cell culture medium into a 37 ℃ cell culture box, incubating the cell culture medium in a dark place for 20 minutes, placing the cell culture medium into a desktop microcentrifuge for 5 minutes at the speed of 300g, carefully removing supernatant, and finally adding GENMED cleaning solution (Reagent C). After mixing, respectively moving 100 microlitres to a black 96-hole plate or a 100 microlitres cuvette, and immediately carrying out the measurement of a 96-hole microplate reader or the cuvette by a fluorescence spectrophotometer.
(6) Sperm DNA damage detection
In the embodiment, the GENMED universal DNA damage comet assay kit is used for detecting sperm DNA damage, and the method specifically comprises the following steps:
a. agar slide preparation
Preparing 1 frosted glass slide with the frosted surface facing upwards, and heating and melting GENMED colloidal fluid (Reagent A) in a microwave oven, wherein attention is paid to avoid overflow; then, the slide glass is put into a constant temperature water tank at 40 ℃ for incubation for 10 minutes, 5 microliters of GENMED colloidal fluid (Reagent A) is removed to the middle of the slide glass, the clean cover glass is covered on the slide glass for light pressing for 5 minutes, and the slide glass is put into a refrigerator at 4 ℃ for standby
b. Sample preparation
Before the experiment, the GENMED matrix solution (Reagent B) is placed in a microwave oven to be heated and melted, and then is placed in a constant-temperature water tank at 40 ℃ for standby. The following operations were then carried out:
preparing a semen sample, transferring the semen sample into a 1.5 ml centrifuge tube, placing the tube into a 4 ℃ micro-desktop centrifuge for centrifugation for 10 minutes at the speed of 300g, adding GENMED cleaning solution (Reagent C), mixing uniformly, transferring 20 microliters to a new 1.5 ml centrifuge tube, adding 50 microliters of GENMED matrix solution (Reagent B) in a 40 ℃ constant-temperature water tank, sucking up and down by using a gun head for mixing uniformly, pushing and removing a cover glass on the prepared slide by using a thumb, and immediately transferring 75 microliters of cell matrix mixture onto the slide.
Cover with new cover glass, cover the cover glass for 5 minutes lightly, put in a refrigerator at 4 ℃ to cool for 10 minutes until the gel is condensed to avoid light irradiation, remove the cover glass with thumb push, put the slide in a 9cm petri dish, add 2 ml of precooled GENMED lysate (Reagent D), put in a refrigerator at 4 ℃ to incubate for 60 minutes, and pour out the lysate.
c. Gel electrophoresis
Removing 2 ml of GENMED electrophoresis liquid (Reagent E) into a 250 ml triangular flask, adding 225 ml of deionized water, fully mixing uniformly, marking as GENMED electrophoresis working liquid, adding the GENMED electrophoresis working liquid into a 9cm culture dish, incubating for 30 minutes at room temperature, pouring the electrophoresis working liquid, placing a glass slide into a horizontal electrophoresis tank with temperature control, adding the GENMED electrophoresis working liquid into the electrophoresis tank to ensure that the GENMED electrophoresis working liquid is just above the glass slide, plugging a power supply, setting the voltage to be 25V or the current to be 300 mA, performing electrophoresis for 30 minutes at 4 ℃, disconnecting the power supply, taking out the glass slide, soaking for 2 times, soaking for 1 second each time, placing the glass slide into the 9cm culture dish, adding GENMED neutralization liquid (Reagent F), incubating for 5 minutes at 4 ℃ in a refrigerator, pouring out the neutralization liquid, and airing in air.
d. Staining analysis
Add 1 μ l of gen med staining solution (Reagent G), cover with new coverslips, incubate at room temperature for 5 minutes, avoid light, observe immediately under fluorescence microscope and record by taking pictures: an excitation wavelength of 546nm and an emission wavelength of 590 nm. The images taken were then analyzed by the software of the fluorescence microscope itself.
3. In vitro artificial fertilization test
In order to accurately determine the fertilization capability of three frozen bovine semen, the example adopts an in-vitro artificial fertilization mode to test three frozen bovine semen, and the specific steps are as follows:
(1) collection of bovine oocytes
The cow ovary is collected from a slaughterhouse in Hebei province, stored in normal saline at 28-30 ℃ and transported to a laboratory within 2 hours. The ovaries were washed 3 times with physiological saline at 37 ℃ containing penicillin and streptomycin. A follicle fluid in a follicle with a diameter of 2-6mm is extracted by a vacuum pump and a No. 18 needle, and a cumulus-oocyte complex (hereinafter abbreviated as COCs) which contains 3 or more layers of cumulus cells and has uniform cytoplasm is selected from the follicle fluid precipitate under a microscope. The COCs are washed for 3 times in a pre-balanced in vitro maturation culture solution and placed in a four-hole plate, 750 microliters of in vitro maturation culture solution (IVM culture solution) is added into each hole to culture 50 COCs, and the culture conditions are as follows: at 38.5 deg.C, 5% CO2And culturing for 22-24h in an incubator with saturated humidity. The in vitro maturation medium comprises: TCM-199(GIBCO), 0.01IU/mL Follicle Stimulating Hormone (FSH),Luteinizing Hormone (LH) at 10IU/mL, estradiol at 1. mu.g/mL (E2).
(2) In vitro fertilization
Mature COCs were washed 3 times in fertilization drops pre-equilibrated for 2h and the peripheral portion of cumulus cells was removed. The COCs were then placed in 50 microliter fertilization drops, each drop containing 15-20 COCs. Finally, the sperm and oocyte were incubated at 38.5 ℃ with 5% CO2And co-incubating for 8-16h in an incubator with saturated humidity.
(3) In vitro embryo culture
Removing peripheral cumulus cells of fertilized eggs 8-16h after in vitro fertilization by using a glass needle, washing the fertilized eggs in the embryo culture solution of the early stage for 3 times, and placing the fertilized eggs into a culture medium at 38.5 ℃ and 5% CO2Culturing for 48h in a saturated humidity incubator, changing the later-stage embryo culture solution, then changing the medium for half at intervals of 48h, and respectively counting the cleavage rate and the blastocyst rate of the early-stage embryo on the 2 nd and 7 th days.
4. Detection of weak sperm-associated gene expression
In the embodiment, a GENMED sperm cell ATP bioluminescence method quantitative detection kit is adopted to detect four genes related to weak sperm, namely SMCP, TEKT3, DNAH1 and TCTE.
After three frozen semen are recovered, RNA is extracted by a liquid nitrogen grinding method, the extracted RNA is reversely transcribed into cDNA, and then Real-time PCR detection is carried out. The specific reaction conditions and reaction systems are referred to the kit instructions.
The detection primers for the four genes are shown in Table 1.
TABLE 1 primers for detection of genes associated with weak sperm
Second, result in
1. Observation result of frozen bovine semen state
The appearance of fresh semen of a Holstein bull is rich milky white and grey white, and sometimes is light yellow, opaque and slightly fishy. Observations of three bovine frozen semen showed that it substantially conformed to the characteristics of the semen of a Holstein species bull.
2. In vitro artificial conception result
In the embodiment, three bovine frozen semen portions are unfrozen and then are subjected to in vitro artificial pregnancy, and the cleavage rate and the blastocyst rate are counted, wherein partial observation results are shown in fig. 1 to 6, fig. 1 to 3 are graphs of cleavage conditions of semen A, semen B and semen C in sequence, and fig. 4 to 6 are graphs of blastocyst development conditions of semen A, semen B and semen C in sequence. The statistical result shows that in the aspect of the cleavage rate, the semen C is 70.77%, the semen B is 62.89%, and the semen A is 51.90%; in terms of blastocyst rate, the semen C is 31.90%, the semen B is 25.14%, and the semen A is 16.43%.
The above results indicate that sperm C has the strongest fertilization ability, followed by sperm B, which has the weakest fertilization ability.
3. Sperm motility test results
The performance of three bovine frozen semen samples was examined using a sperm viagra analyzer, and the results are shown in Table 2.
TABLE 2 statistics of key indicators for three bovine frozen semen
The results in Table 2 show that, in terms of sperm motility, 72% for semen C, 47% for semen B and 44% for semen A, the results are consistent with those of in vitro artificial conception; in the aspects of the tropism, the linearity and the swinging, although the three are C semen > B semen > A semen, the numerical difference between the C semen and the B semen is not obvious enough, and the difference exists with the result of in vitro artificial pregnancy; the test results of the curve speed, the linear speed and the whipping frequency are completely different from the in vitro artificial pregnancy results. Thus, according to the above analysis, sperm motility is used as an indicator of the fertility of frozen bovine semen.
4. Sperm motility rate test result
In this example, trypan blue staining was used to identify normal viable sperm cells, from which sperm viability was counted. The basic principle is that when the cell membrane structure is complete, trypan blue can be excluded, so that the trypan blue can not enter cells; while spermatids with lost activity or incomplete cell membranes have increased permeability of the cell membranes and can be stained blue by trypan blue. It is generally believed that the integrity of the cell membrane is lost, i.e., the cell is thought to have died, as opposed to a neutral red effect. Therefore, live cells and dead cells can be distinguished very easily and rapidly by means of trypan blue staining.
Partial observation results are shown in fig. 7 to 9, fig. 7 is a result of staining of semen a, fig. 8 is a result of staining of semen B, and fig. 9 is a result of staining of semen C. The results of the statistics of the sperm motility rates of the three semen are respectively that the sperm motility rate of the semen C is 90%, the sperm motility rate of the semen B is 70%, and the sperm motility rate of the semen A is 66%. The result shows that the statistical result of the sperm motility rate is consistent with the in vitro artificial conception result; therefore, the sperm motility rate is used as the index of the fertilization capability of the frozen bovine semen.
5. Percentage of sperm acrosome integrity
The staining results of sperm acrosome integrity are shown in fig. 10 to 12, fig. 10 is the staining result of semen a, fig. 11 is the staining result of semen B, and fig. 12 is the staining result of semen C. Statistics show that the average value of the acrosome integrity of three different frozen semen A semen, B semen and C semen is 88.46%, 94.63% and 98.31%, respectively.
6. Results of functional analysis of sperm mitochondria
In this example, different kits were used to measure mitochondrial membrane channel pore, mitochondrial membrane potential, mitochondrial activity and mitochondrial oxidative stress active oxygen fluorescence measurements, respectively.
The detection results of mitochondrial membrane channel pores are shown in fig. 13, and the results in fig. 13 show that the lowest RFU value of semen a indicates the lowest membrane channel pore activity, and the highest membrane channel pore activity of semen C mitochondria. The mitochondrial membrane potential results are shown in fig. 14, and the results in fig. 14 show that the mitochondrial membrane potential results for the three seminal fluids generally tend to be the worst seminal fluid a, the second seminal fluid B, and the best seminal fluid C. The results of indirect measurement of mitochondrial activity of sperm by the MTT method are shown in FIG. 15, and the results in FIG. 15 show that the mitochondrial activity trend of three kinds of semen is not obvious. The mitochondrial oxidative stress reactive oxygen fluorescence measurement results are shown in fig. 16, and the results in fig. 16 show that the general trends are that semen a is worst, semen B is second, and semen C is best. Comparing and analyzing the results of fig. 13 to 16, wherein the results of mitochondrial membrane channel pore activity are consistent with the results of in vitro artificial conception; therefore, it is used as an index of the fertilization ability of the frozen bovine semen. The general trend of the fluorescence measurement results of the mitochondrial membrane potential and the mitochondrial oxidative stress active oxygen is consistent with the results of in vitro artificial pregnancy, but the differentiation is not obvious enough.
7. DNA damage detection result of cow frozen semen sperm
The comet-star kit is used for detecting the tailing of the three frozen bovine semen parts, the result is shown in figure 17, figure 17 is a statistical result of sperm DNA damage of the three semen parts, and A, B, C in the figure are respectively semen A, semen B and semen C. The results in fig. 17 show that the a semen smears the longest, the B semen is the second and the C semen is the shortest, therefore, the a semen DNA damage is considered to be the most severe and the C semen damage the least. The result is consistent with the result of in vitro artificial fertilization; therefore, sperm DNA damage is used as an indicator of the fertility of frozen bovine semen.
8. Results of gene detection related to asthenospermia
The results of statistical analysis of the qPCR relative mRNA expression of the four genes SMCP, TEKT3, DNAH1, TCTE are shown in fig. 18, wherein A, B, C are semen a, semen B, and semen C, respectively. The results in FIG. 18 show that the results of measuring the expression levels of the four genes do not show the same tendency as the development of the embryo in the in vitro artificial pregnancy test.
According to the test results, the indexes with higher consistency with the in-vitro artificial pregnancy test results are used as the indexes of the cattle frozen semen fertilization capability, including the sperm motility rate, the sperm motility, the mitochondrial membrane channel pore motility and the sperm DNA damage, and the four indexes can accurately and effectively reflect the fertilization capability of the cattle frozen semen. Therefore, the method for judging the fertilization capability of the frozen bovine semen by detecting the four indexes of the sperm motility rate, the sperm motility, the mitochondrial membrane channel hole activity and the sperm DNA damage is constructed, a quick, accurate and effective objective analysis method is provided for the detection of the fertilization capability of the frozen bovine semen, the method is particularly suitable for the customs quick detection of imported bovine semen, and the theoretical and application foundation is laid for the development of modern biotechnology.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
SEQUENCE LISTING
<110> Shenzhen customs animal and plant inspection and quarantine technical center
South China university of justice
<120> method for detecting fertilization capability of frozen bovine semen and application thereof
<130>19I28762
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Claims (6)
1. A method for detecting the fertilization capability of frozen bovine semen is characterized in that: the method comprises the steps of detecting the fertilization capability index of the frozen bovine semen on the semen in the semen, and judging the fertilization capability of the frozen bovine semen according to the fertilization capability index of the frozen bovine semen; the indexes of the fertilization capability of the frozen bovine semen comprise sperm motility rate, sperm motility, mitochondrial membrane channel pore activity and sperm DNA damage.
2. The method of claim 1, wherein: the sperm motility detection method comprises the steps of staining sperm by trypan blue, and judging the sperm motility through the staining condition.
3. The method of claim 1, wherein: the sperm motility detection method comprises the step of detecting the sperm motility by using a sperm analyzer.
4. The method of claim 1, wherein: the method for detecting the activity of the channel hole of the mitochondrial membrane comprises the steps of adopting calcein-AM as a fluorescent probe to dye sperms, and judging the activity of the channel hole of the mitochondrial membrane through the fluorescent change of calcein in mitochondria; preferably, the GENMED living cell mitochondrial membrane channel pore fluorescence detection kit is used for detecting the mitochondrial membrane channel pore.
5. The method of claim 1, wherein: the detection method of sperm DNA damage comprises the step of detecting sperm DNA damage by adopting a DNA damage comet assay kit.
6. Use of a method according to any one of claims 1 to 5 for the detection of sperm fertilisation capacity.
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| US20080199430A1 (en) * | 2007-02-16 | 2008-08-21 | Dyn-Bioshaf (2006) Ltd. | Methods and kits for qualifying sperm cells |
| US20150065556A1 (en) * | 2013-08-05 | 2015-03-05 | Whitehead Institute For Biomedical Research | Therapeutic targets for mitochondrial disorders |
| US20170105963A1 (en) * | 2015-10-16 | 2017-04-20 | The Board Of Trustees Of The University Of Illinois | Small molecules that induce intrinsic pathways apoptosis |
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