CN108693101A - A kind of method of bivalve shellfish sperm quality detection - Google Patents
A kind of method of bivalve shellfish sperm quality detection Download PDFInfo
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- CN108693101A CN108693101A CN201810465708.2A CN201810465708A CN108693101A CN 108693101 A CN108693101 A CN 108693101A CN 201810465708 A CN201810465708 A CN 201810465708A CN 108693101 A CN108693101 A CN 108693101A
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- 238000001514 detection method Methods 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 19
- 235000015170 shellfish Nutrition 0.000 title claims abstract description 18
- 239000000975 dye Substances 0.000 claims abstract description 24
- 210000003470 mitochondria Anatomy 0.000 claims abstract description 21
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 20
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 15
- 239000013535 sea water Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 12
- 230000006378 damage Effects 0.000 claims description 11
- 238000005119 centrifugation Methods 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 8
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
- 230000003321 amplification Effects 0.000 claims description 7
- 238000004043 dyeing Methods 0.000 claims description 7
- 238000000386 microscopy Methods 0.000 claims description 7
- 230000002438 mitochondrial effect Effects 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 230000026109 gonad development Effects 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 claims description 2
- 206010016825 Flushing Diseases 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 239000012528 membrane Substances 0.000 abstract description 9
- 230000004083 survival effect Effects 0.000 abstract description 8
- 238000012360 testing method Methods 0.000 abstract description 8
- 238000000684 flow cytometry Methods 0.000 abstract description 4
- 230000003834 intracellular effect Effects 0.000 abstract description 2
- 238000011534 incubation Methods 0.000 abstract 1
- 210000001700 mitochondrial membrane Anatomy 0.000 abstract 1
- 150000007523 nucleic acids Chemical class 0.000 abstract 1
- 102000039446 nucleic acids Human genes 0.000 abstract 1
- 108020004707 nucleic acids Proteins 0.000 abstract 1
- 241000237502 Ostreidae Species 0.000 description 3
- 238000005273 aeration Methods 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013505 freshwater Substances 0.000 description 3
- 210000004907 gland Anatomy 0.000 description 3
- 235000020636 oyster Nutrition 0.000 description 3
- 230000001568 sexual effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 230000033001 locomotion Effects 0.000 description 2
- 241000237509 Patinopecten sp. Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000020637 scallop Nutrition 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019100 sperm motility Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
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- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention provides a kind of bivalve shellfish sperm quality detection method, the double dyes of this method binding fluorescent dyes detect bivalve shellfish sperm quality with flow cytometry, include the following steps:Seminal concentration is adjusted first, then it is dyed with the fluorescent dye of the fluorescent dye of permeable membrane and impermeable membrane and sperm to be measured, incubation at room temperature, Flow cytometry, obtain testing result, the fluorescent dye of the permeable membrane can be combined with mitochondrial membrane, and the fluorescent dye of impermeable membrane can pass through damaged cell membrane, and be combined with the nucleic acid specificity of intracellular.The present invention can obtain multiple parameters result simultaneously by one-time detection, the sperm of different conditions can not only be clearly distinguished and show its ratio, obtain dead sperm rate, survival rate, plasma membrane loss percentage and tool mitochondria activity ratio, the relatively low phenomenon of accurate rate caused by can also avoiding subjective factor, it is ensured that the accuracy of testing result.
Description
Technical field
After being forced the present invention relates to a kind of bivalve shellfish sperm quality detection method more particularly to a kind of sperm,
Detect method and the application of living spermatozoa percentage, membrane integrity and mitochondria activity.
Background technology
Currently, bivalve shellfish sperm quality detection method is microscopy.Sperm is placed under light microscope, sperm is observed
Run duration, movement ratio and movement severity.But the experience due to everyone and criterion difference, then add
The time interval of upper observation sperm survival is too long, and such methods often have larger subjectivity and inaccuracy, easily to experiment
Accuracy impact, be easy to derive a wrong conclusion.Computer aided pass design(CASA)Application, make sperm matter
Quantifier elimination is greatly improved, by the analysis to sperm motility track, speed and dynamic parameter, to identify the matter of sperm
Amount.Although CASA can be measured the parameters such as sperm concentration and vigor, it cannot be detected with certain limitation
The variation of sperm internal structure, especially chilled preservation or the sperm coerced, because its membrane integrity and mitochondria are lived
Property be vulnerable to damage, the quality of sperm is influenced notable.In the research of shellfish sperm quality, two kinds of fluorescence dyes are carried out to sperm
Material dyeing makes the fluorescence of sperm release different colours, and then the random counter under fluorescence microscope, counts the survival rate of sperm, this
Although class method can detect the variation of sperm internal structure, there are large error, and it is time-consuming and laborious.
Therefore, the accuracy of experiment can either be improved, again there is an urgent need to a kind of accurate sperm quality detection method now
Can it is convenient, fast, objectively detect sperm quality.
Invention content
The shortcomings of in order to avoid the traditionally subjectivity, inaccuracy and inexactness of microscopy sperm quality, the present invention
A kind of method of novel bivalve shellfish sperm quality detection is provided.
PI is a kind of film impermeability dyestuff, can be damaged into plasma membrane or death intracellular, and discharges fluorescence, commonly uses PI
Detect the integrality of cytoplasma membrane;Rh123 is a kind of cation, can penetrate plasma membrane and mitochondria and specifically bind, have across
The mitochondria of film potential can discharge green fluorescence, due to Rh123 nontoxicitys, not influence the metabolism of cell, so Rh123 is common
To detect mitochondria activity.Therefore present invention combination Rh123/PI it is bis- dye with flow cytometry come detect plasmalemmae of sperms damage situations and
Mitochondria activity, and the survival ratio of Accurate Determining sperm conveniently and efficiently distinguish each shape in the case where not damaging sperm
The sperm of state, and then objectively and accurately evaluate sperm quality.
In order to achieve the above objectives, in conjunction with above-mentioned principle, the specific technical solution that the present invention takes is:
A kind of method of bivalve shellfish sperm quality detection, includes the following steps:
(1)Sperm is produced:Microscopy is dissected, the supermature male of sexual gland is selected, drawing sperm with suction pipe is placed in sterilizing seawater
In;
(2)Fluorescence dye liquor is prepared:PI and Rh123 are dissolved in ultra-pure water respectively, stored;
(3)Sperm dyes:A certain amount of sperm is taken, two kinds of above-mentioned fluorescence dye liquor mixing are added, places, centrifugation, washs, be resuspended, then
Carry out flow cytomery;
(4)Flow cytomery result:Sperm through PI/Rh123 dyeing and FCM detections is distributed in different quadrants:R1 tables
Show the inactive Necrospermia of destruction of plasma membrane mitochondria, R2 indicates plasmarrhexis but mitochondria is still active, that is, will be dead
The sperm died, R3 indicate that plasma membrane is normal but the sperm without mitochondrial transmembrane potentials, R4 are normal sperm living.
Further, the step(1)In, the use of blood counting chamber adjustment sperm concentration is 107~108A/mL.
Further, the step(2)Specially:PI and Rh123 are dissolved in ultra-pure water respectively, are made into a concentration of
The storing liquid of 1mg/mL is put in -20 DEG C and -4 DEG C preservations respectively according to reagent storage request.
Further, the step(3)Specially:Above two storing liquid is diluted to 100 μ g/mL respectively, is taken certain
Sperm is measured, two kinds of dye liquor mixing are added(The sequencing of dye liquor is added without influence), make the dye liquor final concentration of sample to be tested be
10 μ g/mL, 10 ~ 12min of avoid light place, centrifuges at room temperature(300 × g, 5min), 0.01M PBS(pH7.4)It rinses, repeats
Washing centrifugation 2 ~ 3 times, PBS is resuspended, flow cytomery.
Further, in the above method, flow cytometer parameter is set:Forescatering FSC and lateral scattering SSC is linear
Amplification, fluorescence channel FL1 and FL2 logarithmic amplification survey the fluorescence intensity of each sperm Rh123 and PI, and every part of suspension detection 8000 ~
10000 sperms or so.
The beneficial effects of the invention are as follows:
The bis- dyes of present invention combination Rh123/PI detect plasmalemmae of sperms damage situations and mitochondria activity with flow cytometry, and accurate
The survival ratio for really measuring sperm conveniently and efficiently distinguishes the sperm of each state, Jin Erke in the case where not damaging sperm
Sperm quality is accurately evaluated in sight.And the application of flow cytometer can not only improve the accuracy of data, moreover it is possible to rapidly examine
Survey sperm the extent of damage and its ratio, while also have many advantages, such as it is convenient, fast, accurate.
The present invention can obtain multiple parameters by one-time detection, and the sperm of different conditions can not only be clearly distinguished simultaneously
It shows its ratio, obtains dead sperm rate, survival rate, plasma membrane loss percentage and tool mitochondria activity ratio, can also avoid leading
The relatively low phenomenon of accurate rate caused by sight factor, while reducing influence of the human factor to testing result, it is ensured that testing result
Accuracy.
The present invention has detected survival rate, the death rate and the membrane integrity and mitochondria activity of bivalve shellfish sperm, no
The accuracy of experiment can be only increased substantially, error caused by reducing human factor also has many advantages, such as convenient, fast.It is right
Bivalve shellfish sperm, is especially coerced or the survival rate of the sperm of chilled preservation, membrane integrity and mitochondria activity
It is detected, fully assesses sperm quality.
Description of the drawings
Fig. 1 is flow cytomery result figure.
Specific implementation mode
Below in conjunction with the accompanying drawings and specific embodiment is described in further detail the essentiality content of the present invention.
Embodiment 1:
A kind of bivalve shellfish quality determining method and application, concrete operation step are as follows:
1. sperm is produced:Full oyster is developed in selection, and anatomical lens pick goes out the good male of gonad development, drawn with suction pipe
The preferable sperm of vigor is as sperm stoste.
2. gradient seawater configures:Using sterilizing seawater, sea crystal and aeration fresh water, prepare salinity be 10,15,20,25,
30, sperm stoste is added in gradient seawater 35 gradient seawater, and adjustment sperm concentration is 107~108A/mL.
3. fluorescence dye liquor is prepared:PI and Rh123 are dissolved in ultra-pure water respectively, are made into the storage of a concentration of 1mg/mL
Liquid is put in -20 DEG C and -4 DEG C preservations respectively according to reagent storage request.
4. sperm dyes:Two kinds of storing liquids are diluted to 100 μ g/mL, take salinity stress 15min, 30min, 1h, 2h, 4h
Each 160 μ L of sperm in 1.5 mL centrifuge tubes, each 20 μ L mixing of two kinds of dye liquors is added, makes the dye liquor final concentration of sample to be tested be
10 μ g/mL, avoid light place 10min, centrifuges at room temperature(300 × g, 5min), 0.01M PBS(pH7.4)It rinses, repeated washing
Centrifugation 2 times, PBS are resuspended, flow cytomery.
5. flow cytometer parameter setting:Forescatering FSC and lateral scattering SSC are Linear Amplifer, fluorescence channel FL1 and
FL3 logarithmic amplifications, survey the fluorescence intensity of each sperm Rh123 and PI, and every part of suspension detects 10000 sperms or so.
6.FCM testing results:Sperm through PI/Rh123 dyeing and FCM detections is distributed in different quadrants, and R1 is
Rh123-/PI+, indicate the inactive Necrospermia of destruction of plasma membrane mitochondria;R2 is Rh123+/PI+, indicate plasmarrhexis but line grain
Body is still active, that is, i.e. by dead sperm;R3 is Rh123-/PI-, indicate that plasma membrane is normal but electric without Mitochondrial transmembrane
The sperm of position;R4, that is, Rh123+/PI-, indicate normal sperm living.
Fig. 1 is the quality by stress oyster sperm using bivalve shellfish sperm quality detection method of the present invention detection.FL3 is
Fluorescent dye PI sense channels, FL1 are fluorescent dye Rh123 sense channels, and Number is the sperm number of all quadrants, %Gate
For the ratio shared by all quadrants sperm count, X-Mean is average fluorescent strength, R1 Rh123-/PI+, indicate destruction of plasma membrane line
The inactive Necrospermia of plastochondria;R2 is Rh123+/PI+, indicate plasmarrhexis but mitochondria be still active, that is, will be dead
The sperm died;R3 is Rh123-/PI-, indicate plasma membrane normally but the sperm without mitochondrial transmembrane potentials;R4, that is, Rh123+/PI-,
Indicate normal sperm living.
Embodiment 2
A kind of bivalve shellfish quality determining method and application, concrete operation step are as follows:
1. sperm is produced:Dissect microscopy, select the supermature male oyster of sexual gland, use suction pipe draw the preferable sperm of vigor as
Sperm stoste.
2. environment temperature is arranged:Using heating rod, ice cube, refrigerator setting temperature be -20 DEG C, 4 DEG C, 14 DEG C, 24 DEG C, 34
DEG C, 44 DEG C, 54 DEG C of water bath, adjustment sperm concentration is 107~108Then sperm is put into the water-bath of different temperatures by a/mL
In environment.
3. fluorescence dye liquor is prepared:PI and Rh123 are dissolved in ultra-pure water respectively, are made into the storage of a concentration of 1mg/mL
Liquid is put in -20 DEG C and -4 DEG C preservations respectively according to reagent storage request.
4. sperm dyes:Two kinds of storing liquids are diluted to 100 μ g/mL, take 15min, 30min under condition of different temperatures, 1h,
Each 240 μ L of sperm of 2h are added each 30 μ L mixing of two kinds of dye liquors, keep the dye liquor final concentration of sample to be tested equal in 1.5 mL centrifuge tubes
For 10 μ g/mL, avoid light place 12min, centrifuges at room temperature(300 × g, 5min), 0.01M PBS(pH7.4)It rinses, repetition is washed
Centrifugation 2 ~ 3 times is washed, PBS is resuspended, flow cytomery.
5. flow cytometer parameter setting:Forescatering FSC and lateral scattering SSC are Linear Amplifer, fluorescence channel FL1 and
FL3 logarithmic amplifications, survey the fluorescence intensity of each sperm Rh123 and PI, and every part of suspension detects 8000 ~ 10000 sperms.
6.FCM testing results:Sperm through PI/Rh123 dyeing and FCM detections is distributed in different quadrants, and R1 is
Rh123-/PI+, indicate the inactive Necrospermia of destruction of plasma membrane mitochondria;R2 is Rh123+/PI+, indicate plasmarrhexis but line grain
Body is still active, that is, i.e. by dead sperm;R3 is Rh123-/PI-, indicate that plasma membrane is normal but electric without Mitochondrial transmembrane
The sperm of position;R4, that is, Rh123+/PI-, indicate normal sperm living.
Embodiment 3
A kind of bivalve shellfish quality determining method and application, concrete operation step are as follows:
1. sperm is produced:Microscopy selects the supermature male scallop of sexual gland, and suction pipe is used to draw the preferable sperm of vigor as sperm
Stoste.
2. gradient seawater configures:Using sterilizing seawater, sea crystal and aeration fresh water, prepare salinity be 5,10,15,20,
25, sperm stoste is added in gradient seawater 30,35,40 gradient seawater, and adjustment sperm concentration is 107~108A/mL.
3. fluorescence dye liquor is prepared:PI and Rh123 are dissolved in ultra-pure water respectively, are made into the storage of a concentration of 1mg/mL
Liquid is put in -20 DEG C and -4 DEG C preservations respectively according to reagent storage request.
4. sperm dyes:Two kinds of storing liquids are diluted to 100 μ g/mL, take salinity stress 20min, 40min, 60min, 2h,
Each 160 μ L of sperm of 4h are added each 20 μ L mixing of two kinds of dye liquors, keep the dye liquor final concentration of sample to be tested equal in 1.5 mL centrifuge tubes
For 10 μ g/mL, avoid light place 12min, centrifuges at room temperature(280 × g, 6min), 0.01M PBS(pH7.4)It rinses, repetition is washed
Centrifugation 2 ~ 3 times is washed, PBS is resuspended, flow cytomery.
5. flow cytometer parameter setting:Forescatering FSC and lateral scattering SSC are Linear Amplifer, fluorescence channel FL1 and
FL3 logarithmic amplifications, survey the fluorescence intensity of each sperm Rh123 and PI, and every part of suspension detects 10000 sperms or so.
6.FCM testing results:Sperm through PI/Rh123 dyeing and FCM detections is distributed in different quadrants, and R1 is
Rh123-/PI+, indicate the inactive Necrospermia of destruction of plasma membrane mitochondria;R2 is Rh123+/PI+, indicate plasmarrhexis but line grain
Body is still active, that is, i.e. by dead sperm;R3 is Rh123-/PI-, indicate that plasma membrane is normal but electric without Mitochondrial transmembrane
The sperm of position;R4, that is, Rh123+/PI-, indicate normal sperm living.
Embodiment 4
A kind of bivalve shellfish quality determining method and application, concrete operation step are as follows:
1. sperm is produced:Microscopy selects the good male clam of gonad development, and suction pipe is used to draw the preferable sperm of vigor as sperm
Stoste.
2. gradient seawater configures:Using sterilizing seawater, sea crystal and aeration fresh water, prepare salinity be 10,15,20,25,
30, sperm stoste is added in gradient seawater 35,40,45,50 gradient seawater, and adjustment sperm concentration is 108A/mL.
3. fluorescence dye liquor is prepared:PI and Rh123 are dissolved in ultra-pure water respectively, are made into the storage of a concentration of 1mg/mL
Liquid is put in -20 DEG C and -4 DEG C preservations respectively according to reagent storage request.
4. sperm dyes:Two kinds of storing liquids are diluted to 100 μ g/mL, take the essence of salinity stress 15min, 30min, 1h, 2h
In 1.5 mL centrifuge tubes each 10 μ L mixing of two kinds of dye liquors is added, it is 10 μ g/ to make the dye liquor final concentration of sample to be tested in each 80 μ L of son
ML, avoid light place 10min, centrifuges at room temperature(280 × g, 5min), 0.01M PBS(pH7.4)It rinses, repeated washing centrifugation 2
Secondary, PBS is resuspended, flow cytomery.
5. flow cytometer parameter setting:Forescatering FSC and lateral scattering SSC are Linear Amplifer, fluorescence channel FL1 and
FL3 logarithmic amplifications, survey the fluorescence intensity of each sperm Rh123 and PI, and every part of suspension detects 8000 ~ 10000 sperms.
6.FCM testing results:Sperm through PI/Rh123 dyeing and FCM detections is distributed in different quadrants, and R1 is
Rh123-/PI+, indicate the inactive Necrospermia of destruction of plasma membrane mitochondria;R2 is Rh123+/PI+, indicate plasmarrhexis but line grain
Body is still active, that is, i.e. by dead sperm;R3 is Rh123-/PI-, indicate that plasma membrane is normal but electric without Mitochondrial transmembrane
The sperm of position;R4, that is, Rh123+/PI-, indicate normal sperm living.
Using bivalve shellfish sperm quality detection method of the present invention and application, the lower clam of detection different salinity stress is smart respectively
The quality of son show that the death rate of sperm, plasma membrane are damaged ratio and mitochondria activity ratio, is provided for judgement sperm quality comprehensively
Accurately, reliable experimental data.
Claims (5)
1. a kind of method of bivalve shellfish sperm quality detection, which is characterized in that this approach includes the following steps:
(1)Sperm is produced:Full individual is developed in selection, is dissected microscopy, is selected the good male of gonad development, inhaled with suction pipe
Sperm is taken to be placed in sterilizing seawater;
(2)Fluorescence dye liquor is prepared:PI and Rh123 are dissolved in ultra-pure water respectively, stored;
(3)Sperm dyes:A certain amount of sperm is taken, two kinds of above-mentioned fluorescence dye liquor mixing are added, places, centrifugation, washs, be resuspended, then
Carry out flow cytomery;
(4)Flow cytomery result:Sperm through PI/Rh123 dyeing and FCM detections is distributed in different quadrants:R1 tables
Show the inactive Necrospermia of destruction of plasma membrane mitochondria, R2 indicates plasmarrhexis but mitochondria is still active, that is, will be dead
The sperm died, R3 indicate that plasma membrane is normal but the sperm without mitochondrial transmembrane potentials, R4 are normal sperm living.
2. the method as described in claim 1, which is characterized in that the step(1)In, it is dense using blood counting chamber adjustment sperm
Degree is 107~108A/mL.
3. the method as described in claim 1, which is characterized in that the step(2)Specially:PI and Rh123 are dissolved in respectively
In ultra-pure water, it is made into the storing liquid of a concentration of 1mg/mL, according to reagent storage request, is put in -20 DEG C and -4 DEG C preservations respectively.
4. the method as described in claim 1, which is characterized in that the step(3)Specially:Above two storing liquid is distinguished
100 μ g/mL are diluted to, a certain amount of sperm is taken, two kinds of dye liquor mixing are added, it is 10 μ g/ to make the dye liquor final concentration of sample to be tested
ML, at room temperature 10 ~ 15min of avoid light place, centrifugation, 0.01M PBS, pH7.4 flushings wash repeatedly centrifugation 2 times, and PBS is resuspended,
Flow cytomery.
5. the method as described in claim 1, which is characterized in that in the method, flow cytometer parameter is arranged:Forescatering
FSC and lateral scattering SSC is Linear Amplifer, and fluorescence channel FL1 and FL2 logarithmic amplification surveys the fluorescence of each sperm Rh123 and PI
Intensity, every part of suspension detect 8000 ~ 10000 sperms.
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Cited By (1)
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| CN110426339A (en) * | 2019-07-03 | 2019-11-08 | 佛山科学技术学院 | A method of assessment boar sperm quality |
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Application publication date: 20181023 |