WO2015137466A1 - 精子の検査方法及び装置 - Google Patents
精子の検査方法及び装置 Download PDFInfo
- Publication number
- WO2015137466A1 WO2015137466A1 PCT/JP2015/057380 JP2015057380W WO2015137466A1 WO 2015137466 A1 WO2015137466 A1 WO 2015137466A1 JP 2015057380 W JP2015057380 W JP 2015057380W WO 2015137466 A1 WO2015137466 A1 WO 2015137466A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sperm
- quality
- value
- population
- quality index
- Prior art date
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10004—Still image; Photographic image
Definitions
- the present invention relates to a sperm inspection method and apparatus that can predict fertility, a control program that causes an information processing apparatus to determine the quality of sperm, and a computer-readable medium that encodes the control program.
- Patent Document 1 Patent 5092149
- Non-Patent Document 2 Rodriguez-Martinez H., Reprod Domest Anim. 41, Suppl 2, 2-10 (2006).
- the correlation between these test methods and fertility is not high in all items, and it is not a simple index that reflects fertility from the sperm side. The reason why the correlation between these test methods and fertility is not high is considered to be because the test method does not reflect the behavior of sperm in the female body and the environment at the time of fertilization.
- Non-patent Document 3 Suarez SS.,
- Non-Patent Document 4 since the method and in vitro fertilization test of Non-Patent Document 4 generate a large amount of labor and cost, it is difficult to perform a simple and low-cost test on all semen to be supplied. Moreover, it cannot be said to be a highly reliable test that completely mimics the environment inside the female body. Therefore, it is desired to develop a simple sperm inspection method and apparatus that easily reproduces the fertilization environment in the female body outside the body and reflects the fertility of the sperm.
- the present invention has been made in view of such problems, and an object thereof is to provide a sperm inspection method and apparatus for solving the above problems.
- the present inventors conducted intensive research on sperm quality inspection using semen whose fertility has been found, and as a result, the ratio of sperm with extremely high sperm quality index values and By judging the number of spermatozoa, the quality of spermatozoa highly correlated with fertility could be evaluated.
- the sperm population is stratified based on sperm quality assessment values, and correlated with fertility by assessing sperm quality based on quality assessment values of one or more groups in the stratified population It was possible to evaluate the quality of sperm with a high level. Accordingly, the present invention relates to a sperm inspection method, a sperm inspection device, a control program that causes an information processing device to determine sperm quality, and a recording medium that encodes the control program.
- the method for examining sperm of the present invention comprises a step of suspending a sperm population in a test solution and measuring a quality index value of each sperm, and evaluating the quality of the sperm based on the quality index value. And a process. Further, the step of evaluating the quality of sperm includes stratifying the sperm population based on the value of the quality index, and evaluating based on the value of the quality index of one or a plurality of groups of the stratified group. Features. Further, the quality index value of the stratified group is an average value, a median value, or a mode value of the quality index values for each group.
- the stratification is characterized in that a high quality group is selected based on a value of a quality index.
- the stratification is characterized in that the quality of sperm is evaluated based on the value of the quality index of another group.
- the top quality group is within the top 50%. Further, the top quality group is within the top 20%. Further, the top quality group is within the top 10%.
- the upper quality group is within the upper 5%. Further, the upper quality group is within the upper 3%. Further, the upper quality group is within the upper 1%. Further, the upper quality group is within the upper 0.5%. Further, the upper quality group is within the upper 0.3%.
- the quality index value of the upper quality group is an average value, a median value, or a mode value of the quality index values in the higher quality group.
- the sperm population is a living sperm. Further, the sperm population is characterized in that sperm motility is not less than a certain value or in a certain range.
- the quality index is an index of sperm motility.
- the test solution has a pH of 6.0 to 9.0.
- the test solution has a pH of 7.2 to 8.2.
- the test solution has a pH of 7.4 to 7.8.
- the test solution has an osmotic pressure of 230 to 400 mmol / kg (mOsm / kg).
- the test solution is maintained at 33 to 43 ° C.
- the test solution is maintained at 36 to 40 ° C.
- the test solution contains a buffer, sugar and salt.
- the buffer for the test solution is tris (hydroxymethyl) aminomethane and citric acid.
- the sugar of the test solution is glucose.
- the salt of the test solution is sodium chloride.
- the test solution contains an activator.
- the activator is procaine, caffeine, and / or theophylline.
- the test solution contains a loading agent.
- the loading agent is polyvinylpyrrolidone K-90.
- the suspension time is from immediately after suspension to 30 minutes.
- the index of sperm motility is VAP, VCL, ALH and / or BCF.
- the step of evaluating the quality of the sperm includes a step of determining the quality of the sperm by comparing with a reference value of the quality index of one or a plurality of groups.
- the reference value of the quality index is characterized in that VCL is 150 ⁇ m / second or more.
- the reference value of the quality index is characterized in that VCL is 200 ⁇ m / second or more.
- the step of evaluating the quality of the sperm is characterized by being based on a ratio of sperm or a sperm number exceeding a reference value of a predetermined quality index.
- the reference value of the predetermined quality index is a value selected from the group consisting of VCL 150 ⁇ m / second, 175 ⁇ m / second, and 200 ⁇ m / second.
- the reference value of the quality index is characterized in that VCL is 200 ⁇ m / second or more.
- the ratio of a sperm is 1% or more, It is characterized by the above-mentioned.
- the ratio of a sperm is 0.7% or more, It is characterized by the above-mentioned.
- the ratio of a sperm is 0.3% or more, It is characterized by the above-mentioned.
- the number of sperm is 300,000 sperm or more per straw.
- the number of sperm is 100,000 sperm or more per straw.
- the step of evaluating the quality of the sperm is characterized in that it is obtained from a graph in which quality index values of individual sperm of the sperm group are arranged in order. Further, the step of evaluating the quality of the sperm is characterized in that it is obtained from an approximate line of the graph in which the quality index values of the individual sperm of the sperm population are arranged in order. Further, the step of evaluating the quality of the sperm is characterized in that it is obtained from the slope of the approximate line of the graph in which the quality index values of the individual sperm of the sperm population are arranged in order.
- the step of evaluating the quality of the sperm is characterized in that it is obtained from the coefficients of the approximate lines of the graph in which the quality index values of the individual sperms of the sperm population are arranged in order. Further, the step of evaluating the quality of the sperm is characterized in that it is obtained from an intercept of the approximate line of the graph in which the quality index values of the individual sperm of the sperm population are arranged in order.
- the sperm is a mammalian sperm.
- the sperm is cow sperm.
- the sperm inspection apparatus of the present invention evaluates the quality of sperm based on the sperm quality measurement means for measuring the value of the quality index of each sperm of the sperm population suspended in the test solution, and the value of the quality index. And a sperm quality evaluation means. Further, the sperm quality evaluation means stratifies the sperm group based on a quality index value, and evaluates based on the quality index value of one or a plurality of groups of the stratified group. . Further, the quality index value of the stratified group is an average value, a median value, or a mode value of the quality index values for each group. Further, the stratification is characterized in that a high quality group is selected based on a value of a quality index.
- the stratification is characterized in that the quality of sperm is evaluated based on the value of the quality index of another group.
- the top quality group is within the top 50%. Further, the top quality group is within the top 20%. Further, the top quality group is within the top 10%. Further, the upper quality group is within the upper 5%. Further, the upper quality group is within the upper 3%. Further, the upper quality group is within the upper 1%. Further, the upper quality group is within the upper 0.5%. Further, the upper quality group is within the upper 0.3%.
- the quality index value of the upper quality group is an average value, a median value, or a mode value of the quality index values in the higher quality group.
- the sperm population is a living sperm.
- the sperm population is characterized in that sperm motility is not less than a certain value or in a certain range.
- the quality index is an index of sperm motility.
- the test solution has a pH of 6.0 to 9.0.
- the test solution has a pH of 7.2 to 8.2.
- the test solution has a pH of 7.4 to 7.8.
- the test solution has an osmotic pressure of 230 to 400 mmol / kg (mOsm / kg).
- the test solution is maintained at 33 to 43 ° C.
- the test solution is maintained at 36 to 40 ° C.
- the test solution contains a buffer, sugar and salt. Further, the buffer for the test solution is tris (hydroxymethyl) aminomethane and citric acid.
- the sugar of the test solution is glucose.
- the salt of the test solution is sodium chloride.
- the test solution contains an activator.
- the activator is procaine, caffeine, and / or theophylline.
- the test solution contains a loading agent.
- the loading agent is polyvinylpyrrolidone K-90.
- the suspension time is from immediately after suspension to 30 minutes.
- the index of sperm motility is VAP, VCL, ALH and / or BCF.
- the sperm quality evaluation means includes means for determining the quality of sperm by comparing with a reference value of a quality index of one or a plurality of groups. The reference value of the quality index is characterized in that VCL is 150 ⁇ m / second or more.
- the reference value of the quality index is characterized in that VCL is 200 ⁇ m / second or more.
- the sperm quality evaluation means is characterized in that it is based on a ratio of sperm or a sperm number exceeding a reference value of a predetermined quality index.
- the reference value of the predetermined quality index is a value selected from the group consisting of VCL 150 ⁇ m / second, 175 ⁇ m / second, and 200 ⁇ m / second.
- the reference value of the predetermined quality index is characterized in that VCL is 200 ⁇ m / second.
- the sperm ratio is 1% or more. Further, the ratio of the sperm is 0.3% or more.
- the number of sperm is 300,000 sperm or more per straw.
- the number of sperm is 100,000 sperm or more per straw.
- the sperm quality evaluation means is characterized in that it is obtained from a graph in which quality index values of individual sperm of the sperm population are arranged in order.
- the sperm quality evaluation means is characterized in that it is obtained from the approximate line of the graph in which the quality index values of the individual sperm of the sperm population are arranged in order.
- the sperm quality evaluation means is characterized in that it is obtained from the slope of the approximate line of the graph in which the quality index values of the individual sperm of the sperm population are arranged in order.
- the sperm quality evaluation means is characterized in that it is obtained from the coefficients of the approximate lines of the graph in which the quality index values of the individual sperms of the sperm population are arranged in order. Further, the sperm quality evaluation means is characterized in that it is obtained from the intercepts of the approximate lines of the graph in which the quality index values of the individual sperms of the sperm population are arranged in order.
- the sperm is a mammalian sperm.
- the sperm is cow sperm.
- the control program of the present invention is a control program that causes an information processing device including an output unit and a storage unit to determine the quality of sperm, Storing the value of the quality index of each sperm in the sperm population in the test solution in the storage unit, Stratifying the sperm population based on the stored quality indicator value; Determine the quality of the sperm based on the quality indicator value of the stratified population,
- the information processing apparatus is caused to output the result of sperm quality to the output unit.
- the information processing apparatus further includes an imaging unit, and the following: The imaging unit acquires a plurality of images over time, The quality index is determined based on a plurality of images.
- the determination of sperm quality based on the value of the quality indicator of one or more groups of the stratified population further includes: Calculate the quality decision value from the quality index values of the stratified population, Determining the quality of sperm based on the quality determination value.
- the quality index value of the stratified group is an average value, a median value, or a mode value of the quality index values for each group. Further, a high quality group is selected as the stratified group.
- the stratification is characterized in that the quality of sperm is evaluated based on the value of the quality index of another group.
- the top quality group is within the top 50%. Further, the top quality group is within the top 20%. Further, the top quality group is within the top 10%.
- the upper quality group is within the upper 5%. Further, the upper quality group is within the upper 3%. Further, the upper quality group is within the upper 1%. Further, the upper quality group is within the upper 0.5%. Further, the upper quality group is within the upper 0.3%. Further, the quality index value of the upper quality group is an average value, a median value, or a mode value of the quality index values in the higher quality group.
- the sperm population is a living sperm. Further, the sperm population is characterized in that sperm motility is not less than a certain value or in a certain range. The quality index is an index of sperm motility.
- the test solution has a pH of 6.0 to 9.0.
- the test solution has a pH of 7.2 to 8.2.
- the test solution has a pH of 7.4 to 7.8.
- the test solution has an osmotic pressure of 230 to 400 mmol / kg (mOsm / kg).
- the test solution is maintained at 33 to 43 ° C.
- the test solution is maintained at 36 to 40 ° C.
- the test solution contains a buffer, sugar and salt.
- the buffer for the test solution is tris (hydroxymethyl) aminomethane and citric acid.
- the sugar of the test solution is glucose.
- the salt of the test solution is sodium chloride.
- the test solution contains an activator.
- the activator is procaine, caffeine, and / or theophylline.
- the test solution contains a loading agent.
- the loading agent is polyvinylpyrrolidone K-90.
- the sperm is suspended in the test solution for a period of 30 to 30 minutes immediately after the suspension.
- the index of sperm motility is VAP, VCL, ALH and / or BCF.
- evaluating the quality of the sperm includes determining the quality of the sperm in comparison with a reference value of a quality index of one or a plurality of groups.
- the reference value of the quality index is characterized in that VCL is 150 ⁇ m / second or more.
- the reference value of the quality index is characterized in that VCL is 200 ⁇ m / second or more.
- the sperm quality is evaluated based on a sperm ratio or a sperm count exceeding a reference value of a predetermined quality index.
- the reference value of the predetermined quality index is a value selected from the group consisting of VCL 150 ⁇ m / second, 175 ⁇ m / second, and 200 ⁇ m / second. Further, the reference value of the predetermined quality index is characterized in that VCL is 200 ⁇ m / second. Further, the sperm ratio is 1% or more. Further, the ratio of the sperm is 0.3% or more. The number of sperm is 300,000 sperm or more per straw. The number of sperm is 100,000 sperm or more per straw. The quality of the sperm may be evaluated from a graph in which quality index values of individual sperms of the sperm population are arranged in order.
- the quality of the sperm is evaluated by obtaining from the approximate line of the graph in which the quality index values of the individual sperm of the sperm population are arranged in order.
- the quality of the spermatozoa is evaluated from the slope of the approximate line of the graph in which the quality index values of the individual sperms of the sperm population are arranged in order.
- the quality of the spermatozoa is evaluated from the coefficients of the approximate lines of the graph in which the quality index values of the individual sperms of the sperm population are arranged in order.
- the quality of the spermatozoa is evaluated by obtaining from the intercept of the approximate line of the graph in which the quality index values of the individual sperms of the sperm population are arranged in order.
- the sperm is a mammalian sperm.
- the sperm is cow sperm.
- the present invention it is possible to provide a simple sperm inspection method having a high correlation with fertility by a quality decision value based on the quality index value of the higher quality group.
- fertility can be easily predicted. Therefore, sperm with high fertility can be efficiently supplied by examining the semen to be used in advance.
- development can be promoted more efficiently.
- FIG. 1 is a diagram for explaining parameters used for analysis of sperm motility which can be a quality index.
- FIG. 2 is a configuration diagram of the sperm inspection apparatus according to the embodiment of the present invention.
- FIG. 3 is a flowchart showing an example of an operation for determining sperm quality.
- FIG. 1 is a diagram for explaining parameters used for analysis of sperm motility which can be a quality index.
- FIG. 2 is a configuration diagram of the sperm inspection apparatus according to the embodiment of the present invention.
- FIG. 3 is a flowchart showing an example of an operation for determining sperm quality.
- FIG. 4 is a graph showing the ratio
- FIG. 6A is a graph showing the correlation between the proportion of sperm with a VAP of 50 ⁇ m / sec or more and the fertility rate by measuring three lots for each of 40 sire spermatozoa whose conception rate is known. The correlation between the rate of sperm with VAP of 50 ⁇ m / sec or more and the conception rate is not high.
- FIG. 6A is a graph showing the correlation between the proportion of sperm with a VAP of 50 ⁇ m / sec or more and the fertility rate by measuring three lots for each of 40 sire spermatozoa whose conception rate is known. The correlation between the rate of sperm with VAP of 50 ⁇ m / sec or more and the
- FIG. 6B is a graph showing the correlation between the proportion of sperm having a VCL of 50 ⁇ m / sec or more and the fertility rate by measuring 3 lots of sperm of 40 bulls with known conception rates. The correlation between the proportion of sperm with VCL of 50 ⁇ m / sec or more and the conception rate is not high.
- FIG. 6C is a graph showing the correlation between the proportion of sperm having a VCL of 100 ⁇ m / sec or more and the fertility rate by measuring 3 lots of each of 40 semen spermatozoa whose conception rate is known. The correlation between the rate of sperm with a VCL of 100 ⁇ m / sec or more and the conception rate is not high.
- FIG. 6C is a graph showing the correlation between the proportion of sperm having a VCL of 100 ⁇ m / sec or more and the fertility rate by measuring 3 lots of each of 40 semen spermatozoa whose conception rate is known. The correlation between the rate of sperm with a VCL of 100
- FIG. 6D is a graph showing the correlation between the proportion of sperm with a VCL of 150 ⁇ m / sec or more and the conception rate by measuring three lots for each of 40 sire spermatozoa whose conception rate is known. A correlation can be confirmed between the ratio of sperm with VCL of 150 ⁇ m / sec or more and the conception rate.
- FIG. 6E is a graph showing the correlation between the proportion of sperm with a VCL of 200 ⁇ m / sec or more and the fertility rate by measuring three lots for each of 40 semen of known bulls whose conception rate is known. A high correlation can be confirmed between the proportion of sperm with a VCL of 200 ⁇ m / sec or more and the conception rate.
- FIG. 8A is a graph comparing the proportion of sperm with a VCL of 200 ⁇ m / second or more for each category of fertilization rate by measuring three lots for 29 bulls with known conception rates. Values are expressed as mean values and standard deviations.
- FIG. 8B is a graph comparing the ratio of sperm with a VCL of 200 ⁇ m / sec or more for each conception rate category by measuring three lots for 40 bulls with known conception rates. Values are expressed as mean values and standard deviations. It can be confirmed that those having a high conception rate have a high ratio of sperm having a VCL of 200 ⁇ m / second or more.
- FIG. 9 shows the correlation between the number of spermatozoa with a VCL of 200 ⁇ m / sec or more in one straw and the conception rate for 40 semen of known bulls with a known conception rate.
- FIG. 10A shows the measurement of 3 lots of 29 sire semens with known conception rates, and compares the number of sperm with VCL of 200 ⁇ m / sec or more in one straw for each category of conception rate. It is a graph. Values are expressed as mean values and standard deviations. It can be confirmed that those having a high conception rate have a large number of spermatozoa with a VCL of 200 ⁇ m / second or more in one straw.
- FIG. 10A shows the measurement of 3 lots of 29 sire semens with known conception rates, and compares the number of sperm with VCL of 200 ⁇ m / sec or more in one straw for each category of conception rate. It is a graph. Values are expressed as mean values and standard deviations. It can be confirmed that those having a high conception rate have a large number of spermatozoa with a VCL of 200 ⁇ m / second or more in one straw.
- FIG. 10A shows the measurement of 3 lots of 29 sire semens with known conception rates,
- FIG. 10B shows the measurement of 3 lots of 40 semens with known conception rates, and compares the number of sperm with VCL of 200 ⁇ m / sec or more in one straw for each category of conception rate. It is a graph. Values are expressed as mean values and standard deviations. It can be confirmed that those having a high conception rate have a large number of spermatozoa with a VCL of 200 ⁇ m / second or more in one straw.
- FIG. 11A is a graph showing the ratio of sperm in each group, measured for 3 lots each of 29 sire bulls with known conception rates, divided into 6 groups based on VCL values. is there. Values are shown as average values.
- FIG. 11B is a graph showing the ratio of sperm in each group, measured for 3 lots for each 40 semen of known bulls with a known conception rate, divided into 9 groups based on the VCL value. is there. Values are shown as average values.
- the proportion of sperm with VCL of 175 ⁇ m / sec or more is small.
- the proportion of sperm with a VCL of 75-150 ⁇ m / sec is high.
- FIG. 12A shows the measurement of 3 lots each of 40 semen with known conception rates, and the values of VCL values of the top 75% sperm in descending order for each conception rate category. It is the shown graph. Values are shown as average values.
- FIG. 12B shows the measurement of 3 lots of 40 semens with known conception rates, and the VCL values of the top 10% spermatozoa in descending order for each conception rate category. It is the shown graph. Values are shown as average values. The standard deviation is shown only in the section with a conception rate of 65% or more.
- FIG. 13 shows the measurement of 3 lots for 40 semens with known conception rates, and 3 for a graph in which VCL values of all sperm are arranged in descending order for each conception category. It is the graph which showed the approximate line approximated with the following function. By comparing the values of the y-intercepts of the approximate lines, it is possible to evaluate the fertility of sperm with high accuracy.
- FIG. 14 shows a straight line with respect to a graph in which three lots of each sperm of 40 bulls whose conception rates are known are measured and VCL values of all sperm are arranged in descending order for each category of conception rate. It is the graph which showed the approximate line approximated by. By comparing the values of the slopes of the approximate lines, it is possible to evaluate the fertility of sperm with high accuracy.
- the curve shows the upper and lower limits of the 95% confidence interval when 30 artificial insemination is performed.
- the curve shows the upper and lower limits of the 95% confidence interval when 30 artificial insemination is performed.
- the present invention encodes a sperm inspection method for evaluating sperm quality on the basis of the value of a higher quality index of the sperm population, a control apparatus for causing the information processing apparatus to determine the quality of sperm, and the control program.
- the present invention relates to a computer readable medium. Embodiments of a sperm inspection method and apparatus according to the present invention will be described below.
- Embodiment 1 The sperm inspection method of the present invention comprises the following steps. (1) Measuring the value of the quality index of sperm (S100) This is a step of suspending the sperm population in a test solution and measuring the quality index value of each sperm. (2) Step of evaluating sperm quality (S200) This is a step of evaluating the quality of sperm based on the value of the higher quality index of the sperm population.
- the process of measuring the value of the sperm quality index is further as follows: (1-1) Step of acquiring a plurality of sperm images over time (S101) (1-2) A step of measuring a sperm quality index value based on a plurality of images (S102) May be included. The step (S100) of measuring the value of the quality index can be omitted by performing the step (S199) of reading the value of the quality index acquired and stored in advance.
- the process of assessing sperm quality is further as follows: (2-1) Step of stratifying the sperm population based on the quality index value (S201) (2-2) Step of calculating a quality decision value from the quality index value of the stratified group (S202) (2-3) A step of determining the quality of the sperm from the quality determination value (S203) One or more, preferably all.
- a step of optionally outputting the quality of the sperm (S300) may be included.
- the method according to the first embodiment of the present invention may be performed manually by a sperm inspection engineer or a researcher, or automatically performed by an inspection apparatus or an information processing apparatus installed with the control program of the present invention. It may be performed semi-automatically.
- sperm derived from any animal can be used.
- animals include any mammals including humans, such as livestock animals, pet animals, zoo animals, and laboratory animals.
- domestic animals include cattle, pigs, goats, horses, sheep and the like.
- pet animals include dogs, cats and rabbits.
- zoo animals include pandas and other species that are threatened with extinction.
- experimental animals include mice, hamsters, rats, sea urchins and starfish.
- any sperm derived from the testis, epididymis, ejected semen, stem cells, testicular stem cells, iPS cells, cultured cells and the like can be used.
- the sperm may be stored refrigerated or frozen. Fresh semen and frozen semen are preferable, and sperm that reflects the quality immediately before being used for artificial insemination, in vitro fertilization, microinsemination, and the like.
- the method of obtaining sperm is, for example, a method of collecting the testis and sucking out the sperm if it is a sperm derived from the testis, or a method of collecting the epididymis and sucking out or scraping out the sperm if the sperm is derived from the epididymis If sperm is derived from semen, it is collected after ejaculation into the female body, or collected using electrical stimulation or artificial sputum. Stem cells, testicular stem cells, iPS cells, and cultured cells are collected by cell culture. Methods and the like. The acquired sperm may be further diluted or washed with an aqueous solution or the like even in a state of floating in the seminal plasma immediately after acquisition.
- the sperm population of the present invention includes a plurality of sperm. When the sperm is obtained by the above method, it is usually collected as a sperm population.
- the sperm population also includes a sub-sperm population after selecting or sorting a portion of the sperm population. Select sub-sperm populations, for example, by measuring sperm quality indicators, measuring sperm motility measured with a sperm motility analyzer, etc., with a sperm population within a certain value or range, flow cytometer, nucleo counter, etc. And a sperm population in which the quality of a sperm constituent protein measured by a flow cytometer, a fluorescence microscope, or the like exceeds a certain value or in a certain range.
- Sorting of sub-sperm populations includes, for example, sperm populations after separating X sperm or Y sperm with a flow cytometer, sperm groups after separating motor sperm by the swim-up method or the method using glass beads, etc. Can be taken out. In one aspect, it is preferred that the sperm included in the sperm population is alive.
- the quality index of the present invention is an index for determining the quality of individual sperm in the sperm population.
- Quality indicators include, for example, any molecular biology and life such as sperm motility, sperm viability, mitochondrial activity, acrosome normality, sperm morphology normality, sperm component protein normality assessment by lectin staining or immunostaining, etc.
- the index which can evaluate a cell chemically is mentioned.
- These quality indicators are usually sperm motion analyzers, high-speed cameras, phase contrast microscopes, differential interference microscopes, polarizing microscopes, fluorescence microscopes, confocal laser microscopes, transmission electron microscopes, scanning electron microscopes, etc., flow sites Measurement is performed using a meter, nucleo counter, luminometer, absorbance reader, fluorescence reader, fluorescence deflection reader, chemiluminescence reader, or the like.
- the quality index of the present invention preferably uses sperm motility.
- sperm quality indicators include VSL (linear progression speed, ⁇ m / second), VAP (average speed of cell pathway, ⁇ m / second), VCL (forward curve speed, ⁇ m / second), ALH (head amplitude). , ⁇ m), STR (straight forward, VSL / VAP), LIN (linearity, VSL / VCL) and / or BCF (head frequency, Hz) index such as sperm motion analyzer (CASA), high-speed camera, etc. Each numerical value is determined by the frame rate at the time of shooting.
- the acquired frame rate may be an arbitrary numerical value, and is generally an arbitrary frame rate of 10 to 2000 frames / second used for sperm observation, for example, 30 frames / second, 60 frames / second, 100 frames / second. , 500 frames / second, 1000 frames / second can be used. These parameters are described in FIG. 1 and explained in Non-Patent Document 5 (Kinkawa M., Doctoral Dissertation, http://hdl.handle.net/2261/119 (2005)). As an index of sperm motility, VAP, VCL, ALH and / or BCF can be preferably used.
- VAP is the speed of the trajectory where the sperm has actually moved
- VAP is the speed of the path obtained by smoothing the trajectory.
- VAP when using VAP, for example, 100 ⁇ m / second or more
- VCL when using VCL, for example, VCL is 150 ⁇ m / second or more, preferably 200 ⁇ m / second or more
- ALH when using ALH, for example, 9 ⁇ m or more, preferably
- BCF 12 ⁇ m or more, for example, 10 to 40 Hz, preferably 20 to 30 Hz is used.
- a quality index related to sperm motility can also be used.
- movement of sperm flagella sperm intracellular concentration such as cAMP and calcium
- quality and quantity of sperm constituent proteins such as dynein, adenylate cyclase, adenylate kinase, protein phosphatase, and protein phosphatase
- quality indicators include the acquisition of fertility that occurs in parallel with the transformation and changes in cell membrane properties associated with acrosome reactions.
- test solution of the present invention any solution can be used as long as the quality of each sperm in the sperm population can be evaluated.
- the solution is usually an aqueous solution. If the spermatozoa are derived from the testis, epididymis, injected semen, stem cells, testicular stem cells, iPS cells, cultured cells, etc., the liquid itself collected with the sperm can be used as the test liquid.
- the test solution is preferably an aqueous solution containing a buffer, sugar and / or salt. More preferably, the test solution is a fluid present in the female reproductive tract environment such as the uterus, fallopian tube, and a large portion of the fallopian tube.
- Imitation of the female reproductive tract environment is to imitate the environment in which the sperm reaches fertilization in the female reproductive tract and the fluid present there.
- a water flow caused by cilia a highly viscous environment around the cervical mucus and the ovum, a liquid that mimics components such as oviduct fluid and follicular fluid, and pH.
- the pH of the test solution of the present invention may be any pH as long as the quality of sperm can be evaluated, and is usually 6.0 to 9.0. Preferably it is 7.2 to 8.2, and more preferably 7.4 to 8.2.
- the component concentration in the test solution of the present invention is determined so that the pH of the aqueous solution falls within the above range.
- the osmotic pressure of the test solution of the present invention may be any osmotic pressure as long as the sperm quality can be evaluated, but is usually 230 to 400 mmol / kg (mOsm / kg). This range is specified as a range in which motility of sperm can be maintained by the description of Non-Patent Document 6 (Guthrie et al., Biology of Reproduction 67, 1811-1816 (2002)).
- the osmotic pressure of the aqueous solution is preferably 250 to 350 mmol / kg (mOsm / kg), more preferably 260 to 320 mmol / kg (mOsm / kg).
- the osmotic pressure can be calculated from the solute concentration, the degree of dissociation, and the like, but is determined using an osmometer (osmometer) in consideration of the interaction of substances constituting the solution.
- the component concentration in the test solution of the present invention is determined so that the osmotic pressure of the aqueous solution falls within the above range.
- the test solution of the present invention can contain any buffer as long as it achieves a desired pH.
- Any buffer can be selected as long as it has a buffering action near neutrality.
- Good buffer such as tris (hydroxymethyl) aminomethane, female, hepes, tes, tricine, phosphate buffer, etc. Liquid, citrate buffer, acetate buffer, carbonate buffer and the like. Acids or bases can also be used to achieve the desired pH.
- tris (hydroxymethyl) aminomethane and citric acid are used. More specifically, the concentration of tris (hydroxymethyl) aminomethane is preferably 50 to 300 mM, and more preferably 75 to 200 mM.
- the citric acid concentration is preferably 20 to 100 mM, more preferably 25 to 75 mM.
- the test solution of the present invention can contain any sugar or energy source as long as it is a substance that becomes a sperm energy source.
- sugars and energy sources include glucose, xylose, rhamulose, fructose, mannose, galactose, sucrose, lactose, maltose, trehalose, melibiose, raffinose, merethyrose, stachyose, dextrin, N-acetyl-D-glucosamine, D-glucuronic acid , ATP, ADP and the like.
- glucose is used.
- the glucose concentration is 5 mM to 100 mM, preferably 10 mM to 50 mM.
- the test solution of the present invention can contain any salt as long as it is used for the purpose of adjusting the osmotic pressure.
- the salt for example, chloride salt, sulfate salt, sulfite salt, nitrate salt, acetate salt, gluconate salt, amino acid salt, citrate salt, carbonate salt, bicarbonate salt and the like can be used.
- a chloride salt is used. More preferably, sodium chloride can be used.
- the sodium chloride concentration is 50 to 200 mM, preferably 50 to 150 mM, and more preferably 50 to 100 mM.
- the test solution of the present invention can contain any biologically active substance such as an activator that activates sperm.
- biologically active substances include calcium, magnesium, selenium, zinc, catechin, caffeine, theophylline, pentoxifylline, procaine, lidocaine, bupivacaine, imidazole, sodium pyruvate, hypotaurine, polyphenol, L-glutamine, SOD, vitamin B2, vitamin C, vitamin E, flavonoid, spermine, ⁇ -carotene, glutathione, glutathione peroxidase, glutathione reductase, catalase, carnitine, albumin, transferrin, ceruloplasmin, glucose phosphate D dehydrogenase, cholesterol, fatty acid, phosphatidylcholine, ATP, etc.
- motility or the like of sperm can be activated, and the quality evaluation value of sperm can be made to stand out.
- motility or the like of sperm is usually 0.1 to 40 mM for procaine, 0.1 to 40 mM for caffeine, and 0.1 to 40 mM for theophylline.
- the test solution of the present invention may contain an arbitrary loading agent for the purpose of imparting a load to the sperm motility due to viscosity or the like.
- an arbitrary loading agent for example, polyvinylpyrrolidone, methylcellulose, ficoll, polyacrylamide, polyvinyl alcohol, alginic acid, alginic acid salt, hyaluronic acid, hyaluronic acid salt, gelatin and the like can be used.
- polyvinylpyrrolidone K-90 is used.
- the concentration of polyvinylpyrrolidone K-90 is 0.5 to 16% (W / V), preferably 1 to 8% (W / V).
- the test solution of the present invention may contain any antibiotic for the purpose of preventing bacterial growth.
- antibiotics include penicillin, streptomycin, gentamicin, dibekacin and the like.
- the preparation of the test solution can include a sterilization operation to prevent bacterial growth. Examples of the sterilization operation include treatment with a pore size 0.2 ⁇ m or 0.45 ⁇ m filter, autoclave, and the like.
- any method can be used as long as the test solution can be transferred.
- a manual pipette, an automatic pipette, a dispenser or the like can be used.
- the time for suspending the sperm in the test solution of the present invention may be any time as long as the value of the quality index of the sperm can be measured.
- it is usually immediately after suspension to 6 hours, preferably immediately after suspension to 3 hours, more preferably immediately after suspension to 1 hour, particularly preferably suspension. Immediately after 30 minutes.
- the temperature when suspending the sperm with the test solution of the present invention may be any temperature as long as the quality index value of the sperm can be measured. Usually, it can be measured in the range of 0 to 50 ° C. using an incubator or the like. Preferably, the measurement is carried out while maintaining a temperature around 25 to 45 ° C., more preferably 33 to 43 ° C., particularly preferably 36 to 40 ° C., which is around the body temperature of the animal from which the sperm is derived.
- the step of evaluating the quality of sperm of the present invention includes stratification of the sperm population based on the value of the quality index.
- Stratification means classification into two or more groups based on the value of the sperm quality index.
- the number of groups can be arbitrarily selected, but can be classified into, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 groups.
- the criteria for stratification can be arbitrarily selected based on the value of the quality index to be used, and the ratio of sperm and the number of sperm may be selected to be a fixed number for each group, or as appropriate. You may classify by setting a numerical value.
- the sperm group can be stratified in the form of quality index values in the top 20%, top 10%, top 5%, top 3%, top 1%, etc.
- the quality index value in the sperm group is within the top 80%, top 50%, top 30%, top 10%, top 20-80%, top 10-50%, top 1-5%, etc.
- stratification can be performed based on the quality index value in the sperm population.
- the quality determination value can be calculated from the quality index values in all groups, and the quality of one or more groups arbitrarily selected from each group. It can also be calculated from the index value.
- the quality decision value can be calculated from the average value, median value, or mode value of the quality index of the higher quality group among the stratified groups.
- the value of the quality index of the upper group of the sperm population of the present invention is a sperm obtained by the above-described method of obtaining sperm
- the value of the quality index of the sperm is usually within 50% from the better one in the sperm population.
- the value is preferably within 10%, more preferably within 5%, further preferably within 3%, more preferably within 1%, and even more preferably within 0.3%.
- these values are predicted from a graph obtained by sequentially arranging individual sperm values of the sperm quality index.
- the sperm population may be all sperm suspended in the test solution, or sperm existing in a certain region may be arbitrarily selected. However, the upper scale can be arbitrarily changed depending on the sperm population.
- the sperm having a higher quality index value of the sperm group obtained from injection semen etc. is selectively acquired Therefore, the value of all sperm in the sperm population may be used.
- the quality decision values are as follows: (1) Average value, median value, or mode value of quality index values of the stratified population; (2) the proportion or number of spermatozoa in the stratified population that exceeds or exceeds a certain quality index reference value; and (3) the quality in individual sperm of the stratified population. It is selected from the group consisting of approximate lines, intercepts, coefficients or slopes obtained from a graph in which index values are arranged in order. (1) to (3) may be combined.
- the quality-determined value may be compared between samples of the sperm population, or compared with a reference value of a quality index that has been correlated with sperm quality in advance, such as fertility, or fertility is known in advance. It is possible to evaluate the quality of sperm by applying an estimation formula calculated using a plurality of semen.
- a method for determining the quality of sperm using the quality determination values of (1) to (3) above will be specifically described.
- the quality determination value is an average value, median value, or mode value of the quality index values of the stratified population
- the quality index is VCL
- the stratified population Of these, 10% is selected as the top quality group, but there is nothing intended to limit the quality index and the stratified group to these.
- the process of assessing sperm quality based on the quality indicator value is performed as follows: Selecting the top 10% quality top group of the sperm population based on the quality index value (VCL); Calculating the mean, median, or mode of quality index values (VCL) of the top quality group; Sperm quality is determined based on the mean, median, or mode of VCL.
- the calculated average value, median value, or mode value is compared with a reference value of a quality index that has been previously correlated with fertility, for example, 200 ⁇ m / second, 175 ⁇ m / second, 150 ⁇ m / second, etc.
- the fertility of the sperm population can be determined.
- the calculated average value, median value, or mode value of the VCL is estimated in advance to indicate the relationship between fertility and the average value, median value, or mode value of the VCL of the top 10% higher quality group. By fitting the equation, the quality of the sperm can be determined.
- the quality decision value is the ratio of sperm or the number of sperm that exceeds the reference value of the predetermined quality index in the stratified population or the reference value in a certain range
- VCL is selected as the quality index
- 200 ⁇ m / second is selected as the reference value of the quality index, there is nothing intended to limit the quality index and the reference value to these.
- the process of assessing sperm quality based on the quality indicator value is performed as follows: Selecting a quality upper group having a quality index value (VCL) that exceeds a reference value (200 ⁇ m / sec) of the quality index value (VCL); Calculate the number of sperm or percentage of sperm in the top quality group; Determine sperm quality based on sperm count or sperm ratio. By comparing the calculated sperm count or sperm ratio with the sperm count or sperm ratio that has been previously shown to be correlated with fertility, the fertility of the sperm population can be determined. In addition, by applying the sperm count or sperm ratio to the estimated formula of the sperm count or sperm ratio calculated using a plurality of semen whose fertility is known in advance and fertility, the sperm population Fertility can be determined.
- VCL quality index value
- VCL quality index value
- the quality determination value is an approximate line, intercept, coefficient, or slope obtained from a graph in which the quality index values of individual spermatozoa in the stratified population are sequentially arranged.
- the quality index is VCL. Yes, 10% is selected as the upper quality group, but it is not intended to limit the quality index and the upper group to these.
- the process of assessing sperm quality based on the quality indicator value is performed as follows: Selecting the top 10% quality top group of the sperm population based on the quality index value (VCL); Arrange the quality index values (VCL) of the top quality group in ascending or descending order and calculate the approximate value, intercept, coefficient or slope of the graph obtained by plotting the quality index value on the Y axis and the ranking on the X axis And Determine sperm quality based on approximate line, intercept, coefficient or slope. The intercept, coefficient, or slope can also be obtained from an approximate line.
- the plots on the X axis and Y axis may be reversed.
- the approximate line is an expression for grasping the approximate shape when the values of individual sperm of the sperm quality index are arranged in order, for example, linear approximation, polynomial approximation, exponential approximation, logarithmic approximation, power approximation, moving average Asymptote, tangent, etc.
- Arranging in order means arranging the values of individual spermatozoa in ascending or descending order, and the highest quality group, for example, the top 30% or more, preferably the top 20% or more, more preferably the top 10% or more, even more preferably Approximate lines can be obtained by arranging the upper 8% or more of sperm.
- the approximate line is represented by a straight line, a function, a logarithm, an exponent, a sigmoid curve or the like by calculating a regression equation by performing regression analysis mainly using a statistical method such as a least square method.
- a predicted value of fertility can be obtained.
- the predicted value can also be obtained using the intercept, coefficient, or slope of the approximate line.
- a fertility estimation formula can be calculated by performing multivariate analysis such as multiple regression analysis.
- the stratified group used in (1) to (3) may be considered for the higher quality group, or after considering the value of the quality index in the higher quality group, It is also possible to consider the quality index value in.
- the quality decision value can be calculated based on the value of the quality index in another group as well as the higher quality group.
- the quality determination value is the above (1) to (3)
- the same quality decision value used for the can be used, or different quality decision values can be used.
- An example in which the quality determination value is calculated based on the quality index values in the plurality of groups will be described below.
- the quality decision value is an average value, median value, or mode value of the quality index values of the stratified population
- the quality index is VCL and the stratified sperm population
- the quality index value a group in which the top 80% is divided every 10% is selected, but there is nothing intended to limit the quality index and the sperm population to these groups.
- the process of assessing sperm quality based on the quality indicator value is performed as follows: 8 groups are selected by dividing the top 80% quality top group in the sperm population by 10% based on the quality index value (VCL); Calculating the mean, median, or mode of quality index values (VCL) for each of the eight groups; Sperm quality is determined based on the mean, median, or mode of each VCL.
- Each calculated average value, median value, or mode value should be compared with a reference value of a quality index that has been previously correlated with fertility, for example, 200 ⁇ m / second, 175 ⁇ m / second, 150 ⁇ m / second, etc.
- the fertility of the sperm population can be determined.
- the calculated average value, median value, or mode value of the VCL is determined based on the average value, the median value, or the maximum value of the VCL of eight groups obtained by dividing the entire sperm population every 10%.
- the quality of sperm can be determined using an estimation formula indicating the relationship with the frequent value and an estimation formula obtained by performing multivariate analysis such as multiple regression analysis.
- the quality determination value is a sperm ratio or sperm count exceeding the reference value of a predetermined quality index value or within a certain range in the stratified population.
- the standard value of stratification is 200 ⁇ m / second or more, 150-200 ⁇ m / second, 100-150 ⁇ m / second, 50-100 ⁇ m / second is selected. There is nothing intended to be limited to these.
- the process of assessing sperm quality based on the quality indicator value is performed as follows: Select four groups by dividing the reference value of the quality index (VCL) into 200 ⁇ m / second or more, 150 to 200 ⁇ m / second, 100 to 150 ⁇ m / second, 50 to 100 ⁇ m / second; Calculate the sperm count or sperm ratio for each of the four groups; Sperm quality is determined based on each sperm count or sperm ratio. By comparing the calculated sperm count or sperm ratio with the sperm count or sperm ratio that has been previously shown to be correlated with fertility, the fertility of the sperm population can be determined.
- VCL quality index
- multivariate analysis such as sperm count or sperm ratio, approximate line of sperm count or sperm ratio calculated using multiple semen whose fertility is known in advance and fertility and multiple regression analysis
- the fertility of the sperm population can be determined by applying it to the estimation formula obtained by performing.
- the quality determination value is an approximate line, intercept, coefficient, or slope obtained from one or more graphs obtained by sequentially arranging quality index values in individual sperm of the sperm population in the stratified population.
- the quality index is VCL and the value of the quality index is divided into the top 80% every 10% as a stratified sperm group. It is not intended to be limited to these.
- the process of assessing sperm quality based on the quality indicator value is performed as follows: 8 groups are selected by dividing the top 80% quality top group in the sperm population by 10% based on the quality index value (VCL); Approximate line, intercept, coefficient or slope of graph obtained by arranging quality index values (VCL) for each of 8 groups in ascending or descending order and plotting quality index values on Y axis and ranking on X axis Calculate; Determine sperm quality based on approximate line, intercept, coefficient or slope. The intercept, coefficient, or slope can also be obtained from an approximate line.
- the plots on the X axis and Y axis may be reversed.
- the approximate line is an expression for grasping the approximate shape when the values of individual sperm of the sperm quality index are arranged in order, for example, linear approximation, polynomial approximation, exponential approximation, logarithmic approximation, power approximation, moving average Asymptote, tangent, etc.
- Arranging in order means arranging the values of individual sperm in ascending or descending order.
- the approximate line is represented by a straight line, a function, a logarithm, an exponent, a sigmoid curve or the like by calculating a regression equation by performing regression analysis mainly using a statistical method such as a least square method.
- an estimation formula can be calculated by performing multivariate analysis such as multiple regression analysis.
- the estimation formula calculated in this way is used to estimate the relationship between fertility and the eight groups obtained by dividing the entire sperm population every 10%, and multivariate analysis such as multiple regression analysis.
- the quality of the sperm can be determined using the estimation formula that has been made.
- FIG. The configuration of the sperm inspection apparatus of the present invention is shown in FIG.
- the sperm inspection apparatus 10 includes a sperm quality measurement unit 11 (sperm quality measurement unit) that measures the value of a sperm quality index, an input unit 12 (input unit) that gives measurement data, an instruction of arithmetic processing, and the like.
- Storage unit 13 storage unit for storing the calculation results and calculation results
- sperm quality evaluation unit 14 sperm quality evaluation unit for evaluating sperm quality based on the measured sperm quality index value
- sperm quality evaluation results Is mainly provided with an output unit 15 (output means).
- the sperm quality measurement unit 11 performs a process of measuring the value of the sperm quality index described in the first embodiment.
- the sperm quality measuring unit 11 is a constituent part for measuring the quality index value of each sperm of the sperm population suspended in the test solution.
- the sperm quality measurement unit 11 is a microscope such as a sperm motion analyzer, a high-speed camera, a phase contrast microscope, a differential interference microscope, a polarization microscope, a fluorescence microscope, a confocal laser microscope, a transmission electron microscope, and a scanning electron microscope.
- the sperm quality measurement unit 11 may be configured separately from the sperm inspection apparatus 10 and may input measured data or the like via the input unit 12 using a network or a storage medium.
- the input unit 12 is an interface or the like, and includes operation units such as a keyboard and a mouse. As a result, the input unit 12 can input data measured by the sperm quality measurement unit 11, instructions for arithmetic processing performed by the sperm quality evaluation unit 14, and the like. For example, when the sperm quality measurement unit 11 is outside, the input unit 12 may include an interface unit that can input measured data or the like via a network or a storage medium, separately from the operation unit.
- the storage unit 13 includes a memory device such as a RAM, a ROM, and a flash memory, a fixed disk device such as a hard disk drive, or a portable storage device such as a flexible disk and an optical disk.
- the storage unit 13 is a computer program used for various processes of the information processing apparatus in addition to the data measured by the sperm quality measurement unit 11, the instruction input from the input unit 12, the calculation processing result performed by the sperm quality evaluation unit 14, and the like , Memorize database etc.
- the computer program may be installed via a computer-readable Khan type recording medium such as a CD-ROM or DVD-ROM, or via the Internet.
- the computer program is installed in the storage unit 13 using a known setup program or the like.
- the sperm quality evaluation unit 14 performs the process of evaluating the quality of the sperm described in the first embodiment.
- the sperm quality evaluation unit 14 is a constituent part for evaluating the quality of sperm on the basis of the value of the higher quality index of the sperm group.
- various arithmetic processes are executed on the data measured by the sperm quality measurement unit 11 and stored in the storage unit 13 in accordance with a program stored in the storage unit 13.
- the arithmetic processing is performed by a CPU included in the sperm quality evaluation unit 14.
- the CPU includes a functional module that controls the sperm quality measurement unit 11, the input unit 12, the storage unit 13, and the output unit 15, and can perform various controls.
- Each of these units may be configured by an independent integrated circuit, a microprocessor, firmware, and the like.
- the output unit 15 includes a display device such as a liquid crystal display and output means such as a printer.
- the sperm quality evaluation unit 14 outputs the result of the sperm quality evaluation result value, the fertility index value, the predicted conception rate, and the like.
- Example 1 Examination of pH of test solution Frozen semen produced using the injection semen of breeding bulls by the Japan Livestock Improvement Corporation was thawed according to a conventional method, and centrifuged at 2000 rpm at room temperature for 5 minutes against 10 million sperm After separation and removal of the supernatant, 140.6 mM Tris (hydroxymethyl) aminomethane (Wako Pure Chemical Industries), 45.3 mM citric acid (Wako Pure Chemical Industries), 16.7 mM Glucose (Wako Pure Chemical Industries), 79.2 mM sodium chloride (Wako Pure Chemical Industries), 2 mM calcium chloride (Wako Pure Chemical Industries), 0.3% (w / v) BSA (Wako Pure Chemical Industries), 650 U / ml penicillin G potassium (Manyu Pharmaceutical), 0.7 mg (titer) / ml streptomycin (Meiji Pharmaceutical), 1N sodium hydroxide so that the pH is in increments of 0.2 from 6.0 to 9.0 Or 250 ⁇ l
- a slide chamber (Leja) having a thickness of 20 ⁇ m, mixed with an equal amount of each of the above-described test solutions to which 4% (w / v) polyvinylpyrrolidone K-90 (Wako Pure Chemical Industries) was added, and pre-warmed to 38 ° C. And measured using a sperm motion analysis apparatus Ceros (Hamilton Thorn) at an imaging speed of 1/60 seconds.
- Example 2 Examination of Suspension Time Frozen semen prepared using the injection semen of breeding bulls by the Japan Livestock Improvement Corporation was thawed according to a conventional method, and centrifuged at 2000 rpm at room temperature for 5 minutes. After removing the supernatant, 250 ⁇ l of the test solution described in Example 1 adjusted to pH 7.6 was added to the precipitate. Incubation was performed at 38 ° C. Immediately after suspension (0), after 10, 30, 60 minutes, an equal amount of the above test solution to which 4% (w / v) polyvinylpyrrolidone K-90 was added was mixed and measured by the method described in Example 1. did.
- FIG. 5 shows the sperm ratio (%) of VCL 150 ⁇ m / second or more and 200 ⁇ m / second or more. Immediately after suspending to 30 minutes, sperm with high driving force could be detected.
- the index of sperm motility which is a conventional test example, was expressed as a percentage (%) of sperm having a VAP of 50 ⁇ m / second or more at 38 ° C. using a sperm motility analyzer for sperm after freezing and thawing.
- the ratio of sperm with a VAP of 50 ⁇ m / sec or more is shown in FIG. 6A.
- Conventional indicators of sperm motility did not correlate with conception rate.
- measurement results obtained by changing the cutoff value of VCL are shown in FIGS. 6B to 6E.
- the ratio of sperm with a VCL of 50 ⁇ m / second or more is shown in FIG. 6B
- the ratio of sperm with a VCL of 100 ⁇ m / second or more is shown in FIG. 6C
- the ratio of sperm with a VCL of 150 ⁇ m / second or more is shown in FIG. Shown in In the low conception sample with a conception rate of 40% or less, it was confirmed that the proportion of sperm decreased as the cut-off value increased. Therefore, it has been found that fertility can be predicted by increasing the cut-off value and detecting sperm exhibiting higher driving force.
- FIG. 7 shows the result of calculation using logarithmic approximation for the correlation between the ratio of sperm with VCL of 200 ⁇ m / sec or more and the conception rate. It was confirmed that the ratio of sperm with VCL of 200 ⁇ m / second or higher was highly correlated with the conception rate. From this prediction formula, a markedly low conception was predicted when the ratio of sperm with a VCL of 200 ⁇ m / sec or more was 0.3% or less, and a conception rate of 50% or less was predicted when 0.7% or less. 8A and 8B show a comparison of the ratio of sperm with VCL of 200 ⁇ m / sec or more and the conception rate for each category. The higher the conception rate, the higher the proportion of sperm with a VCL of 200 ⁇ m / sec or higher.
- FIG. 9 shows the result of calculation using logarithmic approximation on the correlation between the number of sperm with VCL of 200 ⁇ m / sec or more per straw and the conception rate.
- the number of sperm with a VCL of 200 ⁇ m / sec or more was confirmed to have a high correlation with the conception rate. From this prediction formula, a markedly low conception was predicted when the number of sperm having a VCL of 200 ⁇ m / sec or more per straw was 100,000 or less, and a conception rate of 40% or less was predicted when the number was 300,000 or less.
- the rate of sperm with a VCL of 200 ⁇ m / sec or higher was remarkably low, and the rate of sperm with a VCL of 100 to 150 ⁇ m / sec was high.
- the structure of each VCL speed category it was possible to detect the fertility of sperm with higher reliability.
- the peak of the VCL histogram is roughly 125 to 175 ⁇ m / sec in the section with a conception rate of 65% or more, 50 to 150 ⁇ m / sec in the section with a conception rate of 55 to 65%, and 50 to 50 in the section with a conception rate of 40 to 55%.
- the peak value tended to be higher as the conception rate was higher at 75 ⁇ m / sec.
- the rate of 0 to 75 ⁇ m / second with a low VCL was similar, but the group with a conception rate of less than 40% was 75 to 150 ⁇ m. It was characterized by the highest rate per second.
- FIG. 12A shows the result of displaying the VCL value on the vertical axis and the top 75% in the sperm population in descending order on the horizontal axis. It was shown that the composition of the sperm population is different for each conception rate.
- FIG. 12B shows the results of displaying the VCL value on the vertical axis, the top 10% in the sperm population in descending order on the horizontal axis, and the standard deviation for sections with a conception rate of 65% or higher.
- the VCL values of the top 0.3-6% exceeded those of low conception cows with a conception rate of less than 40% at a conception rate of 65% or more, 55-65%, and 40-55%.
- the conception rate could be predicted by comparing VCL values of arbitrary values in the upper 0.3 to 6%. Moreover, fertility could be predicted with higher accuracy by comparing higher VCL values such as upper 3%, 2%, 1%, 0.5%, 0.3% or more.
- FIG. 13 shows the results of calculating approximate lines by displaying VCL values on the vertical axis and the top 10% in the sperm population in descending order on the horizontal axis and approximating with a cubic function.
- the y-intercept of the cubic function is 216.57, 216.93, 213.04 in normal cattle with a conception rate of 65% or more, 55 to 65%, and 40 to 55%, respectively, and 200.23 at a conception rate of less than 40%.
- the low conception cows showed low values.
- FIG. 14 shows the result of calculating the approximate line by displaying the VCL value on the vertical axis and the top 20% in the sperm population in descending order on the horizontal axis and approximating with a straight line.
- the slopes of the straight lines are ⁇ 347.4, ⁇ 395.13, ⁇ 421.62 in normal cows with a conception rate of 65% or more, 55 to 65%, and 40 to 55%, respectively, and low conception cows with a conception rate of less than 40%.
- FIG. 15 shows the result of approximation with a straight line, with the estimated conception rate calculated from the estimation formula obtained from the multiple regression analysis on the vertical axis and the actual conception rate on the horizontal axis. A high correlation with the actual conception rate was found by evaluating based on the quality index values of multiple groups of the stratified sperm population.
- the VCL value in the sperm population of one lot of frozen semen was divided into 5 groups of 0 to 75 ⁇ m / second, 75 to 150 ⁇ m / second, 150 to 175 ⁇ m / second, 175 to 200 ⁇ m / second, 200 ⁇ m / second or more, and sperm for each.
- the multiple regression analysis was conducted using 40 bulls whose conception rates were known.
- FIG. 16 shows the result of approximation with a straight line, with the estimated conception rate calculated from the estimation formula obtained from the multiple regression analysis on the vertical axis and the actual conception rate on the horizontal axis. A high correlation with the actual conception rate was found by evaluating based on the quality index values of multiple groups of the stratified sperm population.
- Example 6 Comparison of fresh semen and frozen semen Fresh injected semen collected from slaughtered bulls by the Livestock Improvement Corporation and frozen semen thawed according to conventional methods, centrifuged at 2000 rpm at room temperature for 5 minutes After separation and removal of the supernatant, 250 ⁇ l of the test solution described in Example 2 was added to the precipitate. And it incubated for 10 minutes at 38 degreeC. An equal amount of the above test solution to which 4% (w / v) polyvinylpyrrolidone K-90 was added was mixed and measured by the method described in Example 1.
- the ratio of sperm with a VCL of 200 ⁇ m / sec or more is shown in FIG. Measurement was possible with both fresh semen and frozen semen, and the ratio of sperm with a VCL of 200 ⁇ m / sec or more significantly decreased with frozen semen. From this, it has been confirmed that it can be used as a quality index of sperm in the technical development of a diluted solution or the like when preparing frozen semen.
- the sperm inspection method of the present invention it is possible to obtain the evaluation of sperm having a high correlation with the conception rate by determining the ratio and number of sperm having extremely high sperm quality index values. Quality sperm can be supplied. In addition, since sperm can be supplied that reflects fertility, it is expected that fertility can be improved by, for example, artificial insemination in the field.
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pregnancy & Childbirth (AREA)
- Pathology (AREA)
- Reproductive Health (AREA)
- Gynecology & Obstetrics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Radiology & Medical Imaging (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Quality & Reliability (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Theoretical Computer Science (AREA)
- Medical Informatics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
卵管膨大部に到達した精子は、雌の排卵時期に運動性が顕著に上昇し、超活性化様運動を示したものが受精できると考えられている(非特許文献3:Suarez SS.,Int J Dev Biol.52(5-6),455-62 (2008年))。そこで、実際に雌体内の条件を反映させた検査法も考案されている(非特許文献4:Hamanoら、日本畜産学会北陸支部会報81,63-68 (2000年))。また、実際に精子を卵子と受精させる体外受精試験を用いて検査することもできる。
本発明の精子の検査方法は、精子集団に対して検査液に懸濁を行って個々の精子の品質指標の値を測定する工程と、前記品質指標の値に基づいて精子の品質を評価する工程とを備えることを特徴とする。
また、精子の品質を評価する工程は、品質指標の値に基づき、前記精子集団を層別化し、層別化された集団の1又は複数の群の品質指標の値に基づいて評価することを特徴とする。
また、層別化された集団の品質指標の値が、各集団毎の品質指標の値の平均値、中央値、又は最頻値であることを特徴とする。
また、前記層別化が、品質指標の値に基づいて、品質上位群を選択することを特徴とする。
また、前記層別化が、さらに他の群の品質指標の値に基づいて、精子の品質を評価することを特徴とする。
また、前記品質上位群は、上位50%以内であることを特徴とする。
また、前記品質上位群は、上位20%以内であることを特徴とする。
また、前記品質上位群は、上位10%以内であることを特徴とする。
また、前記品質上位群は、上位5%以内であることを特徴とする。
また、前記品質上位群は、上位3%以内であることを特徴とする。
また、前記品質上位群は、上位1%以内であることを特徴とする。
また、前記品質上位群は、上位0.5%以内であることを特徴とする。
また、前記品質上位群は、上位0.3%以内であることを特徴とする。
また、前記品質上位群の品質指標の値が、品質上位群における品質指標の値の平均値、中央値、又は最頻値であることを特徴とする。
また、前記精子集団は、生存している精子であることを特徴とする。
また、前記精子集団は、精子運動性が一定の値以上や一定の範囲であることを特徴とする。
また、前記品質指標は、精子運動性の指標であることを特徴とする。
また、前記検査液は、pHが6.0~9.0であることを特徴とする。
また、前記検査液は、pHが7.2~8.2であることを特徴とする。
また、前記検査液は、pHが7.4~7.8であることを特徴とする。
また、前記検査液は、浸透圧が230~400mmol/kg(mOsm/kg)であることを特徴とする。
また、前記検査液は、33~43℃に維持することを特徴とする。
また、前記検査液は、36~40℃に維持することを特徴とする。
また、前記検査液は、緩衝剤、糖及び塩を含むことを特徴とする。
また、前記検査液の緩衝剤は、トリス(ヒドロキシメチル)アミノメタン及びクエン酸であることを特徴とする。
また、前記検査液の糖は、グルコースであることを特徴とする。
また、前記検査液の塩は、塩化ナトリウムであることを特徴とする。
また、前記検査液は、活性化剤を含むことを特徴とする。
また、前記活性化剤は、プロカイン、カフェイン、及び/又はテオフィリンであることを特徴とする。
また、前記検査液は、負荷剤を含むことを特徴とする。
また、前記負荷剤は、ポリビニルピロリドンK-90であることを特徴とする。
また、前記懸濁の時間は、懸濁直後~30分間であることを特徴とする。
また、前記精子運動性の指標は、VAP、VCL、ALH及び/又はBCFであることを特徴とする。
また、前記精子の品質を評価する工程は、1又は複数の群の品質指標の基準値と比較して、精子の品質を決定する工程を含むことを特徴とする。
また、前記品質指標の基準値は、VCLが150μm/秒以上であることを特徴とする。
また、前記品質指標の基準値は、VCLが200μm/秒以上であることを特徴とする。
また、前記精子の品質を評価する工程は、所定の品質指標の基準値を超える精子の割合又は精子数に基づくことを特徴とする。
また、所定の品質指標の基準値が、VCL150μm/秒、175μm/秒、及び200μm/秒からなる群から選ばれる値であることを特徴とする。
また、前記品質指標の基準値は、VCLが200μm/秒以上であることを特徴とする。
また、精子の割合が1%以上であることを特徴とする。
また、精子の割合が0.7%以上であることを特徴とする。
また、精子の割合が0.3%以上であることを特徴とする。
また、精子数が1ストローあたり30万精子以上であることを特徴とする。
また、精子数が1ストローあたり10万精子以上であることを特徴とする。
また、前記精子の品質を評価する工程は、前記精子集団の個々の精子における品質指標の値を順に配列したグラフから求めることを特徴とする。
また、前記精子の品質を評価する工程は、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線から求めることを特徴とする。
また、前記精子の品質を評価する工程は、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線の傾きから求めることを特徴とする。
また、前記精子の品質を評価する工程は、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線の係数から求めることを特徴とする。
また、前記精子の品質を評価する工程は、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線の切片から求めることを特徴とする。
また、前記精子は、哺乳類の精子であることを特徴とする。
また、前記精子は、牛精子であることを特徴とする。
また、精子品質評価手段は、品質指標の値に基づき、前記精子集団を層別化し、層別化された集団の1又は複数の群の品質指標の値に基づいて評価することを特徴とする。
また層別化された集団の品質指標の値が、各集団毎の品質指標の値の平均値、中央値、又は最頻値であることを特徴とする。
また、前記層別化が、品質指標の値に基づいて、品質上位群を選択することを特徴とする。
また、前記層別化が、さらに他の群の品質指標の値に基づいて、精子の品質を評価することを特徴とする。
また、前記品質上位群は、上位50%以内であることを特徴とする。
また、前記品質上位群は、上位20%以内であることを特徴とする。
また、前記品質上位群は、上位10%以内であることを特徴とする。
また、前記品質上位群は、上位5%以内であることを特徴とする。
また、前記品質上位群は、上位3%以内であることを特徴とする。
また、前記品質上位群は、上位1%以内であることを特徴とする。
また、前記品質上位群は、上位0.5%以内であることを特徴とする。
また、前記品質上位群は、上位0.3%以内であることを特徴とする。
また、前記品質上位群の品質指標の値が、品質上位群における品質指標の値の平均値、中央値、又は最頻値であることを特徴とする。
また、前記精子集団は、生存している精子であることを特徴とする。
また、前記精子集団は、精子運動性が一定の値以上や一定の範囲であることを特徴とする。
また、前記品質指標は、精子運動性の指標であることを特徴とする。
また、前記検査液は、pHが6.0~9.0であることを特徴とする。
また、前記検査液は、pHが7.2~8.2であることを特徴とする。
また、前記検査液は、pHが7.4~7.8であることを特徴とする。
また、前記検査液は、浸透圧が230~400mmol/kg(mOsm/kg)であることを特徴とする。
また、前記検査液は、33~43℃に維持することを特徴とする。
また、前記検査液は、36~40℃に維持することを特徴とする。
また、前記検査液は、緩衝剤、糖及び塩を含むことを特徴とする。
また、前記検査液の緩衝剤は、トリス(ヒドロキシメチル)アミノメタン及びクエン酸であることを特徴とする。
また、前記検査液の糖は、グルコースであることを特徴とする。
また、前記検査液の塩は、塩化ナトリウムであることを特徴とする。
また、前記検査液は、活性化剤を含むことを特徴とする。
また、前記活性化剤は、プロカイン、カフェイン、及び/又はテオフィリンであることを特徴とする。
また、前記検査液は、負荷剤を含むことを特徴とする。
また、前記負荷剤は、ポリビニルピロリドンK-90であることを特徴とする。
また、前記懸濁の時間は、懸濁直後~30分間であることを特徴とする。
また、前記精子運動性の指標は、VAP、VCL、ALH及び/又はBCFであることを特徴とする。
また、前記精子品質評価手段は、1又は複数の群の品質指標の基準値と比較して、精子の品質を決定する手段を含むことを特徴とする。
また、前記品質指標の基準値は、VCLが150μm/秒以上であることを特徴とする。
また、前記品質指標の基準値は、VCLが200μm/秒以上であることを特徴とする。
また、前記精子品質評価手段は、所定の品質指標の基準値を超える精子の割合又は精子数に基づくことを特徴とする。
また、所定の品質指標の基準値が、VCL150μm/秒、175μm/秒、及び200μm/秒からなる群から選ばれる値であることを特徴とする。
また、前記所定の品質指標の基準値は、VCLが200μm/秒であることを特徴とする。
また、前記精子の割合が1%以上であることを特徴とする。
また、前記精子の割合が0.3%以上であることを特徴とする。
また、前記精子数が1ストローあたり30万精子以上であることを特徴とする。
また、前記精子数が1ストローあたり10万精子以上であることを特徴とする。
また、前記精子品質評価手段は、前記精子集団の個々の精子における品質指標の値を順に配列したグラフから求めることを特徴とする。
また、前記精子品質評価手段は、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線から求めることを特徴とする。
また、前記精子品質評価手段は、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線の傾きから求めることを特徴とする。
また、前記精子品質評価手段は、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線の係数から求めることを特徴とする。
また、前記精子品質評価手段は、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線の切片から求めることを特徴とする。
また、前記精子は、哺乳類の精子であることを特徴とする。
また、前記精子は、牛精子であることを特徴とする。
検査液中の精子集団における個々の精子の品質指標の値を前記記憶部に記憶し、
前記記憶された品質指標の値に基づき、前記精子集団を層別化し、
当該層別化された集団の品質指標の値に基づき精子の品質を決定し、
精子の品質の結果を前記出力部に出力する
ことを前記情報処理装置に実行させることを特徴とする。
また、前記情報処理装置は、撮像部をさらに有し、以下の:
前記撮像部が経時的に複数枚の画像を取得し、
複数の画像に基づいて前記品質指標を決定することを特徴とする。
また、前記層別化された集団の1又は複数の群の品質指標の値に基づき精子の品質の決定が、さらに以下の:
層別化された集団の品質指標の値から、品質決定値を算出し、
前記品質決定値に基づき精子の品質を決定する
ことを含むことを特徴とする。
また層別化された集団の品質指標の値が、各集団毎の品質指標の値の平均値、中央値、又は最頻値であることを特徴とする。
また、前記層別化された集団として、品質上位群を選択することを特徴とする。
また、前記層別化が、さらに他の群の品質指標の値に基づいて、精子の品質を評価することを特徴とする。
また、前記品質上位群は、上位50%以内であることを特徴とする。
また、前記品質上位群は、上位20%以内であることを特徴とする。
また、前記品質上位群は、上位10%以内であることを特徴とする。
また、前記品質上位群は、上位5%以内であることを特徴とする。
また、前記品質上位群は、上位3%以内であることを特徴とする。
また、前記品質上位群は、上位1%以内であることを特徴とする。
また、前記品質上位群は、上位0.5%以内であることを特徴とする。
また、前記品質上位群は、上位0.3%以内であることを特徴とする。
また、前記品質上位群の品質指標の値が、品質上位群における品質指標の値の平均値、中央値、又は最頻値であることを特徴とする。
また、前記精子集団は、生存している精子であることを特徴とする。
また、前記精子集団は、精子運動性が一定の値以上や一定の範囲であることを特徴とする。
また、前記品質指標は、精子運動性の指標であることを特徴とする。
また、前記検査液は、pHが6.0~9.0であることを特徴とする。
また、前記検査液は、pHが7.2~8.2であることを特徴とする。
また、前記検査液は、pHが7.4~7.8であることを特徴とする。
また、前記検査液は、浸透圧が230~400mmol/kg(mOsm/kg)であることを特徴とする。
また、前記検査液は、33~43℃に維持することを特徴とする。
また、前記検査液は、36~40℃に維持することを特徴とする。
また、前記検査液は、緩衝剤、糖及び塩を含むことを特徴とする。
また、前記検査液の緩衝剤は、トリス(ヒドロキシメチル)アミノメタン及びクエン酸であることを特徴とする。
また、前記検査液の糖は、グルコースであることを特徴とする。
また、前記検査液の塩は、塩化ナトリウムであることを特徴とする。
また、前記検査液は、活性化剤を含むことを特徴とする。
また、前記活性化剤は、プロカイン、カフェイン、及び/又はテオフィリンであることを特徴とする。
また、前記検査液は、負荷剤を含むことを特徴とする。
また、前記負荷剤は、ポリビニルピロリドンK-90であることを特徴とする。
また、前記検査液への精子の懸濁の時間は、懸濁直後~30分間であることを特徴とする。
また、前記精子運動性の指標は、VAP、VCL、ALH及び/又はBCFであることを特徴とする。
また、前記精子の品質を評価するは、1又は複数の群の品質指標の基準値と比較して、精子の品質を決定することを含むことを特徴とする。
また、前記品質指標の基準値は、VCLが150μm/秒以上であることを特徴とする。
また、前記品質指標の基準値は、VCLが200μm/秒以上であることを特徴とする。
また、前記精子の品質を評価するは、所定の品質指標の基準値を超える精子の割合又は精子数に基づくことを特徴とする。
また、所定の品質指標の基準値が、VCL150μm/秒、175μm/秒、及び200μm/秒からなる群から選ばれる値であることを特徴とする。
また、前記所定の品質指標の基準値は、VCLが200μm/秒であることを特徴とする。
また、前記精子の割合が1%以上であることを特徴とする。
また、前記精子の割合が0.3%以上であることを特徴とする。
また、前記精子数が1ストローあたり30万精子以上であることを特徴とする。
また、前記精子数が1ストローあたり10万精子以上であることを特徴とする。
また、前記精子の品質を評価するは、前記精子集団の個々の精子における品質指標の値を順に配列したグラフから求めることを特徴とする。
また、前記精子の品質を評価するは、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線から求めることを特徴とする。
また、前記精子の品質を評価するは、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線の傾きから求めることを特徴とする。
また、前記精子の品質を評価するは、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線の係数から求めることを特徴とする。
また、前記精子の品質を評価するは、前記精子集団の個々の精子における品質指標の値を順に配列した前記グラフの近似線の切片から求めることを特徴とする。
また、前記精子は、哺乳類の精子であることを特徴とする。
また、前記精子は、牛精子であることを特徴とする。
本発明の精子の検査方法は、以下の工程から構成される。
(1)精子の品質指標の値を測定する工程(S100)
精子集団に対して検査液に懸濁を行って個々の精子の品質指標の値を測定する工程である。
(2)精子の品質を評価する工程(S200)
精子集団の上位の品質指標の値に基づいて精子の品質を評価する工程である。
(1-1)精子の画像を経時的に複数枚取得する工程(S101)
(1-2)複数枚の画像に基づいて精子の品質指標の値を測定する工程(S102)
を含んでもよい。品質指標の値を測定する工程(S100)は、予め取得され、記憶された品質指標の値の読み出しを行う工程(S199)を行うことで、省略することもできる。
(2)精子の品質を評価する工程は、さらに以下の:
(2-1)品質指標の値に基づき、前記精子集団を層別化する工程(S201)
(2-2)層別化された集団の品質指標の値から、品質決定値を算出する工程(S202)
(2-3)品質決定値から精子の品質を決定する工程(S203)
のうちの1以上、好ましくは全てを含む。
(1)層別化された集団の品質指標の値の平均値、中央値、又は最頻値;
(2)層別化された集団において所定の品質指標の基準値を超える又は一定の範囲の基準値の精子の割合又は精子数;及び
(3)層別化された集団の個々の精子における品質指標の値を順に配列したグラフから得られる近似線、切片、係数又は傾きからなる群から選択される。(1)~(3)は各々組み合わせることもできる。品質決定値を、精子集団のサンプル間同士で比較してもよいし、予め受胎性などの精子の品質と相関が示された品質指標の基準値と比較するか、又は予め受胎性が判明している複数の精液を用いて計算された推定式にあてはめることで、精子の品質を評価することができる。
(1)品質決定値が、層別化された集団の品質指標の値の平均値、中央値、又は最頻値である場合
一例として、品質指標がVCLであり、層別化された集団のうち、品質上位群として10%を選択するが、品質指標及び層別化された集団が、これらのものに限られることを意図するものはない。品質指標の値に基づいて精子の品質を評価する工程は、以下のように行われる:
品質指標の値(VCL)に基づき精子集団の上位10%の品質上位群を選択し;
品質上位群の品質指標の値(VCL)の平均値、中央値、又は最頻値を算出し;
VCLの平均値、中央値、又は最頻値に基づき、精子の品質を決定する。
算出された平均値、中央値、又は最頻値は、予め受胎性との相関が示された品質指標の基準値、例えば200μm/秒、175μm/秒、150μm/秒などと比較することで、その精子集団の受胎性を決定することができる。また、算出されたVCLの平均値、中央値、又は最頻値を、予め受胎性と、上位10%の品質上位群のVCLの平均値、中央値、又は最頻値との関係を示す推定式と当てはめることにより、精子の品質を決定することができる。
一例として、品質指標としてVCLを選択し、品質指標の基準値として200μm/秒を選択するが、品質指標及び基準値がこれらのものに限られることを意図するものはない。品質指標の値に基づいて精子の品質を評価する工程は、以下のように行われる:
品質指標の値(VCL)の基準値(200μm/秒)を超える品質指標の値(VCL)を有する品質上位群を選択し;
品質上位群の精子数又は精子の割合を算出し;
精子数又は精子の割合に基づき、精子の品質を決定する。
算出された精子数又は精子の割合は、予め受胎性との相関が示された精子数又は精子の割合と比較することで、その精子集団の受胎性を決定することができる。また、精子数又は精子の割合を、予め受胎性が判明している複数の精液を用いて計算された精子数又は精子の割合と、受胎性との推定式に当てはめることで、その精子集団の受胎性を決定することができる。
一例として、品質指標がVCLであり、品質上位群として10%を選択するが、品質指標や上位群をこれらのものに限定することを意図するものではない。品質指標の値に基づいて精子の品質を評価する工程は、以下のように行われる:
品質指標の値(VCL)に基づき精子集団の上位10%の品質上位群を選択し;
品質上位群の品質指標の値(VCL)を昇順または降順に配列し、Y軸に品質指標の値、X軸に順位をプロットして得られたグラフの近似線、切片、係数又は傾きを算出し;
近似線、切片、係数又は傾きに基づき、精子の品質を決定する。切片、係数又は傾きは近似線から求めることもできる。なおX軸とY軸へのプロットは逆にプロットしてもよい。近似線は、精子の品質指標の個々の精子の値を順に配列した場合に、概形を把握するための式であり、例えば線形近似、多項式近似、指数近似、対数近似、累乗近似、移動平均、漸近線、接線等が挙げられる。順に配列するとは、個々の精子の値を昇順又は降順に配置することをいい、品質上位群、例えば上位30%以上、好ましくは上位20%以上、より好ましくは上位10%以上、さらにより好ましくは上位8%以上の精子を配列して、近似線を求めることができる。近似線は、主に最小二乗法等の統計学的手法を用いて回帰分析を行って回帰式を算出し、直線、関数、対数、指数、シグモイド曲線等で表す。このような近似線を用いることで、受胎性の予測値を出すことができる。また、予測値は、近似線の切片、係数又は傾きを用いて出すこともできる。さらに、例えば、VCLとALHといった複数の品質指標を基準として用いる場合には、重回帰分析等の多変量解析を行って受胎性の推定式を算出することができる。
一例として、品質指標がVCLであり、層別化された精子集団として品質指標の値が上位80%を10%毎に区切った群を選択するが、品質指標及び精子集団の各群が、これらのものに限られることを意図するものはない。品質指標の値に基づいて精子の品質を評価する工程は、以下のように行われる:
品質指標の値(VCL)に基づき精子集団での上位80%の品質上位群を10%毎に区切って8個の群を選択し;
8個の群毎の品質指標の値(VCL)の平均値、中央値、又は最頻値を算出し;
各々のVCLの平均値、中央値、又は最頻値に基づき、精子の品質を決定する。
算出された各々の平均値、中央値、又は最頻値は、予め受胎性との相関が示された品質指標の基準値、例えば200μm/秒、175μm/秒、150μm/秒などと比較することで、その精子集団の受胎性を決定することができる。また、算出されたVCLの平均値、中央値、又は最頻値を、予め受胎性と、精子集団の全体を10%毎に区切った8個の群のVCLの平均値、中央値、又は最頻値との関係を示す推定式や重回帰分析等の多変量解析を行って出した推定式を用いて、精子の品質を決定することができる。
一例として、品質指標としてVCLを選択し、層別化の基準値が200μm/秒以上、150~200μm/秒、100~150μm/秒、50~100μm/秒の場合を選択するが、品質指標及び層別化の基準値がこれらのものに限られることを意図するものはない。品質指標の値に基づいて精子の品質を評価する工程は、以下のように行われる:
品質指標の値(VCL)の基準値を200μm/秒以上、150~200μm/秒、100~150μm/秒、50~100μm/秒に区切って4個の群を選択し;
4個の群毎の精子数又は精子の割合を算出し;
各々の精子数又は精子の割合に基づき、精子の品質を決定する。
算出された精子数又は精子の割合は、予め受胎性との相関が示された精子数又は精子の割合と比較することで、その精子集団の受胎性を決定することができる。また、精子数又は精子の割合を、予め受胎性が判明している複数の精液を用いて計算された精子数又は精子の割合と、受胎性との近似線や重回帰分析等の多変量解析を行って出した推定式に当てはめることで、その精子集団の受胎性を決定することができる。
一例として、品質指標がVCLであり、層別化された精子集団として品質指標の値が上位80%を10%毎に区切った場合を選択するが、品質指標及び精子集団の各群をこれらのものに限定することを意図するものではない。品質指標の値に基づいて精子の品質を評価する工程は、以下のように行われる:
品質指標の値(VCL)に基づき精子集団での上位80%の品質上位群を10%毎に区切って8個の群を選択し;
8個の群毎の品質指標の値(VCL)を昇順または降順に配列し、Y軸に品質指標の値、X軸に順位をプロットして得られたグラフの近似線、切片、係数又は傾きを算出し;
近似線、切片、係数又は傾きに基づき、精子の品質を決定する。切片、係数又は傾きは近似線から求めることもできる。なおX軸とY軸へのプロットは逆にプロットしてもよい。近似線は、精子の品質指標の個々の精子の値を順に配列した場合に、概形を把握するための式であり、例えば線形近似、多項式近似、指数近似、対数近似、累乗近似、移動平均、漸近線、接線等が挙げられる。順に配列するとは、個々の精子の値を昇順又は降順に配置することをいう。近似線は、主に最小二乗法等の統計学的手法を用いて回帰分析を行って回帰式を算出し、直線、関数、対数、指数、シグモイド曲線等で表す。さらに、例えば、VCLとALHといった複数の品質指標を基準として用いる場合には、重回帰分析等の多変量解析を行って推定式を算出することができる。このようにして算出された推定式を、予め受胎性と、精子集団の全体を10%毎に区切った8個の群との関係を示す推定式や、さらに重回帰分析等の多変量解析を行って出した推定式を用いて、精子の品質を決定することができる。
本発明の精子の検査装置の構成を図2に示す。精子の検査装置10は、精子の品質指標の値を測定する精子品質測定部11(精子品質測定手段)、測定したデータや演算処理の指示等を与える入力部12(入力手段)、測定したデータや演算処理結果等を記憶する記憶部13(記憶手段)、測定した精子の品質指標の値に基づいて精子の品質を評価する精子品質評価部14(精子品質評価手段)、精子品質の評価結果を出力する出力部15(出力手段)を主に備えている。
一般社団法人家畜改良事業団で繋養の種雄牛の射出精液を用いて作製した凍結精液を常法に従って融解し、1000万精子に対して2000rpm、室温、5分間で遠心分離を行い、上清を除去した後、沈殿に対して140.6mMのトリス(ヒドロキシメチル)アミノメタン(和光純薬工業)、45.3mMのクエン酸(和光純薬工業)、16.7mMのグルコース(和光純薬工業)、79.2mMの塩化ナトリウム(和光純薬工業)、2mMの塩化カルシウム(和光純薬工業)、0.3%(w/v)のBSA(和光純薬工業)、650U/mlのペニシリンGカリウム(万有製薬)、0.7mg(力価)/mlのストレプトマイシン(明治製薬)、pHを6.0~9.0まで0.2刻みになるよう1N水酸化ナトリウム又は1N塩酸を用いて調製した検査液を250μl添加した。そして、38℃で10分間インキュベーションを行った。4%(w/v)のポリビニルピロリドンK-90(和光純薬工業)を添加した各々のpHの上記検査液を等量混合し、予め38℃に温めた厚さ20μmのスライドチャンバー(Leja)に滴下し、1/60秒の撮像速度で精子運動解析装置Ceros(Hamilton Thorne)を用いて測定した。
一般社団法人家畜改良事業団で繋養の種雄牛の射出精液を用いて作製した凍結精液を常法に従って融解し、1000万精子に対して2000rpm、室温、5分間で遠心分離を行い、上清を除去した後、沈殿に対してpH7.6に調整した実施例1に記載の検査液を250μl添加した。38℃でインキュベーションを行った。懸濁直後(0)、10、30、60分間経過後、4%(w/v)のポリビニルピロリドンK-90を添加した上記検査液を等量混合し、実施例1に記載の方法で測定した。
受胎率の判明している一般社団法人家畜改良事業団で繋養の種雄牛(n=29又は40)の射出精液を用いて作製した凍結精液を常法に従って融解し、1000万精子に対して2000rpm、室温、5分間で遠心分離を行い、上清を除去した後、沈殿に対して実施例2に記載の検査液を250μl添加した。そして、38℃で10分間インキュベーションを行った。4%(w/v)のポリビニルピロリドンK-90を添加した上記検査液を等量混合し、実施例1に記載の方法で測定した。従来の検査例である精子運動性の指標は、凍結融解後の精子について精子運動解析装置を用いて38℃においてVAP50μm/秒以上の精子の割合(%)で示した。
受胎率の判明している一般社団法人家畜改良事業団で繋養の種雄牛(n=40)の射出精液を用いて作製した凍結精液を常法に従って融解し、1000万精子に対して2000rpm、室温、5分間で遠心分離を行い、上清を除去した後、沈殿に対して実施例2に記載の検査液を250μl添加した。そして、38℃で10分間インキュベーションを行った。4%(w/v)のポリビニルピロリドンK-90を添加した上記検査液を等量混合し、実施例1に記載の方法で測定した。受胎率65%以上(10頭)、55~65%(10頭)、55%以下(10頭)、低受胎牛(受胎率40%未満:10頭)について、各3ロット測定した。
図12Bは、縦軸にVCL値、横軸に精子集団内の上位10%までを降順で表示し、受胎率65%以上の区は標準偏差を表示した結果を示す。上位0.3~6%のVCL値は、受胎率65%以上、55~65%、40~55%において、受胎率40%未満の低受胎牛の値を上回っていた。このことから、上位0.3~6%の任意の値のVCL値を比較することで受胎率を予測できた。また、上位3%、2%、1%、0.5%、0.3%以上といったより上位のVCL値を比較することで、より精度高く受胎性を予測できた。
受胎率の判明している一般社団法人家畜改良事業団で繋養の種雄牛(n=40)の射出精液を用いて作製した凍結精液を常法に従って融解し、1000万精子に対して2000rpm、室温、5分間で遠心分離を行い、上清を除去した後、沈殿に対して実施例2に記載の検査液を250μl添加した。そして、38℃で10分間インキュベーションを行った。4%(w/v)のポリビニルピロリドンK-90を添加した上記検査液を等量混合し、実施例1に記載の方法で測定した。層別化された集団の1又は複数の群の品質指標の値に基づいて評価する。
一般社団法人家畜改良事業団で繋養の種雄牛から採取した新鮮射出精液及び常法に従って融解した凍結精液について、1000万精子に対して2000rpm、室温、5分間で遠心分離を行い、上清を除去した後、沈殿に対して実施例2に記載の検査液を250μl添加した。そして、38℃で10分間インキュベーションを行った。4%(w/v)のポリビニルピロリドンK-90を添加した上記検査液を等量混合し、実施例1に記載の方法で測定した。
Claims (26)
- 精子集団に対して検査液に懸濁を行って個々の精子の品質指標の値を測定する工程、
前記精子の品質指標の値に基づき、前記精子集団を層別化する工程、及び
層別化された集団の1又は複数の群の品質指標の値に基づいて精子の品質を評価する工程
を備えることを特徴とする精子の検査方法。 - 前記層別化された集団のうち、品質上位群の品質指標の値に基づいて、精子の品質を評価することを特徴とする、請求項1に記載の精子の検査方法。
- 前記層別化された集団のうち、さらに他の群の品質指標の値に基づいて、精子の品質を評価することを特徴とする、請求項2に記載の精子の検査方法。
- 前記品質指標は、精子運動性の指標であることを特徴とする請求項1~3のいずれか一項に記載の精子の検査方法。
- 前記検査液は、pHが7.2~8.2であることを特徴とする請求項1~4のいずれか一項に記載の精子の検査方法。
- 前記検査液は、負荷剤を含むことを特徴とする請求項1~5のいずれか一項に記載の精子の検査方法。
- 前記懸濁の時間は、懸濁直後~30分間であることを特徴とする請求項1~6のいずれか一項に記載の精子の検査方法。
- 前記精子の品質を評価する工程が、1又は複数の群の品質指標の基準値と比較して、精子の品質を決定する工程を含む、請求項1~7のいずれか一項に記載の精子の検査方法。
- 前記精子の品質を評価する工程が、前記精子集団の個々の精子における品質指標の値を順に配列したグラフから求める工程を含む、請求項1~8のいずれか一項に記載の精子の検査方法。
- 検査液に懸濁された精子集団の個々の精子の品質指標の値を測定する精子品質測定手段と、前記精子の品質指標の値に基づき、前記精子集団を層別化する層別化手段と、層別化された精子集団の品質指標の値に基づいて精子の品質を評価する精子品質評価手段とを備えることを特徴とする精子の検査装置。
- 前記精子品質評価手段が、前記層別化された集団のうち、品質上位群の品質指標の値に基づいて、精子の品質を評価することを特徴とする、請求項10に記載の精子の検査方法。
- 前記精子品質評価手段が、前記層別化された集団のうち、さらに他の群の品質指標の値に基づいて、精子の品質を評価することを特徴とする、請求項11に記載の精子の検査方法。
- 前記品質指標は、精子運動性の指標であることを特徴とする請求項10~12のいずれか一項に記載の精子の検査装置。
- 前記検査液は、pHが7.2~8.2であることを特徴とする請求項10~13のいずれか一項に記載の精子の検査装置。
- 前記検査液は、負荷剤を含むことを特徴とする請求項10~14のいずれか一項に記載の精子の検査装置。
- 前記懸濁の時間は、懸濁直後~30分間であることを特徴とする請求項10~15のいずれか一項に記載の精子の検査装置。
- 前記精子品質評価手段が、1又は複数の群の品質指標の基準値と比較して、精子の品質を決定する、請求項10~16のいずれか一項に記載の精子の検査装置。
- 前記精子品質評価手段が、前記精子集団の個々の精子における品質指標の値を順に配列したグラフから求めることを特徴とする請求項10~17のいずれか一項に記載の精子の検査装置。
- 出力部と、記憶部とを含む情報処理装置に精子の品質を決定させる制御プログラムであって、
精子集団における個々の精子の品質指標の値を前記記憶部に記憶し、
前記記憶された品質指標の値に基づき、前記精子集団を層別化し、
当該層別化された集団の品質指標の値に基づき精子の品質を決定し、
精子の品質の結果を前記出力部に出力する
ことを前記情報処理装置に実行させることを特徴とする、前記制御プログラム。 - 前記情報処理装置は、撮像部をさらに有し、
前記撮像部が複数枚の経時的画像を取得し、
複数の画像に基づいて前記品質指標を決定する
ことを特徴とする、請求項19に記載の制御プログラム。 - 前記精子の品質を決定が、前記層別化された集団のうち、品質上位群の品質指標の値に基づいて、精子の品質を評価することを特徴とする、請求項19又は20に記載の制御プログラム。
- 前記精子の品質を決定が、前記層別化された集団のうち、さらに他の群の品質指標の値に基づいて、精子の品質を評価することを特徴とする、請求項21に記載の制御プログラム。
- 前記品質指標は、精子運動性の指標であることを特徴とする請求項19~22のいずれか一項に記載の制御プログラム。
- 前記精子の品質を評価するが、1又は複数の群の品質指標の基準値と比較して、精子の品質を決定することを含む、請求項19~23のいずれか一項に記載の制御プログラム。
- 前記精子の品質を評価するが、前記精子集団の個々の精子における品質指標の値を順に配列したグラフから求めることを含む、請求項19~24のいずれか一項に記載の制御プログラム。
- 請求項19~25のいずれか一項に記載の制御プログラムをコードするコンピュータ可読媒体。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2016507833A JP6747760B2 (ja) | 2014-03-12 | 2015-03-12 | 精子の検査方法及び装置 |
EP15762103.8A EP3118321B1 (en) | 2014-03-12 | 2015-03-12 | Sperm inspection method and device |
US15/124,998 US10593036B2 (en) | 2014-03-12 | 2015-03-12 | Sperm inspection method and device |
CA2942278A CA2942278C (en) | 2014-03-12 | 2015-03-12 | Sperm inspection method and device |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014-049033 | 2014-03-12 | ||
JP2014049033 | 2014-03-12 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2015137466A1 true WO2015137466A1 (ja) | 2015-09-17 |
Family
ID=54071903
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2015/057380 WO2015137466A1 (ja) | 2014-03-12 | 2015-03-12 | 精子の検査方法及び装置 |
Country Status (5)
Country | Link |
---|---|
US (1) | US10593036B2 (ja) |
EP (1) | EP3118321B1 (ja) |
JP (1) | JP6747760B2 (ja) |
CA (1) | CA2942278C (ja) |
WO (1) | WO2015137466A1 (ja) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11333613B2 (en) * | 2015-04-07 | 2022-05-17 | The Boeing Company | Apparatus and methods of inspecting a wire segment |
JP6932893B2 (ja) | 2016-03-16 | 2021-09-08 | 一般社団法人家畜改良事業団 | 精子用希釈液及びそれを用いた精子の保存方法 |
CN110930345B (zh) * | 2018-08-31 | 2023-05-30 | 赛司医疗科技(北京)有限公司 | 一种精子的尾部识别方法 |
WO2022192436A1 (en) * | 2021-03-09 | 2022-09-15 | Thread Robotics Inc. | System and method for automated gamete selection |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2009288189A (ja) * | 2008-05-30 | 2009-12-10 | Chiba Univ | 精子機能の検査方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5897988A (en) | 1998-01-21 | 1999-04-27 | Yale University | Process and system for selection of mature sperm by surface membrane determinants for assisted reproduction |
JP2005074586A (ja) * | 2003-09-01 | 2005-03-24 | Seoul National Univ Industry Foundation | 改選された卵細胞質内精子注入法(Animprovedmethodofintracytoplasmicsperminjection) |
WO2012061578A2 (en) * | 2010-11-03 | 2012-05-10 | The University Of North Carolina At Chapel Hill | Sperm motility analyzer and related methods |
-
2015
- 2015-03-12 EP EP15762103.8A patent/EP3118321B1/en active Active
- 2015-03-12 CA CA2942278A patent/CA2942278C/en active Active
- 2015-03-12 JP JP2016507833A patent/JP6747760B2/ja active Active
- 2015-03-12 US US15/124,998 patent/US10593036B2/en active Active
- 2015-03-12 WO PCT/JP2015/057380 patent/WO2015137466A1/ja active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2009288189A (ja) * | 2008-05-30 | 2009-12-10 | Chiba Univ | 精子機能の検査方法 |
Non-Patent Citations (4)
Title |
---|
A. IJAZ ET AL.: "Effect of Washing and Capacitating Media pH on Bull Sperm Motility, Acroemme Integrity, and Ability to Penetrate Zona-Free Hamster Oocytes", J DAIRY SCI, vol. 72, 1989, pages 2691 - 2699, XP008184403 * |
HIROAKI SHIBAHARA ET AL.: "Prediction of Human Sperm Fertilizing Ability by the Hyperactivated Motility Patterns", JOURNAL OF MAMMALIAN OVA RESEARCH, vol. 20, 2003, pages 29 - 33, XP055224154 * |
J. F. SMITH ET AL.: "Liner Collection Cone and pH Effects on Postthaw Motility, Staining, and Acrosomes of Bovine Spermatozoa", J DAIRY SCI, vol. 74, no. 4, 1991, pages 1310 - 1313, XP008184402 * |
MASASHI KINUGAWA ET AL.: "Ushi Seishi Undosei ni Motozuku Seieki Hinshitsu Hyokaho no Kochiku", THE JOURNAL OF REPRODUCTION AND DEVELOPMENT, vol. 58, 2012, pages 183 * |
Also Published As
Publication number | Publication date |
---|---|
US10593036B2 (en) | 2020-03-17 |
US20170091926A1 (en) | 2017-03-30 |
EP3118321A4 (en) | 2017-11-08 |
JP6747760B2 (ja) | 2020-08-26 |
EP3118321B1 (en) | 2021-09-29 |
EP3118321A1 (en) | 2017-01-18 |
CA2942278A1 (en) | 2015-09-17 |
CA2942278C (en) | 2023-08-22 |
JPWO2015137466A1 (ja) | 2017-04-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Yániz et al. | CASA-Mot in mammals: an update | |
Rijsselaere et al. | New techniques for the assessment of canine semen quality: a review | |
Petrunkina et al. | Determinants of sperm quality and fertility in domestic species | |
van der Horst et al. | Current perspectives of CASA applications in diverse mammalian spermatozoa | |
Rijsselaere et al. | Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer | |
THURSTON et al. | Morphologically distinct sperm subpopulations defined by Fourier shape descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation | |
Anel et al. | Improvement strategies in ovine artificial insemination | |
Yeste et al. | Evaluation of sperm motility with CASA-Mot: which factors may influence our measurements? | |
Holt et al. | Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality | |
Łukaszewicz et al. | Efficacy of evaluation of rooster sperm morphology using different staining methods | |
Gundogan et al. | Influence of sperm concentration on the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm during liquid storage | |
Maroto-Morales et al. | Current status and potential of morphometric sperm analysis | |
Rijsselaere et al. | Computer‐assisted sperm analysis in dogs and cats: An update after 20 years | |
Jasko | Evaluation of stallion semen | |
Simonik et al. | Computer assisted sperm analysis–the relationship to bull field fertility, possible errors and their impact on outputs: a review | |
WO2015137466A1 (ja) | 精子の検査方法及び装置 | |
Koonjaenak et al. | Seasonality affects post-thaw plasma membrane intactness and sperm velocities in spermatozoa from Thai AI swamp buffaloes (Bubalus bubalis) | |
Waberski et al. | Assessment of sperm motility in livestock: Perspectives based on sperm swimming conditions in vivo | |
Sancho et al. | The boar ejaculate: sperm function and seminal plasma analyses | |
Henning et al. | Cluster analysis reveals a binary effect of storage on boar sperm motility function1 | |
Petrunkina et al. | Functional significance of responsiveness to capacitating conditions in boar spermatozoa | |
RU2700454C1 (ru) | Способ прогнозирования качества спермы хряков | |
Singh et al. | Computer-assisted sperm analysis (CASA) in veterinary science: A review | |
Rijsselaere et al. | Use of the Sperm Quality Analyzer (SQA II‐C) for the assessment of dog sperm quality | |
Jo et al. | Effect of antibodies binding to Y chromosome-bearing sperm conjugated with magnetic nanoparticles on bull sperm characteristics |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15762103 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2016507833 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2942278 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15124998 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2015762103 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2015762103 Country of ref document: EP |