Patent Application: US-91476501-A

Abstract:
a method for the simultaneous determination of the total concentration of sperm cells and the proportion of live sperm cells in a semen sample is provided . by selective staining of live , dead and / or dying sperm cells , for example using one or more fluorochromes , the detection means can distinguish between live , dead and / or dying sperm cells , for example by detection of the fluorescent response from the sperm cells . the selective staining may be performed at room temperature and in concentrations below standard concentrations . the determination may be performed by selective counting of cells of each fluorescent quality for example by using a flow cytometer having internal concentration standard means . furthermore , the likelihood of fertilizing a female animal with an insemination dose , taken from a semen sample having a known total concentration of sperm cells and a known proportion of live sperm cells , is predicted on the basis of the thus determined total concentration of sperm cells and the proportion of live sperm cells .

Description:
in a first experiment , sperm concentration and viability in bull ejaculates have been determined by selective staining of the live and dead sperm cells in a semen sample and subsequently , analysing the semen samples with flow cytometry according to a preferred embodiment of the invention . for comparison purposes , the sperm concentration has also been determined using prior art methods , i . e . by determination of sperm concentration with a spectrophotometer ( l &# 39 ; aiglon , imv , cedex , france ) and a particle counter ( sysmex f - 820 , sysmex , almind , denmark ), and further using manual counting using a makler chamber ( sefi medical , haifa , israel ) and a phase contrast microscope . the analyses using flow cytometry for simultaneous determination of sperm concentration and viability in bull ejaculates at ai station were carried out using two different facscount flow cytometers ( bdis europe , erembodegem - aalst , belgium ) equipped with a blue ( 488 nm ), and a green ( 543 nm ) laser , respectively . sperm was either stained with a combination of sybr - 14 and pi for analysis with the blue ( 488 nm ) excitation wavelength or stained with a combination of mpr71292 and ehd2 for analysis with the green ( 543 nm ) excitation wavelength . the data obtained from the flow cytometers was subsequently analysed with attractor ™ software ( bdis europe , erembodegem - aalst , belgium ). in addition to the determination of sperm concentration , the flow cytometric analyses also provided an estimate of sperm viability . in general , a good agreement was found between the above - mentioned methods . the short staining period ( 2½ – 5 min ) in combination with minimal concentration of sybr - 14 , reduced the toxicity of this dye , thus , facilitating determination of sperm viability with high precision and accuracy . furthermore , the used protocol allowed for the detection of live , dead as well as dying sperm . the semen samples to be used in the experiments were collected from 25 bulls giving a total of 52 ejaculates . the semen samples were collected during 4 days from bulls with differences in both semen concentration and quality . an aliquot of 2 – 3 ml of the raw semen was placed in a tube ( nunc intermed cat # 347880 , lifetechnologies , roskilde , denmark ) and placed on a swelab - 820 mixer ( bie & amp ; berntsen , rødovre , denmark ) and mixed continuously . all processing and analyses were carried out within 30 minutes after semen collection . two samples of the raw semen were diluted 1 : 40 with a hamilton microlab a503 autodiluter ( struers kebo lab ., albertslund , denmark ) and using a phosphate buffered saline ( pbs , ph 7 . 4 , 300 mosm with 0 . 1 % bsa ). the diluted samples were placed on a mixer ( adams nutator , nc . nielsen , brøndby , denmark ). prior to evaluation , spermatozoa were immobilized by adding 7 . 5 μl 4 % ( w / v ) formaldehyde and the makler chamber was loaded with a 7 . 5 μl subsample . the lid of the makler chamber was placed in position and touched gently with the tip of a fingernail to ensure that it rested firmly on the pillars ( newton rings ). a total of 25 squares was counted under phase contrast microscopy using 200 × magnification . after counting , the chamber was washed in distilled water , dried and reloaded with a subsample from the same sample . subsequently , this procedure was applied for the second subsample and a total of 4 measurements ( 2 samples × 2 replicates ) was obtained per ejaculate . a 20 μl sample of the raw semen was diluted to a total volume of 4 ml ( 1 : 200 ) in a cuvette for the l &# 39 ; aiglon spectrophotometer . dilution was done with a cavro autodiluter ( z 069 , imv ). following dilution , a small piece of foil was placed over the cuvette and the diluted sample was mixed gently by hand . after resting approximately 10 sec ., the cuvette was placed in the spectrophotometer and the sperm concentration was measured . the cuvette was again covered with foil and mixed gently by hand , and after a resting period the cuvette was once more measured in the spectrophotometer . following this measurement , a second sample was diluted and measured . thus , a total of 4 measurements was obtained per ejaculate . a two - step dilution is necessary in order to count sperm concentration on the sysmex f - 820 particle counter . the first step was dilution of 20 μl raw semen to a volume of 10 ml ( 1 : 500 ) and in the second step , 100 μl of the diluted semen was diluted to a total volume of 10 ml and placed in a sample - cup ( 1 : 100 , resulting in a combined dilution of 1 : 50 , 000 ). both dilution steps were performed with an ad - 270 autodiluter ( sysmex , almind , denmark ). the sample - cup was covered with a small piece of foil and mixed gently by hand and rested for approximately 10 sec . before counting on the sysmex . after counting , the sample - cup was again covered with foil , mixed and counted once more . the sample was then discarded and a second sample was diluted , treated and counted according to this procedure . a total of 4 counts was obtained per ejaculate . a two - step dilution is necessary for the flow cytometric analyses . first , the sample is diluted 250 × in a pbs medium containing 0 . 1 % bsa , ( nunc intermed cat # 3478880 , lifetechnologies , roskilde , denmark ), and second a 20 μl sample is transferred to a sperm count tube containing approximately 100 , 000 beads using reverse manual pipetting . the dyes were added to the sperm count tubes less than 12 hours before using the tubes in the analysing procedure . as mentioned above , two different flow cytometers were used , one being a facscount equipped with a green ( 543 nm ) laser and the second being a facscount equipped with a blue ( 488 nm ) laser . to each sperm count tube to be analysed in the first facscount ( 543 nm ), 2 μl of mpr71292 and 2 μl of ehd - 2 were added to provide final concentrations of 100 nm and 5 μm , respectively . to each sperm count tube to be analysed in the second facscount ( 488 nm ), 5 μl of sybr - 14 and 5 μl of pi were added to provide for final concentrations of 50 nm and 12 μm , respectively . prior to opening the sperm count tubes , the tubes were whirlmixed for 3 sec in an upside - down position and for 3 sec in a normal position . whirlmixing were repeated for two sec after addition of the sperm and before each analysis . after addition of sperm , the sperm count tubes were placed on a mixer ( adams nutator , n . c . nielsen , brøndby , denmark ) for 4 min at room temperature with no light reaching the tube . after 4 min , the first replicates were analysed simultaneously on the two flow cytometers and the second replicates were analysed after a 6 min staining period . tubes were analysed twice to provide for a second replicate rather than pipetting two subsamples from the tubes . a total of 4 analysis was carried out on each flow cytometer per ejaculate . during the flow cytometric analyses , data were stored on a floppy disk in the facscount , and after the measurements , data from the floppy disk were analysed using attractor ™ ( bd ) software at the royal veterinary and agricultural university , section for reproduction . data obtained from the five methods ( makler chamber , spectrophotometer ( l &# 39 ; aiglon ), particle counter ( sysmex ), and flow cytometers ( facscount with green or blue laser ) were subject to analyses of variance to point out significant effects of date , bull , sample , replicate , combinations of these effects as well as residual variation . viability data from the facscount analyses were also subject to analyses of variance . the following statistical model ( 1 ) was used : ( 1 ) y i = α i , date + b i , bull + c i , bull * date + d i , date * bull * sample + ε i y 1 = result for the method α i , date = effect of date ( systematic ) b i , bull = effect of bull c i , bull * date = effect of ejaculate d i , date * bull * sample = effect of sample ε i = residual error i = the number of observation ( measurement ), 1 , . . . 208 capital letters in the above model indicate random effects . however , estimates for overall sample and replicate differences were made in a statistical model with systematic effect of sample , replicate and date . data from the four methods ( spectrophotometer , particle counter , flow cytometer with green or blue laser ) were subject to regression analyses to compare the results of these methods with results obtained with the makler chamber . regression analyses were based on average values for the four measurements per ejaculate for each of the methods . at first , regression analyses were made without corrections , but due to the large variation of the makler chamber data , regression analyses were also made using a measurement error model which takes the imprecision of the makler chamber into account . all statistical analyses were performed with sas version 6 . 12 ( sas , 1990 ). as can be seen from fig3 – 6 showing the regression lines for the results from each of the four methods against the manual counting from the makler chamber , the concentration of the sperm cells vary from 200 * 10 6 to 3000 * 10 6 . this variation is a variation between bulls as well as a variation between ejaculates from each bull as also indicated in the statistical method above . the data obtained with the makler chamber appeared more imprecise than anticipated from earlier studies and the coefficient of variation was 21 . 3 %. the results of the analyses of variance for data obtained with the spectrophotometer are shown in table 1 . the instrumental coefficient of variation ( cv %) is 2 . 10 % and the large variation between samples within ejaculates ( sample variation ) make the method significantly more imprecise , and the 95 % confidence interval for determination of sperm concentration with the l &# 39 ; aiglon spectrophotometer is ± 169 × 10 6 sperm / ml when a single measurement is made ( one sample , one measurement ). a small overall variation in concentration was detected between the two samples with sample 1 being 48 . 5 ± 15 × 10 6 sperm / ml lower than sample 2 . the reason for this difference is unclear . table 1 also shows the results for the sysmex particle counter . the coefficient of variation with this method is high , 6 . 1 %. in the measurements two sets of counts were used where the discrimination on particle size differed in the two replicates , respectively , in - and excluding a population of smaller particles in the “ sperm count ” ( see fig2 a and 2b ). if these observations are excluded , the coefficient of variation for the sysmex is 4 . 0 %. the sample variation with the sysmex particle counter is small and the 95 % confidence interval of the method for a single measurement per ejaculate is ± 118 × 10 6 sperm / ml ( two sets of counts mentioned above are excluded in this calculation , if included the 95 % confidence interval is ± 182 × 10 6 sperm / ml ). table 1 also shows the results for the sysmex particle counter . the coefficient of variation with this method is high , 6 . 1 %. in the measurements two sets of counts were used where the discrimination on particle size differed in the two replicates , respectively , in - and excluding a population of smaller particles in the “ sperm count ” ( see fig1 and 2 ). if these observations are excluded , the coefficient of variation for the sysmex is 4 . 0 %. the sample variation with the sysmex particle counter is small and the 95 % confidence interval of the method for a single measurement per ejaculate is ± 118 × 10 6 sperm / ml ( two sets of counts mentioned above are excluded in this calculation , if included the 95 % confidence interval is ± 182 × 10 6 sperm / ml ). the results of the facscount with green laser is shown in table 1 . the coefficient of variation is 4 . 3 % and the 95 % confidence interval for a single measurement per ejaculate is ± 182 × 10 6 sperm / ml . it should be noted that no significant differences are observed between replicates or samples . the results of the facscount with blue laser ( table 1 ) have a higher coefficient of variation ( 6 . 0 %) but due to less variation between samples the 95 % confidence interval for a single measurement per ejaculate is ± 184 × 10 6 sperm / ml . a higher sperm concentration was observed for replicate 2 ( 54 . 7 ± 11 × 10 6 sperm / ml higher than replicate 1 ). the reason for this difference was unclear . the results for determination of sperm viability with facscount ( green laser ) are shown in table 2 . coefficient of variation for the method is 1 . 2 %. the 95 % confidence interval for determination of sperm viability based on one measurement per ejaculate is ± 4 . 0 %. a small decrease in sperm viability over time was observed (− 0 . 27 ± 0 . 07 %/ min ). this decrease is much less than decreases observed in trials using a staining time of more than 10 minutes . with a staining time of 3 – 5 minutes , the accuracy of the method is only affected slightly . the results of the facscount ( blue laser ) for determination of sperm viability are shown in table 2 . the coefficient of variation is equal ( cv = 1 . 1 %) to that of the facscount with green laser but due to a slightly larger sample variation , the 95 % confidence interval for a single measurement per ejaculate is 6 . 2 %. sperm viability did not decrease over time (− 0 . 15 ± 0 . 07 %/ min ) which are a likely effect of the reduced concentration of the sybr - 14 stain ( 50 nm )). the regression lines for the four methods after correction for imprecision of the “ golden standard ” are shown in fig3 to 6 and the results of the regression analyses are shown in table 3 . the concentrations of sperm cells in the semen samples vary from 200 * 10 6 to 3000 * 10 6 sperm cells pr . ml . the slopes of all regression lines are not significantly different from 1 and the intercepts for the different regression lines are not significantly different from 0 . it appears from table 3 , that the error of the facscount analyses are higher than for the spectrophotometer and particle counter . correlation coefficients from the regression analyses without correction showed a slightly lower correlation for the analyses with facscount where the correlation for respectively the green and blue laser were 0 . 91 and 0 . 9 versus 0 . 945 for particle counter and 0 . 96 for spectrophotometer . this is in contrast to results obtained earlier where the correlation coefficients for respectively particle counter , spectrophotometer and facscount were 0 . 93 , 0 . 93 and 0 . 97 . in theory , the correlation for the facscount ( either laser ) should be higher than those of the other methods because the flow cytometric method identifies sperm from a staining of dna . however , since bull semen contains a very little amount of debris , the difference between the results obtained using flow cytometry and the results obtained using other techniques is small . sperm viability : the two methods for determination of sperm viability are highly accurate and only for the combination of 292 and ehd2 a slight decrease in viability (− 0 . 27 ± 0 . 07 %/ min ) was observed . since analyses can be performed after a 2½ min staining time , viability can be determined precisely as well as accurately . it may be desirable to stain two samples per ejaculate since sample difference is a significant source of variation . with two samples per ejaculate , the 95 % confidence interval for the determination of sperm viability will be from 2 . 8 % ( green laser ) to 4 . 4 % ( blue laser ). using the described techniques , sperm viability can be determined with a far higher degree of precision and accuracy than possible with microscopic evaluation of sperm motility . this implies that the fertility of a semen sample can be predicted more accurately from sperm viability data . the correlation between sperm viablity and fertility is demonstrated in experiment 3 sperm concentration : determination of sperm concentration with facscount ( either laser ) is in the present investigation very close to the results achieved using the particle counter ( sysmex ) or the spectrophotometer ( l &# 39 ; aiglon ) and the correlation between facscount methods are very high ( fig7 ). in both flow cytometric methods 5000 events were sampled per analysis , and it is believed that still better results may be obtained by a sampling of 10000 events . a significant source of variation for the flow cytometric determination of sperm concentration is the difference between samples . this difference relates both to true difference between samples and to difference between sperm count tubes . the variation between samples may be reduced by applying a fully automated dilution procedure thereby reducing the operator dependent factor to a minimum . furthermore , the results of the method may be improved by a determination of the sperm concentration for an ejaculate based on a mean value obtained from analyses of two sperm count tubes . thereby , also the influence of variation in bead number between individual sperm count tubes could be reduced , and furthermore the sperm viability would be determined more precisely if it was based on two samples . in a second experiment , sperm concentration and viability in boar ejaculates have been determined according to a preferred embodiment of the present invention . live and dead sperm cells in boar semen samples have been stained selectively , and subsequently the semen samples have been analysed with facscount flow cytometers equipped with either a 488 nm or a 543 nm laser . a total of 58 ejaculates was analysed on each of the two flow cytometers and on a spectrophotometer ( corning 254 ). furthermore , the sperm were counted in a thoma haemacytometer using phase contrast microscopy at 200 × magnification . the results of this experiment show that determination of sperm concentration with the two flow cytometric techniques results in a higher precision and accuracy than obtained with the corning 254 spectrophotometer . gel - particles , present in large number in boar semen , increase the turbidity of boar semen and make spectrophotometric measurements unreliable . it is a significant advantage of the flow cytometric technique that it provides for a more precise and accurate sperm count and simultaneously provides for a precise determination of sperm viability . a total of 58 boar ejaculates has been analysed . two subsamples from each ejaculate were diluted 250 × in a pbs medium containing 0 . 1 % bsa and a 60 μl aliquot was transferred to a sperm count tube . the staining times for sybr - 14 / pi as well as for mpr71292 / ehd2 were 4 mins and the tubes were subsequently analyzed on two facscount flow cytometers equipped with a 488 nm laser and a 543 nm laser , respectively . the 58 ejaculates were collected from 40 boars . from each ejaculate a 3 ml aliquot was taken and placed in a tube ( nunc intermed cat # 347880 , lifetechnologies , roskilde , denmark ) and placed on a swelab - 820 mixer ( bie & amp ; berntsen , rødovre , denmark ) and mixed continuously . all the subsequent analyses were carried out within 30 mins after semen collection . raw semen was diluted in a 5 % w / v sodium - chloride solution . the dilution ratio varied according to the sperm concentration determined using the spectrophotometric method in order to achieve an appropiate number of sperm cells in the thoma haemacytometer . thus , the most concentrated samples were diluted 72 ×, whereas the ‘ thin ’ samples only were diluted 16 ×. samples were placed on a mixer ( adams nutator , n . c . nielsen , brøndby , denmark ) during counting . the lid of the thoma haemacytometer was placed in position on the two pillars ( newton rings ) and the two counting areas were filled with a capillary tube . in each half of the counting chamber , 5 large fields of each 6 small squares were counted . the counting chamber was filled twice by each of the two persons involved in the counting . for spectrophotometric determination of sperm concentration , 0 . 25 ml of raw semen was diluted with 9 . 75 ml edta solution in a cuvette . the sperm concentration was subsequently measured and the cuvette was mixed gently by hand and re - measured . a subsequent sample was treated likewise , thus , 4 measurements were made per ejaculate . the dilution of samples for the flow cytometric analyses were performed as described for bull semen ( experiment 1 ), apart from a volume of 60 μl of the 250 × diluted sample being transferred to each sperm count tube using reverse manual pipetting . the dye concentrations and the protocol of analysis are identical to those mentioned above . the coefficient of variation for the flow cytometric determination of sperm concentration were 3 . 2 % and 3 . 1 % for facscount flow cytometers equipped with a 488 nm laser and a 543 nm laser , respectively . for comparison , the coefficient of variation for the spectrophotometric measurements were 5 . 9 % indicating a larger imprecision of this technique . furthermore , the data for the spectrophotometric measurements was more randomly distributed around the regression line than the data obtained using the flow cytometric measurements . in the measurement error model , the standard deviation was 40 for the spectrophotometric method which should be compared to a standard deviation of 20 for the results obtained using the two flow cytometers . the coefficient of variation for the determination of sperm viability was 1 . 4 % for both flow cytometric techniques . determination of sperm concentration in boar semen is difficult due to a large amount of gel - particles making the spectrophotometric readings inaccurate . likewise , the results obtained using particle counters , such as the sysmex used for bull semen , will provide inaccurate readings due to the large amount of gel - particles with a size corresponding to the size of the spermatozoa , and furthermore , the flow system of a particle counter is often obstructed by large gel - particles also present in the boar semen . the determination of sperm concentration using flow cytometric methods is significantly more accurate and precise than spectrophotometric measurements and , furthermore , obstruction of the flow system due to large particles is a rare event . the high precision of the flow cytometric methods when used on boar semen is explained by the large amount of gel particles present in boar semen , making the prior art methods highly inaccurate . still further , the flow cytometric method provides an objective and precise determination of sperm viability , the method being significantly more precise than conventional , subjective microscopic assessment of sperm motility . an important application of the flow cytometric methods described above is the possibility to predict the fertility according to the predetermined sperm concentration and sperm viability . to assess the value of this technique for the selection of semen samples for artificial insemination ( ai ), a large scale fertility trial with bull semen have been performed . in the present experiment there has been used sybr - 14 in a concentration of 50 nm and pi in a concentration of 12 μm . four ejaculates from each of 157 bulls ( holstein or jersey breeds ) have been analyzed fresh and frozen - thawed using flow cytometric methods and computer assisted sperm analysis ( casa ). furthermore , the fresh semen was subjected to morphological evaluation of the semen , and standard routine evaluation of the semen samples were carried out in the involved bull stations . a total of 121 . 232 inseminations with insemination doses provided from the 4 × 157 ejaculates were performed in danish cattle herds . preliminary data from this extensive trial shows that the flow cytometric determination of sperm viability explains 30 to 50 % of the variation in fertility after insemination . in contrast , routine evaluation only explained approximately 10 % of this variation and computerized determination of sperm motility was only slightly better than the microscopic evaluation used rutinely . in conclusion , the flow cytometric determination of sperm viability allows a more precise selection of semen samples for ai and this technique is of great value to cattle breeders worldwide . semen from a total of 157 bulls were collected 4 times each with an interval of one week . semen volume was assessed and the sperm concentration was determined using particle counter . sperm motility and wave motion was determined using a phase contrast microscope at 200 × and 100 × magnification , respectively . morphological evaluation was performed on two eosin - nigrosin stained smears per ejaculate . computer analysis of sperm motility were performed with a htm ivos system ( christensen and stryhn 1997 ). to avoid time - dependent bias , 20 fields were acquired randomly in two makler chambers . flow cytometric analyses were performed using modified facscount flow cytometers . in total , 121 . 232 inseminations were performed randomly in danish cattle herds . fertility data and data from the semen analyses were analyzed using mixed linear models . previous studies have shown a limited value of the subjective evaluation of sperm motility with regard to prediction of fertility of a semen sample . staining of bull sperm with sybr - 14 and pi and subsequent analysis on a facscount flow cytometer as performed according to a preferred embodiment of the present invention , provides an objective and precise determination of viability , and furthermore a precise determination of sperm concentration . as described above , this method allows for simultaneous detection of live , dead and dying sperm , and preliminary data from the first statistical anlyses show a high correlation to fertility in vivo . approximately 30 to 50 % of the variation in fertility after insemination can be explained from the viability parameter . in contrast , microscopic routine evaluation of sperm motility only explains approximately 10 % of the variation . sperm viability determined using flow cytometric methods represents the first technique that makes it possible to improve fertility significantly by culling poor semen samples in the lab of a bull station . because of the high precision of this technique , it is possible to point out ejaculates where an unacceptable result after insemination can be anticipated . such ejaculates can therefore be discarded and the overall fertility can be improved . the same correlation appears likely for a range of different species , but the precise value of the technique can only be clarified in fertility trials for each species / breed . at present , such a trial is in progress with boar semen . donoghue a m , garner d l , donoghue d j , johnson l a , 1995 : viability assessment of turkey sperm using fluorescent staining and flow cytometry . poultry sci , 74 : 1191 – 1200 . donoghue a m , thistlethwaite d , donoghue d j , kirby j d , 1996 : a new method for rapid determination of sperm concentration in turkey semen . poultry sci ., 75 : 785 – 789 . dumont p , coupet h , gary f , 1996 : some aspects of evaluation of semen quality and processing in france . proc . 8th european ai - 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