male labrador retrievers ( n=3 ) in our breeding colony were used in this study .
five hundred 
 microliters of ejaculate was placed into an eppendorf tube ( 1.7 ml , 17401 , sorenson 
 bioscience , salt lake city , ut , usa ) , and then centrifuged for 5 min at 3,000 rpm .
the 
 supernatant was removed , and 1 ml of 0.01 m pbs ( phosphate buffered saline ; 0.35 g / l 
 nah2po4 , 1.28 g / l na2hpo4 , 8.00 g / l nacl , 
 wako , tokyo , japan ) was added .
the suspension obtained was thoroughly mixed using a 
 pipette and subsequently centrifuged for 5 min at 3,000 rpm .
the supernatant was removed , 
 and the pellet was resuspended with 1 ml of 0.01 m pbs .
the washing procedures were 
 repeated twice . the sperm suspension with 200 l of 0.01 m pbs
was fixed by addition of 
 400 l of 3:1 methanol : acetic acid fixative and left at room temperature for 10 min .
then , 
 the tube containing sperm suspension was filled with the fixative , and then centrifuged 
 for 5 min at 3,000 rpm .
the sperm concentration was adjusted to 5  10 
 cells / ml , and 10 l of the suspension was then placed on a defatted glass slide .
the concentration of sperm on the slide was 
 examined using a light microscope ( alphaphot-2 ys2 , nikon , tokyo , japan ) .
when the sperm 
 density was unsatisfactory or the sample was contaminated with dust , the suspension was 
 diluted , concentrated or washed with the fixative and centrifuged to get an appropriate 
 concentration .
preparations were dehydrated on a hot plate at 70 c for 60 min 
 ( hardening ) .
for decondensation of sperm nuclei , the specimen was treated with 1 m naoh for 3 , 4 or 5 
 min at room temperature , rinsed with pbs for 2 min , and then washed with distilled water 
 several times .
the specimen was dehydrated by running it through an ethanol concentration 
 series ( 70% for 5 min and 100% for 30 min ) .
after air - drying , the specimen was placed on a 
 hot plate at 70 c for 60 min . for a single staining of the y - chromosome of canine sperm , 5 l of y chromosome probe 
 labeled with digoxigenin and salmon sperm dna , canine suppression dna , and 50% 
 formamide/10% dextran sulfate/2
x ssc mixture ( doy-10 , chromosome science laboratory , 
 sapporo , japan ) was added to the specimen , and then covered with a cover glass .
the 
 preparation was transferred onto a hot plate set at 70 c for 5 min .
the edges of the 
 preparation were sealed with rubber cement ( sagisaka , tokyo , japan ) .
thereafter , the cover glass was removed , and the specimen 
 was washed several times with 50% formamide in 2xssc at 37 c for 20 min and with 1xssc 
 alone at room temperature for 15 min .
hybridization of the digoxigenin - labelled y probe 
 was visualized with anti - digoxigenin cy3 ( chromosome science laboratory ) .
the slides were 
 counterstained with dapi ( 4 ' , 6-diamino-2-phenylindole , dihydrochloride , d1306 , 
 invitrogen , carlsbad , ca , usa ) and mounted with antifade solution ( afm-01 , chromosome 
 science laboratory ) . for double staining of x and y chromosome in canine spermatozoa ,
sex chromosome probes 
 x - labelled with spectrumgreen and y - labelled with cy3 ( dxy-10 , chromosome science 
 laboratory ) were used . the decondensation condition of sperm nuclei was selected according 
 to the results of a single staining experiment .
sperm samples were analyzed using a fluorescence microscope ( eclipse 80i , nikon ) equipped 
 with dapi ( uv-2a , excitation 380420 nm , absorption 450 nm ) , spectrumgreen ( b2-a , 
 excitation 450490 nm , absorption 520 nm ) and cy3 ( g-2a , excitation 510560 nm , absorption 
 590 nm ) filters ( nikon ) .
digital images were acquired by a peltier - cooled ccd camera 
 ( ds-5mc - l1 , nikon ) using a software for digital photo editing ( paint.net v3.5.10 ) .
fish 
 signals were analyzed visually using an ocular grid by counting random microscope fields 
 and scoring 500600 spermatozoa per sample .
spermatozoa were scored only if they were 
 intact and non - overlapped , had a clearly defined border and had not decondensed to more 
 than twice the size of a non - decondensed sperm head , which could produce large and 
 sometimes fragmented fish signals .
semen samples were collected as described above , and seminal plasma was removed by 
 centrifugation at 2000 rpm for 15 min .
the sorted spermatozoa were obtained following the 
 general procedure described by schenk et al .
briefly , spermatozoa 
 were diluted with talp   at a sperm 
 concentration of 2  10 cells / ml , and then stained with hoechst 33342 
 ( 81.0113.4 m , h3570 , invitrogen ) and incubated for 3540 min at 35 c. an equal volume of 
 talp containing 2.67% of purified egg yolk and 0.002% of food red ( fd & c # 40 , cas 
 25856 - 17 - 6 , invitrogen ) was added to the sperm suspension .
the stained spermatozoa were sorted with a flow cytometer ( moflo - sx , 
 dakocytomation , fort collins , co , usa ) operating at 40 psi and with a laser power of 175 
 mw .
all experiments were carried out in accordance with the guidelines for the care and use 
 of animals approved by obihiro university of agriculture and veterinary medicine .
a chi - squared test was used to investigate the deviation from the expected ratio of 50:50 
 ( x : y ) .
male labrador retrievers ( n=3 ) in our breeding colony were used in this study .
five hundred 
 microliters of ejaculate was placed into an eppendorf tube ( 1.7 ml , 17401 , sorenson 
 bioscience , salt lake city , ut , usa ) , and then centrifuged for 5 min at 3,000 rpm .
the 
 supernatant was removed , and 1 ml of 0.01 m pbs ( phosphate buffered saline ; 0.35 g / l 
 nah2po4 , 1.28 g / l na2hpo4 , 8.00 g / l nacl , 
 wako , tokyo , japan ) was added .
the suspension obtained was thoroughly mixed using a 
 pipette and subsequently centrifuged for 5 min at 3,000 rpm .
the supernatant was removed , 
 and the pellet was resuspended with 1 ml of 0.01 m pbs .
the washing procedures were 
 repeated twice . the sperm suspension with 200 l of 0.01 m pbs
was fixed by addition of 
 400 l of 3:1 methanol : acetic acid fixative and left at room temperature for 10 min .
then , 
 the tube containing sperm suspension was filled with the fixative , and then centrifuged 
 for 5 min at 3,000 rpm .
the sperm concentration was adjusted to 5  10 
 cells / ml , and 10 l of the suspension was then placed on a defatted glass slide .
the concentration of sperm on the slide was 
 examined using a light microscope ( alphaphot-2 ys2 , nikon , tokyo , japan ) .
when the sperm 
 density was unsatisfactory or the sample was contaminated with dust , the suspension was 
 diluted , concentrated or washed with the fixative and centrifuged to get an appropriate 
 concentration .
preparations were dehydrated on a hot plate at 70 c for 60 min 
 ( hardening ) .
for decondensation of sperm nuclei , the specimen was treated with 1 m naoh for 3 , 4 or 5 
 min at room temperature , rinsed with pbs for 2 min , and then washed with distilled water 
 several times .
the specimen was dehydrated by running it through an ethanol concentration 
 series ( 70% for 5 min and 100% for 30 min ) .
after air - drying , the specimen was placed on a 
 hot plate at 70 c for 60 min .
for a single staining of the y - chromosome of canine sperm , 5 l of y chromosome probe 
 labeled with digoxigenin and salmon sperm dna , canine suppression dna , and 50% 
 formamide/10% dextran sulfate/2 x ssc mixture ( doy-10 , chromosome science laboratory , 
 sapporo , japan ) was added to the specimen , and then covered with a cover glass . the 
 preparation was transferred onto a hot plate set at 70 c for 5 min .
the edges of the 
 preparation were sealed with rubber cement ( sagisaka , tokyo , japan ) .
thereafter , the cover glass was removed , and the specimen 
 was washed several times with 50% formamide in 2xssc at 37 c for 20 min and with 1xssc 
 alone at room temperature for 15 min .
hybridization of the digoxigenin - labelled y probe 
 was visualized with anti - digoxigenin cy3 ( chromosome science laboratory ) .
the slides were 
 counterstained with dapi ( 4 ' , 6-diamino-2-phenylindole , dihydrochloride , d1306 , 
 invitrogen , carlsbad , ca , usa ) and mounted with antifade solution ( afm-01 , chromosome 
 science laboratory ) . for double staining of x and y chromosome in canine spermatozoa , sex chromosome probes 
 x - labelled with spectrumgreen and y - labelled with cy3 ( dxy-10 , chromosome science 
 laboratory ) were used . the decondensation condition of sperm nuclei was selected according 
 to the results of a single staining experiment .
sperm samples were analyzed using a fluorescence microscope ( eclipse 80i , nikon ) equipped 
 with dapi ( uv-2a , excitation 380420 nm , absorption 450 nm ) , spectrumgreen ( b2-a , 
 excitation 450490 nm , absorption 520 nm ) and cy3 ( g-2a , excitation 510560 nm , absorption 
 590 nm ) filters ( nikon ) .
digital images were acquired by a peltier - cooled ccd camera 
 ( ds-5mc - l1 , nikon ) using a software for digital photo editing ( paint.net v3.5.10 ) .
fish 
 signals were analyzed visually using an ocular grid by counting random microscope fields 
 and scoring 500600 spermatozoa per sample .
spermatozoa were scored only if they were 
 intact and non - overlapped , had a clearly defined border and had not decondensed to more 
 than twice the size of a non - decondensed sperm head , which could produce large and 
 sometimes fragmented fish signals .
semen samples were collected as described above , and seminal plasma was removed by 
 centrifugation at 2000 rpm for 15 min .
the sorted spermatozoa were obtained following the 
 general procedure described by schenk et al .
briefly , spermatozoa 
 were diluted with talp   at a sperm 
 concentration of 2  10 cells / ml , and then stained with hoechst 33342 
 ( 81.0113.4 m , h3570 , invitrogen ) and incubated for 3540 min at 35 c. an equal volume of 
 talp containing 2.67% of purified egg yolk and 0.002% of food red ( fd & c # 40 , cas 
 25856 - 17 - 6 , invitrogen ) was added to the sperm suspension .
the stained spermatozoa were sorted with a flow cytometer ( moflo - sx , 
 dakocytomation , fort collins , co , usa ) operating at 40 psi and with a laser power of 175 
 mw . control samples consisted of unsorted spermatozoa from the same dogs . all experiments were carried out in accordance with the guidelines for the care and use 
 of animals approved by obihiro university of agriculture and veterinary medicine .
a chi - squared test was used to investigate the deviation from the expected ratio of 50:50 
 ( x : y ) .
 abstract effective preselection of sex has been accomplished in several species of livestock and 
 also in humans using the flow cytometric sperm sorting method . 
 a guaranteed high sorting 
 accuracy is a key prerequisite for the widespread use of sperm sexing . 
 the standard 
 validation method is flow cytometric remeasurement of the dna content of the sexed sperm . 
 since this method relies on the same instrument that produced the original sperm 
 separation , 
 it is not truly independent . therefore , to be able to specifically produce 
 either male or female offspring in the dog , we developed a method of direct visualization 
 of sex chromosomes in a single sperm using fluorescence in situ 
 hybridization ( fish ) as a validation method . 
 denaturation of canine spermatozoa by 
 immersion in 1 m naoh for 4 min yielded consistent hybridization results with over 97% 
 hybridization efficiency and a good preservation of sperm morphology . 
 there was no 
 significant difference between the theoretical ratio ( 50:50 ) and the observed ratio of x- 
 and y - chromosome - bearing spermatozoa in any of the three dogs . 
 in addition , the mean 
 purities of flow - sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for 
 the x chromosome fraction and 89.6% for the y chromosome fraction . 
 this sorting was 
 evaluated by using the dual color fish protocol . 
 therefore , our results demonstrated that 
 the fish protocol worked reliably for both unsorted and sexed sperm samples . 
Methods
Animals and semen preparation
Decondensation
FISH
Fluorescence microscopy and quantitative evaluation
Flow cytometric sperm sorting
Statistical analysis