Apparatus and method for producing high purity x-chromosome bearing and/or y-chromosome bearing populations of spermatozoa

Abstract

A flow cytometer apparatus and method for differentiating X-chromosome bearing sperm and Y-chromosome bearing sperm having differing amounts of a light emission material coupled to nuclear their DNA. The apparatus may include an irradiation source which generates an irradiation beam responsive to the light emission material coupled to stained sperm cells, optics to focus the irradiation beam to a beam pattern having a height about equal to the length of said sperm cells along the longitudinal axis to about three times the length of said sperm cells along the longitudinal axis, and a detector responsive to said light emitted from said light emission material in response to the irradiation beam. The method may include the steps of establishing a fluid stream, producing an irradiation beam directed at the fluid stream, shaping the irradiation beam to have a beam height between about equal to the length of said sperm cells along the longitudinal axis and about three times the length of said sperm cells along the longitudinal axis at the fluid stream, and detecting emissions from sperm cells passing through the beam pattern.

We claim: 
     
         1 . A flow cytometer for differentiating sperm cells, wherein the sperm cells have an associated light emission material coupled to nuclear DNA and wherein the light emissions material stains X-chromosome bearing sperm and Y-chromosome bearing sperm in differing amounts comprising:
 a) an irradiation source which generates an irradiation beam responsive to the light emission material coupled to stained sperm cells in a fluid stream;   b) optics to focus the irradiation beam, wherein said optics focus a beam pattern having a height of about equal to the length of said sperm cells along the longitudinal axis to about three times the length of said sperm cells along the longitudinal axis; and   c) a detector responsive to said light emitted from said light emission material in response to the irradiation beam.   
     
     
         2 . The flow cytometer as claimed in  claim 1 , wherein the optics focus the beam pattern to have a height of between about 9 micrometers and about 20 micrometers. 
     
     
         3 . The flow cytometer as claimed in  claim 1 , wherein the optics focus the beam pattern to have a height of about 20 micrometers. 
     
     
         4 . The flow cytometer as claimed in  claim 1 , wherein the optics focus the beam pattern to have a width of about 160 micrometers. 
     
     
         5 . The flow cytometer as claimed in  claim 1 , wherein the detector comprises a first detector located at 0 degrees and a second detector located at 90 degrees relative to the path of the irradiation beam. 
     
     
         6 . The flow cytometer as claimed in  claim 1 , wherein said detector is deferentially responsive to said light emitted from said light emission material based upon said particle orientation characteristics. 
     
     
         7 . The flow cytometer as claimed in  claim 1 , further comprising an analyzer coupled to said detector. 
     
     
         8 . The flow cytometer as claimed in  claim 7 , wherein the analyzer differentiates between said sperm cells based upon orientation of said sperm cells within said fluid stream. 
     
     
         9 . The flow cytometer as claimed in  claim 7 , further comprising a droplet charger coupled to said analyzer, wherein said droplets receive a charge differentially based upon said difference in amount of said stain bound the nuclear DNA of X-chromosome sperm cells and the nuclear DNA of Y-chromosome bearing sperm cells. 
     
     
         10 . The flow cytometer as described in  claim 7 , further comprising a droplet separator, wherein said droplet separator separates said droplet based upon charge of said droplet. 
     
     
         11 . The flow cytometer as claimed in  claim 10 , further comprising at least one collection container in which droplets containing said X-chromosome bearing sperm cells are collected as an X-chromosome bearing population. 
     
     
         12 . The flow cytometer as claimed in  claim 10 , further comprising at least one collection container in which droplets containing Y-chromosome bearing sperm cells are collected as a Y-chromosome bearing population. 
     
     
         13 . The flow cytometer as claimed in  claim 1 , wherein said sperm cells are selected from a group consisting of bovine sperm cells, equine sperm cells, and ovine sperm cells. 
     
     
         14 . A method of sorting sperm having an associated light emission material coupled to nuclear DNA which stains X-chromosome bearing sperm and Y-chromosome bearing sperm in differing amounts comprising:
 a) establishing a fluid stream;   b) producing an irradiation beam directed at the fluid stream;   c) shaping the irradiation beam to have a beam height between about equal to the length of said sperm cells along the longitudinal axis and about three times the length of said sperm cells along the longitudinal axis at the fluid stream; and   d) detecting emissions from sperm cells passing through the beam pattern.   
     
     
         15 . The method as claimed in  claim 14 , wherein the irradiation beam is shaped to a beam pattern to having a height of between about 9 micrometers and about 20 micrometers. 
     
     
         16 . The method as claimed in  claim 14 , wherein the irradiation beam is shaped to beam pattern to having a height of about 20 micrometers. 
     
     
         17 . The method as claimed in  claim 14 , wherein the irradiation beam is shaped to beam pattern to having a width of about 160 micrometers. 
     
     
         18 . The method as claimed in  claim 14 , wherein the step of detecting emissions comprises detecting emissions at 0 degrees position relative to the path of the irradiation beam and detecting emissions at a 90 degrees position relative to the path of the irradiation beam. 
     
     
         19 . The method as claimed in  claim 14 , wherein the light emitted from said light emission material varies based upon said particle orientation characteristics. 
     
     
         20 . The method as claimed in  claim 19 , further comprising the step of analyzing the light emitted from the light emissions materials to determine the orientation of sperm cells. 
     
     
         21 . The method as claimed in  claim 20 , further comprising the step of analyzing the light emitted from the light emissions materials to differentiate X chromosome bearing sperm from Y chromosome bearing sperm. 
     
     
         22 . The method as claimed in  claim 21 , wherein the step of differentiating X chromosome bearing sperm from Y chromosome bearing sperm is performed on orientated sperm. 
     
     
         23 . The method as claimed in  claim 22 , wherein one or both of the detected X chromosome bearing sperm and Y chromosome bearing sperm are collected in collection containers.