Collection Systems for Cytometer Sorting of Sperm

Abstract

Improved flow cytometer system particularly adapted to use for sex-selected sperm sorting include enhanced sheath fluid and other strategies which minimize stress on the sperm cells, including a 2.9 percent sodium citrate sheath solution for bovine species and a hepes bovine gamete media for equine species. Improved collection systems and techniques for the process are described so that commercial applications of sperms samples as well as the resulting animals may be achieved.

1 . A process for storing unsorted spermatozoa, the process comprising 
 a. forming a sperm dispersion, the sperm dispersion comprising spermatozoa, a composition that induces sperm immotility, and an antibiotic, and    b. storing the sperm dispersion.    
     
     
         2 . A process for storing sorted spermatozoa, the process comprising 
 a. forming a sperm dispersion, the sperm dispersion comprising spermatozoa and a composition that induces sperm immotility,    b. sorting the sperm dispersion into separate populations, wherein the spermatozoa of one of the populations comprises at least about 65% X chromosome bearing sperm cells or at least about 65% Y chromosome bearing sperm cells, and    c. storing the one population at a temperature of about −4° C. to about 30° C.    
     
     
         3 . A process for inseminating a female mammal, the process comprising 
 inseminating a female mammal with a sperm dispersion, the sperm dispersion comprising immotile spermatozoa and a composition that induces sperm immotility.    
     
     
         4 . A process for providing a fresh sperm dispersion for inseminating a female mammal, the process comprising: 
 a. forming a sperm dispersion, the sperm dispersion comprising spermatozoa and a composition that induces sperm immotility,    b. placing the sperm dispersion in a container for shipment to a remote location, and    c. shipping the sperm dispersion in the container to a remote location within about 24 hours after forming the sperm dispersion.    
     
     
         5 . A combination comprising: 
 a. an elongated container for use in the insemination of a female mammal, and    b. a sperm dispersion, the sperm dispersion comprising immotile spermatozoa and a composition that induces sperm immotility, and wherein the sperm dispersion is contained in the elongated container.    
     
     
         6 - 54 . (canceled)  
     
     
         55 . A process for providing a fresh sperm dispersion for inseminating a female mammal, the process comprising: 
 a. forming a sperm collection, the sperm collection comprising spermatozoa and a composition that induces sperm immotility,    b. placing the sperm collection in a container for shipment to a remote location, and    c. shipping the sperm collection in the container to a remote location within about 24 hours after forming the sperm collection.    
     
     
         56 . A sperm cell suspension comprising viable spermatozoa, the spermatozoa having a motility more characteristic of epididymal sperm than endogenous ejaculated spermatozoa of the same species, the concentration of spermatozoa in the suspension being less than about 1×10 6  or at least about 1×10 8  spermatozoa per ml.  
     
     
         57 . A sperm cell suspension comprising viable, immotile sperm, the concentration of spermatozoa in the suspension being less than about 1×10 6  or at least about 1×10 8  spermatozoa per ml.  
     
     
         58 . A sperm cell suspension comprising viable sperm, a motility inhibiting amount of potassium, and sodium, the concentration of spermatozoa in the suspension being less than about 1×10 6  or at least about 1×10 8  spermatozoa per ml and the molar ratio of potassium to sodium being greater than 1:1, respectively.  
     
     
         59 . The suspension of  claim 58  wherein the molar ratio of potassium to sodium is greater than 1.25:1, respectively.  
     
     
         60 . The suspension of  claim 58  wherein the molar ratio of potassium to sodium is greater than 1.5:1, respectively.  
     
     
         61 . The suspension of  claim 58  wherein the molar ratio of potassium to sodium is greater than 1.75:1, respectively.  
     
     
         62 . The suspension of  claim 58  wherein the molar ratio of potassium to sodium is greater than 2:1, respectively.  
     
     
         63 . A sperm cell suspension comprising viable, immotile sperm and a DNA-selective dye.  
     
     
         64 . The suspension of  claim 63 , wherein the dye is a DNA selective fluorescent dye.  
     
     
         65 . The suspension of  claim 63 , wherein the dye is a UV excitable or a visible light excitable dye.  
     
     
         66 . The suspension of  claim 63 , wherein the dye is selected from the group consisting of Hoechst 33342, Hoechst 33258, SYBR-14, and bisbenzimide-BODIPY® conjugate 6-{[3-((2Z)-2-{[1-(difluoroboryl)-3,5-dimethyl-1H-pyrrol-2-yl]methylene}-2H-pyrrol-5-yl)propanoyl]amino}-N-[3-(methyl{3-[({4-[6-(4-methylpiperazin-1-yl)-1H,3′H-2,5′-bibenzimidazol-2′-yl]phenoxy}acetyl)amino]propyl}amino)propyl]hexanamide.  
     
     
         67 . The sperm suspension of  claim 57 , wherein the suspension comprises a source of carbonate.  
     
     
         68 . The sperm suspension of  claim 57 , wherein the suspension comprises NaHCO 3 , KHCO 3 , and C 6 H 8 O 7 ·H 2 O.  
     
     
         69 . The sperm suspension of  claim 57 , wherein the suspension is formed from a buffer comprising 0.097 moles/L of NaHCO 3 , 0.173 moles/L of KHCO 3 , 0.090 moles/L C 6 H 8 O 7 ·H 2 O in water.  
     
     
         70 . The sperm suspension of  claim 57 , wherein the concentration of spermatozoa in the suspension is at least 1.25×10 8  spermatozoa per ml.  
     
     
         71 . The sperm suspension of  claim 57 , wherein the concentration of spermatozoa in the suspension is at least 1.5×10 8  spermatozoa per ml.  
     
     
         72 . The sperm suspension of  claim 57 , wherein the concentration of spermatozoa in the suspension is at least 1.75×10 8  spermatozoa per ml.  
     
     
         73 . The suspension of  claim 57 , wherein the concentration of spermatozoa in the suspension is less than about 9.0.×10 5  spermatozoa per ml.  
     
     
         74 . The suspension of  claim 57 , wherein the concentration of spermatozoa in the suspension is less than about 7×10 5  spermatozoa per ml.  
     
     
         75 . The suspension of  claim 57 , wherein the concentration of spermatozoa in the suspension is less than about 5×10 5  spermatozoa per ml.  
     
     
         76 . The suspension of  claim 57 , wherein the concentration of spermatozoa in the suspension is less than about 2×10 5  spermatozoa per ml.  
     
     
         77 . The suspension of  claim 57 , wherein the concentration of spermatozoa in the suspension is less than about 1×10 5  spermatozoa per ml.  
     
     
         78 . A process for staining sperm cells, the process comprising forming a staining mixture containing intact viable sperm cells, a motility inhibiting amount of potassium, and a DNA selective dye.  
     
     
         79 . The process of  claim 78  wherein the staining mixture is under an atmosphere having an enriched partial pressure of CO 2  relative to air.  
     
     
         80 . A process of forming a sperm cell suspension for use in cell sorting, the process comprising combining a sperm cell source with a composition which inhibits the motility of sperm cells to form a sperm cell suspension, the concentration of sperm cells in the suspension being less than about 1×10 6  or at least 1×10 8  sperm cells per milliliter.  
     
     
         81 . The process of  claim 80 , wherein the cell suspension comprises a DNA-selective dye.  
     
     
         82 . The suspension of  claim 68 , further comprising a DNA-selective dye.  
     
     
         83 . A sperm cell suspension comprising viable, immotile spermatozoa, the spermatozoa having a DNA-selective dye associated with their DNA.  
     
     
         84 . The suspension of  claim 83 , wherein the dye is a UV excitable or a visible light excitable dye.  
     
     
         85 . The sperm suspension of  claim 83 , wherein the suspension comprises NaHCO 3 , KHCO 3 , and C 6 H 8 O 7 ·H 2 O.  
     
     
         86 . The sperm suspension of  claim 83 , wherein the suspension is formed from a buffer comprising 0.097 moles/L of NaHCO 3 , 0.173 moles/L of KHCO 3 , 0.090 moles/L C 6 H 8 O 7 ·H 2 O in water.  
     
     
         87 . A sperm cell suspension comprising viable spermatozoa and a composition which down-regulates carbohydrate uptake by the spermatozoa, the concentration of spermatozoa in the suspension being less than about 1×10 6  or at least about 1×10 8  spermatozoa per ml.  
     
     
         88 . A sperm cell suspension comprising viable sperm, potassium and optionally sodium, the concentration of spermatozoa in the suspension being less than about 1×10 6  or at least about 1×10 8  spermatozoa per ml and the molar ratio of potassium to sodium being greater than 1:1, respectively.