Use of a Composition which Regulates Oxidation/Reduction Reactions Intracellularly and/or Extracellularly in a Staining or Sorting Process

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 staining mixture comprising viable spermatozoa, a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly, and a DNA selective dye, the concentration of the composition in the staining mixture being greater than 50 μM when the composition is pyruvate.  
     
     
         2 . The staining mixture of  claim 1 , wherein the composition is selected from the group consisting of pyruvate, vitamin K, lipoic acid, glutathione, flavins, quinones, superoxide dismutase, superoxide dismutase mimics, and any combinations thereof.  
     
     
         3 . The staining mixture of  claim 2 , wherein the composition is selected from the group consisting of pyruvate, vitamin K, lipoic acid, and combinations thereof.  
     
     
         4 . The staining mixture of  claim 1 , wherein the composition comprises pyruvate at a concentration selected from the group consisting of about 2.5 mM, about 10 mM, about 15 mM, about 25 mM, and about 50 mM.  
     
     
         5 . The staining mixture of  claim 1 , wherein the composition comprises vitamin K at a concentration selected from the group consisting of about 10 μM, about 50 μM, about 75 μM, and about 100 μM.  
     
     
         6 . The staining mixture of  claim 1 , wherein the composition comprises lipoic acid at a concentration selected from the group consisting of about 0.1 mM, about 0.5 mM, about 0.75 mM, about 1.0 mM, and about 1.5 mM.  
     
     
         7 . The staining mixture of  claim 1 , wherein the DNA selective dye is a DNA selective fluorescent dye.  
     
     
         8 . The staining mixture of  claim 2 , wherein the DNA selective dye is a DNA selective fluorescent dye.  
     
     
         9 . The staining mixture of  claim 1 , wherein the dye is a UV excitable or a visible light excitable dye.  
     
     
         10 . The staining mixture of  claim 2 , wherein the dye is a UV excitable or a visible light excitable dye.  
     
     
         11 . The staining mixture  claim 1 , 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.  
     
     
         12 . The staining mixture  claim 2 , 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.  
     
     
         13 . A process for staining sperm cells, the process comprising forming a staining mixture containing intact viable sperm cells, a composition which regulates oxidation/reduction reactions intracellularly and/or extracellularly, and a DNA selective dye, the concentration of the composition in the staining mixture being greater than 50 μM when the composition is pyruvate.  
     
     
         14 . The process of  claim 13 , wherein the DNA-selective 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.  
     
     
         15 . The process of  claim 13 , wherein the staining mixture is subjected to a temperature of about 4° C. to about 30° C.  
     
     
         16 . The process of  claim 14 , wherein the staining mixture is subjected to a temperature of about 30° C. to about 39° C.  
     
     
         17 . The process of  claim 14 , wherein the staining mixture is subjected to a temperature of about 40° C. to about 50° C.  
     
     
         18 . The process of  claim 13 , wherein the composition comprises pyruvate at a concentration selected from the group consisting of about 2.5 mM, about 10 mM, about 15 mM, about 25 mM, and about 50 mM.  
     
     
         19 . The process of  claim 13 , wherein the composition comprises vitamin K at a concentration selected from the group consisting of about 10 μM, about 50 μM, about 75 μM, and about 100 μM.  
     
     
         20 . The process of  claim 13 , wherein the composition comprises lipoic acid at a concentration selected from the group consisting of about 0.1 mM, about 0.5 mM, about 0.75 mM, about 1.0 mM, and about 1.5 mM.  
     
     
         21 . The staining mixture of  claim 1 , wherein the staining mixture further comprises a buffer that inhibits sperm motility.  
     
     
         22 . The staining mixture of  claim 2 , wherein the staining mixture further comprises a buffer that inhibits sperm motility.  
     
     
         23 . The staining mixture of  claim 21 , wherein the buffer is a carbonate-based buffer.  
     
     
         24 . The staining mixture of  claim 22 , wherein the buffer is a carbonate-based buffer.  
     
     
         25 . The staining mixture of  claim 21 , wherein the buffer comprises 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.  
     
     
         26 . The staining mixture of  claim 22 , wherein the buffer comprises 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.