Method of cryopreserving selected sperm cells

Abstract

The present invention provides a method of cryopreserving sperm that have been selected for a specific characteristic. In a preferred embodiment, the method is employed to freeze sex-selected sperm. Although the cryopreservation method of the invention can be used to freeze sperm selected by any number of selection methods, selection using flow cytometry is preferred. The present invention also provides a frozen sperm sample that has been selected for a particular characteristic, such as sex-type. In preferred embodiments, the frozen sperm sample includes mammalian sperm, such as, for example, human, bovine, equine, porcine, ovine, elk, or bison sperm. The frozen selected sperm sample can be used in a variety of applications. In particular, the sample can be thawed and used for fertilization. Accordingly, the invention also includes a method of using the frozen selected sperm sample for artificial insemination or in vitro fertilization.

1 - 55 . (canceled) 
     
     
         56 . A method of cryopreserving selected sperm cells comprising:
 a) obtaining selected sperm cells;   b) cooling the selected sperm cells;   c) extending the selected sperm cells with an initial extender;   d) isolating the selected sperm cells;   e) extending the isolated sperm cells with a final extender to produce a sperm suspension; and   f) freezing said sperm suspension.   
     
     
         57 . The method of  claim 56 , wherein the step of obtaining selected sperm cells further comprises:
 a) staining a mixed population of sperm cells with a flourochrome dye which binds to sperm DNA;   b) differentiating X chromosome bearing sperm from Y chromosome bearing sperm through flow cytometry based on an assessment of the amount of DNA in each cell; and   c) separating sperm to produce a selected sperm cells which are enriched with respect to the presence of either X chromosome bearing sperm or Y chromosome bearing sperm.   
     
     
         58 . The method of  claim 56 , wherein the frozen sperm suspension, when thawed, comprises a sufficient number of sperm cells to fertilize an egg. 
     
     
         59 . The method of  claim 56 , wherein the final extender comprises one selected from the group consisting of: an egg-yolk-Tris extender containing glycerol; egg-yolk-TES-Tris extender containing glycerol; an egg-yolk-sodium citrate extender containing glycerol; and a milk extender containing glycerol. 
     
     
         60 . The method of  claim 56 , wherein the final extender comprises a cold shock treatment, an energy source, an antibiotic, a component which maintains osmolality and buffers pH, and a cryoprotectant. 
     
     
         61 . The method of  claim 60 , wherein the energy source is selected from the group consisting of: a saccharide, a glucose, a fructose, and any combination thereof. 
     
     
         62 . The method of  claim 60 , wherein the cold shock treatment is selected from the group consisting of: egg yolk, an egg yolk extract, milk, a milk extract, casein, albumin, lecithin, and any combination thereof. 
     
     
         63 . The method of  claim 60 , wherein the cryoprotectant is selected from the group consisting of glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol, and any combination thereof. 
     
     
         64 . The method of  claim 63 , wherein the cryoprotectant comprises about 5% to about 7% glycerol. 
     
     
         65 . The method of  claim 60 , wherein the component which maintains osmolality and buffers pH is selected from the group consisting of: a buffer comprising a salt, a buffer containing a carbohydrate, and any combination thereof. 
     
     
         66 . The method of  claim 60 , wherein the component which maintains osmolality and buffers pH is selected from the group consisting of sodium citrate, Tris[hydroxymethyl]aminomethane, TES N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, monosodium glutamate, milk, HEPES buffered medium, and any combination thereof. 
     
     
         67 . The method of  claim 56 , wherein, prior to freezing, the sperm suspension is divided into individual insemination samples. 
     
     
         68 . The method of  claim 67 , wherein the individual insemination samples comprises between about one million and about three million selected sperm cells. 
     
     
         69 . The method of  claim 56 , wherein the sperm cells are selected from the group consisting of: bovine sperm cells, equine sperm cells, and porcine sperm cells. 
     
     
         70 . The method of  claim 56 , wherein the step of cooling the selected sperm cells comprises the step of reducing the temperature of said sperm cells to about 5° Celsius. 
     
     
         71 . The method of  claim 70 , wherein the step of reducing the temperature of said sperm cells to about 5° Celsius comprises reducing the temperature for a period between about 60 minutes and about 240 minutes. 
     
     
         72 . The method of  claim 56 , wherein the initial extender comprises a cold shock treatment, an energy source, an antibiotic, and a component which maintains osmolality and buffers pH. 
     
     
         73 . The method of  claim 56 , wherein the selected sperm cells are sorted into an initial extender that lacks glycerol and wherein the step of extending the selected sperm cells further comprises extending the selected sperm cells in an initial extender having a cryoprotectant. 
     
     
         74 . The method of  claim 73 , wherein the step of extending the selected sperm cells with an initial extender having a cryoprotectant further comprises extending applying to equal volumes of the initial extender having a cryoprotectant about 15 minutes apart. 
     
     
         75 . A frozen separated cell sample in ccordance with the method of  claim 56 .