<sod>However, flow cytometry is the only currently used technique able to determine the sex of future progeny by measuring DNA content of individual sperm cells. It evaluates if they contain the larger [[X chromosome]] (giving rise to a female offspring) or smaller [[Y chromosome]] (leading to male progeny). It then allows separation of X and Y sperm.<ref>{{Cite journal|last=Johnson|first=L. A.|last2=Welch|first2=G. R.|last3=Rens|first3=W.|date=1999-01-01|title=The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI|url=https://www.ncbi.nlm.nih.gov/pubmed/15526798|journal=Journal of Animal Science|volume=77 Suppl 2|pages=213–220|issn=0021-8812|pmid=15526798}}</ref> During the early to mid-1980s, [[Glenn Spaulding]] was the first to sort viable whole human and animal spermatozoa using a flow cytometer, and utilized the sorted motile rabbit sperm for artificial insemination. Subsequently, the first patent application disclosing the method to sort "two viable subpopulations enriched for x- or y- sperm" was filed in April 1987 as US Application Serial Number 35,986 and later became part of US Patent 5,021,244; and the patent included the discovery of [[haploid]] expression (sex-associated membrane proteins, or SAM proteins) and the development of monoclonal antibodies to those proteins.<ref>US Patent 5,021,244, column 9, Sorting Sperm; http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=5021244.PN.&OS=PN/5021244&RS=PN/5021244</ref> Additional applications and methods were added, including antibodies, from 1987 through 1997.<ref>US Patent 5,021,244; 5,346,990; 5,369,012; 5,439,362; 5,496,722; 5,648,468; 5,660,997; PCT/US1989/002069</ref> At the time of the patent filing, both [[Lawrence Livermore National Laboratories]] and the [[United States Department of Agriculture|USDA]] were only sorting fixed sperm nuclei,<ref>L. A. Johnson, J. P. Flook, M. V. Look, D. Pinkel, Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. Gamete Research Volume 16, Issue1, pages 1-9, January 1987; http://onlinelibrary.wiley.com/doi/10.1002/mrd.1120160102/abstract</ref> after the Application Serial Number 35,986 patent filing a new technique was utilized by the USDA where "sperm were briefly sonicated to remove tails".<ref>L. A. Johnson, J. P. Flook and M. V. Look, Flow cytometry of X and Y chromosome-bearing sperm for DNA using an improved preparation method and staining with Hoechst 33342. Gamete Research Volume 17, Issue 3, July 1987, Pages: 203–212; http://onlinelibrary.wiley.com/doi/10.1002/mrd.1120170303/abstract</ref> <eod>
<sod>However, flow cytometry is the only currently used technique able to determine the sex of future progeny by measuring DNA content of individual sperm cells. It evaluates if they contain the larger [[X chromosome]] (giving rise to a female offspring) or smaller [[Y chromosome]] (leading to male progeny). It then allows separation of X and Y sperm.<ref>{{Cite journal|last=Johnson|first=L. A.|last2=Welch|first2=G. R.|last3=Rens|first3=W.|date=1999-01-01|title=The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI|url=https://www.ncbi.nlm.nih.gov/pubmed/15526798|journal=Journal of Animal Science|volume=77 Suppl 2|pages=213–220|issn=0021-8812|pmid=15526798}}</ref> The so-called ''[[Beltsfield Sperm Sexing Technology]]'' was developed by USDA in conjunction with Lawrence Livermore National Laboratories, relying on the DNA difference between the X- and Y- chromosomes.<ref>Garner DL, Seidel GE. History of commercializing sexed semen for cattle. Theriogenology 2008;69: 886-895.</ref> Prior to flow cytometric sorting, semen is labeled with a fluorescent dye called [[Hoechst 33342]] which binds to the DNA of each spermatozoon. As the X chromosome is larger (i.e. has more DNA) than the Y chromosome, the "female" (X-chromosome bearing) spermatozoa will absorb a greater amount of dye than its male (Y-chromosome bearing) counterpart. As a consequence, when exposed to UV light during flow cytometry, X spermatozoa fluoresce brighter than Y- spermatozoa.  As the spermatozoa pass through the flow cytometer in single file, each spermatozoon is encased by a single droplet of fluid and assigned an electric charge corresponding to its chromosome status (e.g. X-positive charge, Y-negative charge).  The stream of X- and Y- droplets is then separated by means of electrostatic deflection and collected into separate collection tubes for subsequent processing.<ref>Seidel GE, Jr., Garner DL. Current status of sexing mammalian spermatozoa. Reproduction 2002;124: 733-743.</ref> <eod>