 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.Additional applications and methods were added, including antibodies, from 1987 through 1997.At the time of the patent filing, both Lawrence Livermore National Laboratories and the USDA were only sorting fixed sperm nuclei, 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".