Flow cytometric sorting of non-human primate sperm nuclei

Abstract

Pre-determination of the sex of offspring has implications for management and conservation of captive wildlife species, particularly those with single sex-dominated social structures. Our goal is to adapt flow cytometry technology to sort spermatozoa of non-human primate species for use with assisted reproductive technologies. The objectives of this study were to: (i) determine the difference in DNA content between X- and Y-bearing spermatozoa (ii) sort sperm nuclei into X- and Y-enriched samples; and (iii) assess the accuracy of sorting. Spermatozoa were collected from two common marmosets ( Callithrix jacchus), seven hamadryas baboons ( Papio hamadryas) and two common chimpanzees ( Pan troglodytes). Human spermatozoa from one male were used as a control. Sperm nuclei were stained (Hoechst 33342), incubated and analyzed using a high-speed cell sorter. Flow cytometric reanalysis of sorted samples (sort reanalysis, 10,000 events/sample) and fluorescence in situ hybridization (FISH; 500 sperm nuclei/sample) were used to evaluate accuracy of sorting. Based on fluorescence intensity of X- and Y-bearing sperm nuclei, the difference in DNA content between X and Y populations was 4.09 ± 0.03, 4.20 ± 0.03, 3.30 ± 0.01, and 2.97 ± 0.05%, for marmoset, baboon, chimpanzee and human, respectively. Sort reanalysis and FISH results were similar; combined data revealed high levels of purity for X- and Y-enriched samples (94 ± 0.9 and 93 ± 0.8%, 94 ± 0.7 and 94 ± 0.5%, 91 ± 0.9 and 97 ± 0.6%, 94 ± 0.6 and 94 ± 0.9%, for marmoset, baboon, chimpanzee and human, respectively). These data indicate the potential for high-purity sorting of spermatozoa from non-human primates.