PARENT SESSION
Sex Determination
158
PRELIMINARY DEVELOPMENTS OF SPERM SORTING TECHNOLOGY IN NON-HUMAN PRIMATES.
O'Brien, Justine1, He, Liwei1, Stojanov, Tomas2, Birrell, Alex3, Maxwell, Chis1, Evans, Gareth1, 1 2 3
ABSTRACT- Pre-determination of the sex of offspring has implications for conservation and management of captive endangered species, particularly those with single sex-dominated social structures. Our goal is to adapt flow cytometry technology to sort sperm of non-human primate species for use in artificial insemination (AI). The aim of this study was to (i) sort sperm heads into X- and Y-enriched samples; (ii) assess the efficiency of sorting and (iii) determine the difference in DNA content between X- and Y-bearing sperm heads. Sperm were collected from 7 baboons (Papio hamadryas) by electroejaculation, and from 2 marmosets (Callithrix jacchus) 15 h and 59 h post-mortem. Human sperm were used as a control. Sperm heads were stained (Hoechst 33342), incubated and analysed using a high speed cell sorter (SX MoFlo®, Cytomation Inc.). Reanalysis of sorted samples (10,000 events/sample) and fluorescence in-situ hybridization (FISH; 500 sperm heads/sample) were used to evaluate efficiency of sorting. Sort reanalysis and FISH results were similar; combined data revealed high levels of purity for X- and Y-enriched samples (94-96% and 93-96%, 96-98% and 92-96%, 92-94% and 91-94% for baboon, marmoset and human, respectively). FISH of unsorted samples resulted in X:Y ratios of 51:49, 53:47 and 51:49 for the baboon, marmoset and human, respectively. Based on fluorescence intensity of X- and Y-bearing sperm heads, the difference (mean ± sem) in DNA content between X and Y populations was 4.20 ± 0.03%, 4.09 ± 0.03% and 2.97 ± 0.05%, for baboon, marmoset and human, respectively. These data indicate the potential for high purity sorting of whole sperm from non-human primates. The larger difference in DNA content between X- and Y-bearing sperm heads in the baboon and marmoset compared to humans should enable more efficient sorting of whole sperm and provide adequate numbers for their cryopreservation and use in AI. (Supported by Zoological Parks Board of NSW, Australian Research Council and XY Inc.)
KEY WORDS: sex predetermination, sperm sorting, baboon, marmoset