Short Communication Quantification of the DNA Difference, and Separation of X- and Y-Bearing Sperm in Alpacas (Vicugna pacos) KM Morton, M Ru¨ckholdt, G Evans and WMC Maxwell Centre for Advanced Technologies in Animal Genetics and Reproduction (ReproGen), Faculty of Veterinary Science, The University of Sydney, Sydney, NSW, Australia Contents Sperm sexing is an emerging reproductive technology which has been successfully used to produce offspring of a pre- determined sex in domestic and wildlife species but has yet to be applied to New World camelids. The aims of the present study were to (i) optimize the Hoescht 33342 (H33342) staining concentration for the flow cytometric separation of X and Y chromosome-bearing alpaca (Vicugna pacos) sperm nuclei, (ii) separate alpaca sperm nuclei into high purity (>90%) populations bearing the X- and Y-chromosome and (iii) determine the DNA difference between X- and Y-bearing sperm in alpacas. Semen was collected from alpacas and sperm nuclei stained with H33342, incubated and analysed using a high-speed cell sorter (SX-MoFlo Ò ). H33342 staining concen- trations of 36, 54, 72 or 90 lM did not affect the proportion of correctly oriented sperm nuclei (43.3 ± 3.9, 46.4 ± 3.7, 44.5 ± 4.0 and 51.1 ± 2.5% respectively) nor the speed of sorting (1381 ± 160, 1386 ± 123, 1371 ± 133 and 1379 ± 127 sperm nuclei ⁄ s). Sort reanalysis determined high levels of purity for X- and Y-enriched populations (96.6 ± 0.7% and 96.1 ± 1.1% respectively). The DNA difference, based on fluorescence intensity (determined by the SX-MoFlo Ò ), was 3.8 ± 0.06%. These data demonstrate for the first time that alpaca sperm nuclei can be separated into high purity populations and the potential for applying sperm sexing technology to New World camelids. Introduction The separation of sperm into X- and Y-chromosome bearing populations by flow cytometry (‘sperm sexing’) is an emerging reproductive technology that has resulted in the birth of pre-sexed offspring in a number of species (Maxwell et al. 2004). The application of sexing technology for a new species involves the development of species-specific protocols for sperm staining, sorting and post-sorting preserva- tion. Preliminary development of this technology, the separation of X- and Y-chromosome bearing sperma- tozoa is currently under development in a number of domestic and wildlife species (Maxwell et al. 2004). There is considerable interest from livestock produc- ers in the production of offspring of a pre-determined sex. Pre-selecting the sex of offspring can increase the rate of genetic progress of flocks and herds and has advantages for management and efficient production of livestock, particularly the production of females place- ment dairy heifers and male and female crossbred lines in the pig industry. Camelids contribute to many economies through meat, milk and fibre production (Tibary et al. 2006) and there is considerable scope for the application of sex-preselection in the various camelid industries. The camel dairy industry would benefit from increased proportions of female replacement dairy camels. Given the differential prices for male and female llamas, alpacas and guanacos, fibre producers would have the option of producing offspring of the sex they desire. Sperm sexing technology can also be used as a potential population management strategy for wildlife species with a single sex dominated social structure (Maxwell et al. 2004) and could potentially be applied to populations of threatened camelids such as the vicuna. However, sperm-sexing technology has yet to be applied to camelids, despite considerable interest. The aims of this study were to (i) optimize the Hoescht 33342 (H33342) staining concentration for the flow cytometric separation of X and Y alpaca sperm nuclei, (ii) separate alpaca sperm nuclei into high purity (>90%) populations bearing the X- and Y-chromo- some and (iii) determine the DNA difference between the X- and Y-bearing sperm in alpacas. Materials and Methods Animals and experimental design Procedures described herein were approved by The University of Sydney’s Animal Ethics Committee. Semen was collected from adult male alpacas main- tained under field conditions supplemented with alpaca pellets and Lucerne hay (owing to drought conditions) at the Animal Reproduction Unit, The University of Sydney, Cobbitty, NSW, Australia. Sperm nuclei were stained with the Hoechst fluoro- chrome 33342 (H33342; Sigma-Aldrich, Sydney, NSW, Australia) and subjected to flow cytometric separation. Sperm nuclei rather than whole sperm were used for all analyses to ensure a more accurate determination of the DNA content of the X- and Y-bearing sperm by removing interference from the sperm midpiece and tail. Semen from each male was processed separately and the whole procedure was replicated on four separate occasions. Semen collection Ejaculates were collected from five adult male alpacas (n = 4 ejaculates per male) using a rubber sheep artificial vagina (IMV Technologies, L’Aigle, France) Reprod Dom Anim 43, 638–642 (2008); doi: 10.1111/j.1439-0531.2007.00965.x ISSN 0936-6768 Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag