Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number of species. However, the fertility of sex-sorted sperm is reduced and the full effects of sperm-sexing remain to be elucidated. The purpose of the present study was to investigate the potential effects of sex-sorted sperm on mRNA expression patterns of developmentally important genes employing in vitro produced bovine embryos. Bovine embryos were produced in vitro with unsorted and sex-sorted sperm and mRNA expression patterns were determined for glucose-3 transporter (Glut-3), glucose-6-phosphate dehydrogenase (G6PD), X-inactive specific transcript (X-ist) and Heat shock protein 70.1 (Hsp) using semi-quantitative endpoint reverse transcriptase-PCR in male and female, day-7 and 8 embryos. The relative abundance (RA) of Glut-3 was higher for day-7 male than female embryos, and day-7 embryos derived from unsorted compared with sex-sorted sperm. The RA of G6PD was higher for embryos derived from unsorted than sex-sorted sperm, and for day-8 female compared with male embryos. The RA of Xist was higher for female than male embryos, and for day-7 female embryos derived from unsorted than sex-sorted sperm. Hsp RA was higher for female compared with male embryos, was similar for day-7 and 8 embryos, and unsorted and sex-sorted sperm derived embryos. These results demonstrate differential expression of developmentally important genes between male and female embryos, and embryos derived from unsorted and sex-sorted sperm.

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IN recenit years, sperm sexing by high-speed flow cytometry hlas become well established in several domestic animal species, but the output of sorted spermatozoa is limited to about 12 million per hour. In cattle, the regular artificial insemination (Al) dose contains 10 to 20 million unsorted spermatozoa. However, it was recently shown that two million sperimiatozoa are sufficient to obtain acceptable pregnianicy rates in heifers wvhenl the semen is deposited into the tip of the uterine horn (Seidel and others 1999). In pigs, comparatively high numbers of spernmatozoa are needed for Al, and it is usual for three billion spermatozoa to be used in the insemination dose. For this reason, piglets of predetermined sex have so far only been produced by surgical insemination (JohInsonI 1991 ), in vitro fertilisation (Rath and others 1997, 1999, Abeeydera and others l998) or intracytoplasniic sperm injection (Probst and others 2002). Recently, a new inseminatioin device has been developed for deep intrauterine inscminiatioin in SOWS (Martinez and others 2000), which allowvs sciemcin to be deposited deep in the uterine horn, close to the uterotubal junctioin. This short communication describes a study in which cycling sows were inseminated, xvith unsorted spermatozoa or spermatozoa which had been sex selected, usinlg the new insemination device for Al. Semen was collected from a boar of proven fertility and wvas diluted with Androhep (Minitdb) to 150 million spermatozoa/ml. Samples of 1 ml were transferred into Eppendorf tubes and 10 pl of the fluorochrome Hoechst 33342 (bis-benzimide, 5mg/ml; Sigma) was added to the semen to enable distinction between X and Y chromosome-bearing sperm. The samples were placed into ani aluminiuLm block heated to 37°C and were incubated for one hour. The incubation was stopped and 1 pl of food dye (FD&c Red number 40; Warner Jenkinson) was added to each sample. The food dye enters only spermatozoa witlh damaged acrosomes and diminishes their fluorescence, such that these spermnatozoa can be excluded from the sperm sorting process. The samples were filtered through a 20 Mm nylon filter into a 5 nml plastic tube. Sperm sorting was performed in a high-speed flow cytometer (MoFlo; Cytomation) using an ultraviolet argon laser (BeamLok 2065; SpectraPhysics) with a wavelength of 351 nm at 150 mNA. Dulbecco's phosphate buffered solution (Sigma) was used as the sheath fluid at 52 psi. The event rate was set to 20,000 cells/second and the sorting rate was 3300 spermatozoa/second. Sorted samples wvere collected into 10 ml conical plastic tubes which had been prefilled with 0 5 ml of a 2 per cent N-Tris(hydroymethyl) methly-2-amiinoethane sulphonic acid-Tris-egg yolk extender (Johnson 1991), supplemented with a 5 pl frozen thawed seminal plasma. The seminal plasma was prepared from the sperm-poor fractioin of an ejaculate which had been centrifuged, directly after collection, at 2800 g for 20 minutes. The supernatant was assessed to be free of spermatozoa by microscopic examination and aliquots of...

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