Preliminary Study On Flow Cytometrically Sorting The X And Y Chromosome-Bearing Mammalian Sperm

The present study was to investigate the factors affecting sperm sorting and preliminarily set up the procedure of flow cytometrically sorting the X and Y chromosome-bearing mammalian sperm.Ejaculates of bull, goat and buffalo were collected from the herd bull station and the pasture. After morphology and motility examinations, the semen was diluted in modified TALP and incubated with Hoechst33342 at 35 C for 60 min to stain the sperm DNA. FD&C #40 was added after incubation to quench Hoechst33342 in the dead sperm. And then the X and Y chromosome-bearing sperm was sorted by the flow cytometer based on DNA differences.The motility showed no significant difference when the bull sperm had been incubated for 0 h and 4 h in neat semen, TALP and TALP plus Hoechst33342 before sorting (P>0.05), but significantly lower in TALP plus Hoechst33342 than in the two other diluents for 8 h (P<0.05) while the best in neat semen for 24 h (P<0.01). The orientation of the sonicated buffalo sperm could reach 69% after calibrating the flow cytometer with goat sperm nuclei, and the CV of the X or Y sperm DNA peak is approximately 1.0% while the CV of the standard fluorescent beads is about 1.93%, which meant the flow cytometer was at its optimal conditions for sperm sorting. The motility of goat and buffalo sperm decreased significantly as the sheath pressure increased from 10spi to 50psi, 69 %, 67%, 56% , 22%, 8% and 73%, 49%, 22%, 6%,3% at 10psi, 20psi, 30psi, 40psi, and 50psi respectively. Sorting reanalysis was carried out for the sorted buffalo sperm, the accuracy of sorted X and Y sperm was 94% and 89%. The sorted buffalo sperm retained higher motility in 5C than in 20 C after storage for 20 h (P<0.01), 67% and 49% respectively.The results above show that the method and procedure of sorting mammalian sperm by flow cytometer has preliminary been set up.