title: Flow cytometric sperm sorting: effects of varying laser power on embryo development in swine.
abstract: This study was conducted to determine fertilization rate and embryo development using the Beltsville Sperm Sexing Technology with two different laser power outputs, 25 and 125 milliwatts (mW). Freshly ejaculated boar semen was diluted; one aliquot was not stained or sorted (nonsort) and a second aliquot was stained with Hoechst 33342 and sorted as a complete population, not separated into X and Y populations (all-sort). Ovulation controlled gilts were surgically inseminated with 2 x 10(5) spermatozoa (44-46 hr after human chorionic gonadotropin (hCG)) into the isthmus of each oviduct, one oviduct receiving nonsort and the other all-sort at 25 or 125 mW. A total of 426 embryos were flushed from oviducts at slaughter 43 hr after laparotomy and prepared for determination of fertilization and cleavage rates using confocal laser microscopy for analysis of actin cytoskeleton and chromatin configuration. The percentage of fertilized eggs and embryos was less for the 25 mW all-sort compared to nonsort or the 125 mW all-sort (77.9 vs. 96.3 and 96.2%, P < 0.05). The percentage of fragmented embryos was greater for the 25 mW all-sort than the nonsort (15.2 vs. 4.5%, P < 0.05), but did not differ significantly from 125 mW all-sort mean (7.2%). The percentage of normal embryos (80.4% overall) did not differ (P > 0.05) among treatments. However, the rate of embryo development was slower (P < 0.05) after insemination with the 25 mW all-sort spermatozoa compared to nonsort spermatozoa. Embryos in the 3-4 and 5-9 cell stages for the 25-mW all-sort and nonsort were 78 and 20% vs. 49 and 50%, respectively. The embryo percentages for the 125 mW (3-4 and 5-9 cell stages, 59 and 35%) did not differ significantly (P > 0.05) from the nonsort or 25 mW all-sort. We conclude that the use of 125 mW laser power for sorting boar spermatozoa is advantageous to maintain high resolution separation and has no detrimental effect on embryo development compared to 25 mW.
authors.affiliation: Germplasm and Gamete Physiology Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, MD, USA. dave@anri.barc.usda.gov
authors.forename: H D
authors.initials: HD
authors.lastname: Guthrie
authors.forename: L A
authors.initials: LA
authors.lastname: Johnson
authors.forename: W M
authors.initials: WM
authors.lastname: Garrett
authors.forename: G R
authors.initials: GR
authors.lastname: Welch
authors.forename: J R
authors.initials: JR
authors.lastname: Dobrinsky
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D004307:Dose-Response Relationship, Radiation; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology; D005314:Embryonic and Fetal Development / Q000502:physiology*; D005260:Female; D005306:Fertilization / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D007834:Lasers / Q000009:adverse effects*; D008297:Male; D018613:Microscopy, Confocal; D010063:Ovum / Q000502:physiology; D013094:Spermatozoa / Q000145:classification* / Q000166:cytology / Q000502:physiology / Q000528:radiation effects*; D013552:Swine / Q000196:embryology / Q000502:physiology