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Title: FLOW CYTOMETRIC SPERM SORTING: EFFECTS OF VARYING LASER POWER ON EMBRYO DEVELOPMENT IN SWINE

Author
item Guthrie, Howard
item Garrett, Wesley
item Welch, Glenn
item Dobrinsky, John

Submitted to: Journal of Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/2/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: Sex preselection has been a long sought after goal of the livestock producer because it could result in a significant reduction in production costs. The only proven method of sex-preselection is the Beltsville Sperm Sexing Technology which is based on the relative difference in DNA content of the X- and Y-chromosome bearing spermatozoa providing a significant shift in the sex ratio of offspring from 50 to 85-95% male or female with precision and repeatability. However, use of chromosome selected spermatozoa reduces the fertilizing capacity of spermatozoa. The present study was conducted to test the hypothesis that reduction of laser power output from 125 to 25 mW would attenuate the loss of fertilizing capacity following separation of X- and Y-chromosome bearing sperm. In contrast to our original hypothesis, we found that the rates of fertilization and embryo development were negatively affected to a greater extent at the lower laser power output. When spermatozoa from the 25 mW sort were used for fertilization, the percentage embryos reaching the 5-9 cell stage 43 hours after surgical insemination was less for the 25 mW sort compared to the no sort group. We conclude that the higher laser power (125 mW) is more effective than lower doses (25 mW) for sorting swine sperm for use to produce offspring. This is important to scientific and commercial application because high resolution sorting can be done at without additional stress to the sorted spermatozoa.

Technical Abstract: This study was conducted to determine fertilization rate and embryo development using spermatozoa that had been sorted using two different laser power outputs, 25 and 125 milliwatt (mW). Freshly ejaculated boar semen was was stained with Hoechst 33342 and sorted as a complete population. Ovulation controlled gilts were surgically inseminated with 2 x105 spermatozoa (44 hr after hCG) into the isthmus of each oviduct; one oviduct receiving no-sort spermatozoa and the other spermatozoa sorted at 25 or 125 mW. Embryos were flushed from oviducts at slaughter 43 h after laparotomy and prepared for determination of fertilization and cleavage rate using confocal laser microscopy analysis of actin cytoskeleton and chromatin configuration. At slaughter, 426 potential embryos were flushed from oviducts. The percentage of fertilized eggs and embryos was less for the 25 mW sort compared to no-sort or the125 mW sort (77.9 vs 96.3 and 96.2%, p < 0.05). The percentage of normal embryos (80.4%) did not differ among treatments. However, the rate of embryo development varied among sperm treatments. The percentage of embryos reaching the 5-9-cell stage for the 25 mW sort was less than that for the no-sort (20 vs 49.6%, p < 0.05), but did not differ significantly for 125 mW sort (35.1%). We conclude that while low laser power output produced adequate resolution of the X- and Y- chromosome bearing spermatozoa, the higher laser power125 mW is more effective for sorting swine sperm for use to produce offspring.