350 COMPARATIVE TIME-LAPSE STUDIES ON PORCINE EMBRYOS FERTILIZED IN VITRO WITH FLOW CYTOMETRICALLY SEX SORTED SPERMATOZOA

Abstract

The objective of the present time-lapse studies was to compare developmental characteristics of porcine embryos after in vitro fertilization with flow cytometrically sexed spermatozoa during an in vitro culture period. Immature oocytes were matured (n = 851) and fertilized in vitro using 50 spermatozoa of either sex per oocyte. Additionally, in vivo-matured oocytes (n = 700) were derived from hormonally stimulated (eCG/hCG) prepuberal gilts, which were slaughtered 38 h after hCG treatment. Potential zygotes were introduced into the time-lapse system (50 µL-microdrops, NCSU-23) 18 h after the onset of in vitro fertilization. The onset and duration of cell cycles and blastomere rotation as well as collapses and re-expansion of cytoplasm were investigated. At the end of the culture period (168 h), embryos were labeled with Hoechst 33342 to identify the number of cell nuclei. The time-lapse system consisted of an incubation chamber installed on an inverted phase-contrast microscope and gassed with 5% CO₂ in maximally humidified air. Computer controlled positioning of the chamber allowed the capture and storage of digital images of individual embryos every 30 min over a 7-day period. Converted time values were tested by ANOVA or ANOVA on ranks. In total, 700 in vivo-matured oocytes were fertilized in vitro (Y-sperm: 342; X-sperm: 358). Cleavage rates were 45.6% for male and 40.2% for female embryos. Out of these, 45.5% developed to male and 62.5% to female blastocysts, respectively. The onset and duration of cell cycles differed significantly at the 2-cell and morula stages (P < 0.01). The onset and number of rotations as well as collapses and re-expansion of cytoplasm were not different. Mean cell numbers of blastocysts were equal for both sexes (male: 35.2; female: 38.8). In parallel, 851 in vitro-matured oocytes were fertilized in vitro (Y-sperm: 431; X-sperm: 422). Cleavage rates were 45.5% for male and 49.3% for female embryos. Out of these, 54.1% developed to male and 56.7% to female blastocysts, respectively. The onset and duration of cell cycles and rotations as well as collapses were not significantly different between sexes. Mean cell numbers of blastocysts were equal for both sexes (male: 29.5; female: 27.7). Comparison between male embryos derived from in vivo (VV) or in vitro (VT)-matured oocytes showed a delay of the onset of the second cell cycle for VV (P < 0.001) and at the blastocyst stage for VT (P < 0.001). Accordingly, in these stages the duration of the cell cycle was shortened (P < 0.001). The onset and duration of rotations as well as collapses and re-expansion of cytoplasm were not different. Similar data were obtained for the female embryos. The data suggest slight sex related differences (onset of cell cycles), but these might be masked in embryos produced from in vitro-matured oocytes due to their higher individual variability. The experiment also shows the advantages of a time-lapse system to identify dynamic cell processes. It might also be a useful tool to precisely correlate cell cycle events to activation of certain marker genes.