234 PREVALENCE OF MEMBRANE-DAMAGED CELLS IN BOVINE EMBRYOS AFTER CULTURE TO THE BLASTOCYST STAGE IN DEFINED MEDIUM

Abstract

Autocrine factors are believed to be the reason why embryos cultured in a group show superior development compared with a single culture in many species. A widespread concern with group culture is that degenerating embryos may have a negative effect on the other embryos. Previously, we were able to identify some proteins in embryo-conditioned medium by using tandem mass spectrometry, predominantly stress-related proteins. Our hypothesis was that poor-quality embryos with membrane-damaged cells passively released these identified proteins into the medium. Therefore, we wanted to investigate what percentage of membrane-damaged cells occurred in an average population of Day 8 bovine embryos. Bovine presumed zygotes (n = 606) were cultured individually in 20-µL drops of modified SOF supplemented with insulin, transferrin, and selenium and 0.1 mg mL^–1 of polyvinylpyrrolidone. At 8 days post-insemination, blastocyst development was evaluated, and subsequently, all embryos were stained with Hoechst/propidium iodide. In this way, we were able to detect the total cell number (TCN) and the percentage of membrane-damaged cells (MDC ratio) because propidium iodide will stain only the nuclei of membrane-damaged cells pink. Embryo-conditioned medium was collected individually and kept at –80°C until further analysis. At 8 days post-insemination, a blastocyst rate of 27.5% was observed. After determination of the TCN per embryo, we divided the embryos in two groups: 207 embryos had a TCN of more than 64 cells (normal development; range: 64 to 313 cells) and 399 embryos had a TCN of fewer than 64 cells (arrested development; range: 1 to 61 cells). In the first group, 76.3% (138 embryos) had an MDC ratio of less than 10%; however, only 11.1% (23 embryos) showed no membrane-damaged cells at all. In the group with arrested development, only 17.8% (71 embryos) of the embryos had an MDC ratio of less than 10%, and only 13.5% (54 embryos) had no membrane-damaged cells. Of the delayed embryos, 48.6% (194 embryos) showed an MDC ratio of more than 50%, and 34.5% (138 embryos) displayed even 100% of membrane-damaged cells. In conclusion, by use of Hoechst/propidium iodide staining, we were able to detect the TCN and MDC ratio of the embryos. We observed that only a small percentage of the embryos had an MDC ratio of 0%, even if the embryos had more than 64 cells. Almost half of the arrested embryos with fewer than 64 cells had a MDC ratio of more than 50%, indicating that poor-quality embryos consist predominantly of membrane-damaged cells that can passively release proteins into the medium, with a possible negative effect on the surrounding embryos in group culture. It must be noted, however, that for logistical reasons, these results were obtained in a single culture. Further research will be done to identify the candidate autocrine factors and proteins released into the medium by poor-quality embryos by analysing embryo-conditioned medium pooled according to the results of the Hoechst/propidium iodide staining.