Ratio of proliferating cell nuclear antigen-positive nuclei to total cell number is higher in day 7 than in day 8 vitrified in vitro-produced bovine embryos

The aim of the present study was to find a reliable functional criterion fo r the evaluation of the proliferation potential of bovine in vitro-produced embryos. We used immunocytochemical detection of proliferating cell nuclea r antigen (PCNA) combined with propidium iodide (PI) staining and subsequen t confocal laser scanning microscopy together with routine morphological ev aluation under a stereomicroscope to study fresh Day 7, 8, and 9, and cryop reserved Day 7 and 8 embryos. The ratio of PCNA/PI-positive nuclei was equa l in fresh Day 7 and Day 8 embryos and significantly lower in Day 9 embryos . In general, Day 7 embryos tolerated the cryopreservation treatments bette r than Day 8 embryos. Vitrification in normal straws was especially detrime ntal to Day 8 embryos. In fresh Day 7 and 8 embryos, the PCNA results were in agreement with stereomicroscopic evaluation. However, in Day 9 fresh and in Day 7 and 8 treated embryos, the missing PCNA revealed disorders that w ere not observed under morphological evaluation. PCNA immunocytochemistry i s an effective method to obtain information about the functional state of n uclei. The ratio of PCNA-positive nuclei can provide more information and n umerical data about the developmental potential of bovine embryos after cry opreservation.