Produção de zebrafish ginogenético mitótico e meiótico: comparação do desenvolvimento da prole

Abstract

The development of inbred animals, due to genetic homogeneity, represents an important tool for biomedical research. To generate homozygous Zebrafish, it is possible to disrupt the completion of meiosis II or mitosis in haploid embryos. To carry out these interferences in the cell cycle, there are different methodologies based on pressure shock, with varying degrees of complexity and success rates. To simplify and improve the success rate, this work proposes to develop and test a new methodology, with simpler procedures, based on the combination of sperm irradiation by UV-C for a shorter time followed by a thermal shock at different temperatures. Thus, the development of descendants generated by meiotic (Mei) or mitotic (Mit) gynogenesis, through combinations of different irradiation parameters (28.5 mJ/cm2, 105 mJ/cm2 and 210 mJ/cm2) and temperatures (Mei: 40.40, 40.60 or 40.90°C; Mt: 41.40, 41.90 or 42.45°C), was compared. To confirm the success of gynogenesis, a compilation of non-molecular methodologies was made, adding the use of embryonic metaphase counts. The results showed that the irradiation of 28.5mJ/cm2 for a short period guarantees haploid embryos and good fertilization rate. Regarding temperature, the shock at 40.6°C applied from 4 minutes to 6 minutes post fertilization reached an average of 42.08% of healthy animals at the end of 120 hours post fertilization. In mitotic gynogenesis, 41.9°C showed an average of 11.91% of healthy larvae in 120 hours post fertilization when the shock was applied between 18 and 20minutes post fertilization. Conclusion: the proposed methodology was effective in generating gynogenetic embryos and meiotic gynogenesis generated offspring with higher viability, malformation and health rates than the mitotic technique. Retention of the second polar body requires lower temperature (~1.3°C) and is linked to the developmental stage. Whereas, to interrupt a mitotic cycle, it was necessary to use a higher temperature.