Cryopreservation of zebrafish (Danio rerio) embryos | |
| Author | Sawitri Silakes |
| Call Number | AIT Diss. no.AQ-11-01 |
| Subject(s) | Zebra danio--Germplasm resources--Cryopreservation Fishes--Embryos |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Technical Science in Aquaculture and Aquatic Resources Management |
| Publisher | Asian Institute of Technology |
| Abstract | Since only vitrification is likely to result in successful cryopreservation of teleost embryos, requiring high level of cryoprotectant penetration into the cell, cavitation level ultrasound was used to enhance cryoprotectant permeation into zebrafish ( Danio rerio ) embryos. Three types of embryos (dechorionated, soft chorion, and normal embryos) at three developmental stages (90% epiboly, bud, and 4-somite stages) were used in this study. Three successive experiments were carried out in this research. The first experiment evaluated the effect of cavitation level ultrasound on different types and stages of developing embryos treated with different voltages and durations to determine post- treatment survival. The second experiment assessed the effects of ultrasound on survival and methanol permeation into the three types and at three stages of embryos. The third experiment attempted to fast freeze and thaw embryo and examine the morphology and survival post methanol immersion. To determine the effect of different ultrasound levels on zebrafish embryos, embryos were exposed to four different ultrasound voltages: 420V, 440V, 480V and 520V for 1, 2 and 3 min durations. Various ultrasound levels and exposure duration resulted in comparable survival between ultrasound treat ed and control groups. Ultrasound at 520 V for up to 3 min exposure time resulted in similar survival percentages of intact or soft chorion embryos at all three developmental stages. Dechorionated embryos at 90% epiboly stage on the other hand, could tole rate only 480V for 1-3 min duration. In the second experiment, three types of embryos were exposed to methanol levels that were optimized for survival during the first set of experiments. Dechorionated embryos were exposed to 20% methanol, so ft chorion embryos were exposed to 30% methanol, and intact embryos were exposed to 40% methanol for 3 minutes with or without ultrasound treatment. High performance liquid chromatography (HPLC) was used to measure methanol levels in the treated embryos. The total MeOH recovery from zebrafish embryo samples spiked with MeOH was 100.62 ± 3.99%. The overall penetration of methanol into zebrafish embryo ranged from 85.31 ± 8.06 μmol (in soft chorion embryos at 90% epiboly stage treated with ultrasound) to 3.13± 0.96 μmol (in dechorionated embryos at 90% epiboly st age treated without ultrasound). Ultrasound clearly helped to improve the permeation le vel of methanol into zebrafish embryos. Significantly higher amount of methanol was found in soft chorion embryos. Dechorionated embryos had the highest relative difference in methanol levels between ultrasound and controls. Although , ultrasound significantly affected survival, survival tended to be generally high (64 ± 2% to 100%). Although embryos appeared to be intact after fast freezing-thawing, no embryos survived the freezing-thaw procedures. It was difficult to ascertain the cause of mortality since the study did not evaluate physiological parameters. Even Though ultrasound clearly enhanced permeation of methanol into zebra fish embryos, the increased methanol level might not have been enough to achieve vitrification. Further studies using novel, high sensitivity and non-invasive methods are needed to achieve greater cryoprotectant penetration for successful vitrification. |
| Year | 2011 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Aquaculture and Aquatic Resources Management (AQ) |
| Chairperson(s) | Bart, Amrit N. |
| Examination Committee(s) | Gallardo, Wenresti G.;Preeda Parkpian |
| Scholarship Donor(s) | Ministry of Agriculture and Cooperatives, Thailand |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2010 |