Submitted to: Society for Cryobiology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/28/2002
Publication Date: 8/5/2002
Citation: Rajamohan, A., Leopold, R.A., Harris, M. 2002. Vitrification of mexican fruit fly embryos [abstract]. Society for Cryobiology Meeting. Abstracts/Cryobiology 45:247.
Technical Abstract: The vitrification procedures developed for most cells and embryos involve rapid cooling in either liquid nitrogen or nitrogen slush, as was the case in the procedures developed for Drosophila embryos (Steponkus et al., 1990; Mazur et al., 1992). However, these procedures were not suitable for cryopreserving embryos of the tephritid fruit flies (Wang et al., 2000). To obtain survival, these embryos required vitrification at a higher temperature before being exposed to liquid nitrogen. Our present study was focused on understanding these processes and optimizing the procedure that we currently use to cryopreserve three species of tephritid embryos. Mexican fruit fly (Anastrephaludens) embryos, loaded with 10% ethylene glycol and dehydrated in ethylene glycol-based vitrification solutions, were either held for 1 min in nitrogen vapor at about -120°C or were directly plunged into liquid nitrogen. Larval hatch was ?11 %, when the samples were quenched in liquid nitrogen and was as high as 62% for samples prechilled in nitrogen vapor before plunging into the liquid nitrogen. Cryomicroscopic observations on the nature of phase changes occurring during chilling and warming showed that 0.5 -1.0 ~l amounts of some of our vitrification solutions would fracture if they were exposed to temperatures lower than -140°C. Differential scanning calorimetric studies indicated that these mixtures have glass transition points (Tg) between -120 and -130°C. Further examination of the vitrification solutions at, above, and below Tg revealed a marked reduction in fracturing when the solution contained 5% polyethylene glycol. Solutions containing 40% ethylene glycol, with or without 0.5 M trehalose tended to fracture, when directly exposed to temperatures below -140°C. Fracturing was also observed upon removal from liquid nitrogen and when samples were held at a temperature below the Tg. Embryos after dehydration in the vitrification solution containing 40% ethylene glycol and rapid exposure to -191°C did not show any immediate major internal or external damage wh~n examined under low magnification. However, since fracturing of the cryoprotectant outside the embryos is very noticeable and hatching after recovery is much reduced, we suggest that subtle damage to the embryos is occurring and is caused by thermal stress when the samples are cooled rapidly to temperatures below -140°C. Annealing the samples before liquid nitrogen storage and before thawing at a temperature just below the Tg significantly improves the hatching percentage of these insect embryos.