Zebrafish embryos (Danio rerio) using microinjection

Abstract

Low membrane permeability is one of the major obstacles to the successful cryopreservation of zebrafish embryos. The aim of the present study was to explore if this could be overcome by yolk modification with different cryoprotectants by micro-injection. Initial investigation of two cryoprotectants, methanol and sucrose, was undertaken to determine their suitability for micro-injection supplementation of the yolk mass. Intact zebrafish embryos at 50% epiboly stage were injected with Hanks' solution, 5.2 M methanol or 1.3 M sucrose yielding approximate final concentrations of 2.0 and 0.5 M of the cryoprotectants within the yolk sac respectively. After micro-manipulation, the embryos were cultured at 28 degree C for three days and their survival assessed at the hatching stage. All micro-manipulations performed in the present study resulted in a significant decrease in embryo survival (P < 0.05). Embryos micro-injected with methanol or sucrose were also subjected to a cooling procedure. They were placed in 3M methanol + 0.5 M sucrose at room temperature for 30 min and then cooled from 20 degree C to 0 degree C at 2 degree/min, from 0 degree C to -7.5 degree/min at 1 degree/min, seeded at -7.5 degree C and held for 10 min, before cooling at 0.3 degree/min to - 20 degree C or until full crystallization in all embryos. The processes of extra- and intracellular crystallization were studied by cryomicroscopy. The temperature of intracellular crystallization did not differ significantly between control and injected embryos. However, it was found that intracellular crystallization did not always happen instantly after extracellular crystallization.

Figure 1

The survival of embryos at hatching stage after different treatments: control-without treatment; punctured- punctured without injection; Hanks' - injection of approximately 33 nl of Hanks'; methanol – injection of 33nl of 5.2 M methanol; sucrose - injection of 33 nl of 1.3 M sucrose at 50% epiboly stage. 10 – 20 embryos were used for each treatment and the experiments were repeated at least 3 times.

Figure 2

A typical appearance of an embryo 2 hours after injection immediately after cooling-thawing procedure.

Figure 3

The images of a sucrose-injected embryo (24 hours after injection) during cooling under cryomicroscope: a-image of the embryo before cooling b-extracellular crystallization c-the beginning of intracellular crystallization (see arrow) d-full intracellular crystallization e-after thawing (this embryo was alive 3 hours after freezing-thawing)

Figure 4

Temperature of intracellular crystallization in control and methanol or sucrose injected embryos. Control – embryos cooled at heart-beat stage; methanol – embryos cooled at heart beat stage, 24 hours after injection of 5.2 M methanol; sucrose - embryos cooled at heart beat stage 24 hours after injection of 1.3 M sucrose. At least 10-20 embryos were used for each treatment and the experiments were repeated at least 3 times.

Figure 5

The images of a methanol-injected embryo during cooling. a - before cooling; b - spreading of extracellular ice; and c - spreading of intracellular crystallization

Figure 6

The images of a sucrose-injected embryo during cooling under cryomicroscope. a, b- images of the embryo before cooling; c- ECC has occurred signs of the beginning of ICC (darkening of embryonic part see arrow); d – propagation of ICC from 2 different epicenters (see arrows); e- full ICC in embryo; f, g, h - embryo immediately after thawing.