Cryopreservation and in vitro fertilization at the zebrafish international resource center

Abstract

In recent decades, laboratories throughout the world generated several thousand mutant, transgenic, and wild-type zebrafish lines and more lines continue to be produced. At the same time, relatively little effort has been expended to develop reliable, high-throughput, standardized, long-term cryopreservation storage methods, even though laboratories and the research community as a whole struggle to maintain the large number of lines alive. Safe and reliable methods for maintaining these valuable genetic resources are vital for future biomedical research.Cryopreservation is the most efficient method for large-scale, long-term storage of important genetic materials. It extends the time offspring can be produced from individual fish, reduces the need to maintain live populations, and can prevent catastrophic loss of irreplaceable research lines. Cryopreservation is also the most cost-effective alternative for maintaining genetic resources because it reduces costs for animal and facility maintenance, personnel, and space. In addition, it provides novel opportunities to develop new types of research using large numbers of lines. For example, several genetic strategies, such as TILLING-or enhancer and gene trapping-depend on the use of cryopreservation to bypass generations of live organisms until a strain is revived for research.This chapter describes and discusses the current cryopreservation method used at the Zebrafish International Resource Center. This method is derived from the initial protocol developed for zebrafish over 20 years ago that has recently been refined.

Fig. 1. Zebrafish sperm cryopreservation tools

(A) Tools to make powdered dry ice from liquid CO₂. A fire extinguisher cone is attached to the liquid output of a liquid CO₂ tank (arrows). At the bottom: cooler, safety gloves, goggles, ear protection, isolation pad, and PVC tubing to compact dry ice powder. (B) The aspirator tube assembly. The aspirator is a soft rubber tube adaptor that has a mouthpiece on one end and a capillary holder on the other. (C) Sponge fish holder. A 35 × 10 mm Petri dish with a sponge that is cut to hold males. (D) The sponge needs to be moistened before fish are placed in it. (E) Examples of empirically determined sperm quality. On the right, milky, opaque milt indicates high density of sperm cells whereas lower quality sperm (left) is watery and more transparent. The observed difference in opacity correlates only loosely with sperm cell density counts using a hemocytometer and does not correlate well with post-thaw fertilization rates. Scale bars: (C, D) 1 cm; (E) 1mm.

Fig. 2. Preparation of the cryopreservation workspace

(A) Assistant’s side, (B) squeezer’s side, (C) box with powdered dry ice and Falcon tubes (foreground), Styrofoam container with liquid nitrogen fiberglass tray insert and color-coded screwcap cryovials. (A) On the assistant’s side 1. fish tank; 2. data records sheet; 3. Tricaine stock solution; 4. Pasteur Pipette; 5. timer; 6. recovery dish; 7. anesthetic dish; 8. Stack of paper towels; 9. Sponge fish holder (see also Fig. 1 C, D); 10. spoon; 11. screwcaps for cryovials (in cups and on table); 12. paper wipes; rack with 15 ml Falcon tubes and lids. (B) On the squeezer’s side: 14. aspirator tube assembly; 15. forceps; 16. dissection scope; 17. row of tubes containing cryoprotectant solution without Methanol; 18. row of tubes containing cryoprotectant solution with Methanol; 19. micropipetter; 20. 20 µL glass capillaries, prelabeled at 1.67 cm; 21. watchglasses; 22. micropipette tips; 23. pre-labeled empty cryovials; 24. waste container for glass and sharp objects. (C) 25. Container with powdered dry ice and inserted Falcon Tubes on the left; 26. liquid nitrogen and immersed color-coded cryovials. (D) Profile of 15 ml Falcon Tube containing a 2 ml cryovial with sample, submerged in dry ice.

Fig. 3. Visual assessment of egg quality

(A) Anesthetized female is squeezed gently to release eggs into 35 mm Petri dish. (B, C) “Good” quality batches of eggs. Eggs are yellowish in incident light and appear translucent with transmitted light. (D) Lower quality batch of eggs. Note the opaque, white eggs intermingled with normal looking ones. Scale bars: (A) 1 cm; (B–D) 1 mm.