The fish were from Ekkwill Water-life Resources. They are referred to as the EKK strain. After four hours of normal looking development, cell blebbing occurs. Sperm are collected into Hank's Balanced Salts. Are there any zebra fish embryologist that might provide us an insight?
Can you elaborate on some of your rearing conditions? What is in your embryo media, what are you rearing them in (vials, plates, petri dishes), and what temperature are you using? Is your mortality 100%?
It sounds like you are fertilizing in vitro. I'm not familiar with collecting sperm in HBSS but I am familiar with strip spawning. Are you strip spawning the oocytes and then applying your sperm in HBSS?
We are strip spawning. We have tried every possible combination. We hold the eggs in HBSS, and then transfer them to tank water and apply semen directly to them. We also apply semen to eggs that have not been held, but are collected and tank water is then added. Fertilization rates are near 100%. Growth to epiboly is around 80%. Growth beyond this stage is 10%. Thanks.
Rather than using tank water to grow them, maybe try raising them in E3 medium, recipe here http://cshprotocols.cshlp.org/content/2011/10/pdb.rec66449.full?text_only=true
Are you incubating at 28degrees C? often you get epiboly issues if the water temperature gets too cold
I'm having the same problem at the moment. Have you resolved your problem? If so, what did you do?
The problem persists. My suspicion is that the fish are too inbred. These EKK strains are possibly the issue. We got some from the pet store and these gave better results. We are still working on this issue.
Fungal and bacterial contamination of the fish, sperm and eggs seem to be the cause. I am halfway to solving this. We are incorporating the state-of-the-art non-toxic anti-fungicides. A professor in our chemistry department is synthesizing our compounds (lucky).
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