raising dechorionated embryos

Chi-Bin Chien chi-bin.chien at hsc.utah.edu
Fri Nov 19 14:36:51 EST 1999


Hi Pam,

>Has anyone had luck in raising zebrafish embryos that have been
>dechorionated?  I've been getting about 60% to live past the critical day
>8-10 period but hope to find a way to increase this percentage.

Do you think that the dechorionation procedure is having late 
effects on survival? That is, are you having special problems with 
dechorionated embryos, or are your naturally-hatched embryos 
also dying?

You should be careful with the young guys, as their yolks are quite
delicate and can be scratched or stick to plastic. Keep them in 
glass dishes until ~24 hpf.

>The protocol I am currently using is as follows:
>    - dechorionate embryos at 10 hpf
>    - raise them in embryo medium
>    - from day 3 to day 5, gradually replace the embryo medium with fish
>    water - begin feeding on day 5

We usually keep them in embryo medium until day 5, then switch 
to system water. Gradual replacement is not necessary, in my 
experience. You don't mention what you're using to feed, and when 
you start water changes. We have had good luck with the Tetra AZ 
baby powder, raising them in a 2-liter Marine Biotech tank, starting 
water changes at about 8-10 days pf.

I'm appending our baby-raising protocol. Hope that this helps.

--Chi-Bin Chien

1) Bleach the embryos at 1 day postfertilization (pf), and add 
Pronase to assist hatching (10 ul per 100 mm dish of a 30 mg/ml 
stock). Put into a new 100 mm petri dish after bleaching.

2) Raise embryos to 5 days pf in a 28.5 degree Celsius incubator.

3) At day 5, transfer embryos to an MBI 2-liter tank, in system 
water (we have also sometimes used E3). We usually put 60 
embryos in each tank. These tanks just sit on the shelf for the next 
few days.

4) Feed a little bit of baby powder (Tetra AZ New (0-160 micron), 
the same stuff that we used in Tuebingen) once or twice a day. 
This powder floats on the surface and the babies swim to the 
surface to eat it. They cannot eat very much yet, so it is important 
not to overfeed.

5) At about 8-10 days pf, put the baby tanks into the water system. 
To keep them from being washed out, we put a piece of 100 micron 
nylon mesh behind the MBI drain plug. This is checked twice daily 
for clogging. We continue to feed them twice a day with the baby 
powder.

The babies are *not* yet put on constant water. We just turn on the 
water (a fast drip) just before feeding, for about half an hour. This is 
enough to exchange about one tank volume of water. It is important 
to change the water, as babies are very sensitive to water quality.

6) At about 12-14 days pf, the babies are usually large enough to 
start feeding artemia. We feed them just a few brine shrimp to see 
if they eat them. For the next few days we also continue to feed 
powder as well, in case there are some fish that are a little too 
small to eat brine shrimp yet.

7) By three weeks, the babies should be large enough to put on a 
slow, constant water drip. We feed them brine shrimp twice a day, 
enough that their stomachs are bulging.

8) By five weeks, we start mixing some ground-up flake food with 
the brine shrimp.

9) At two months, the juveniles are large enough to go onto full 
water flow, and get fed the same as adults. (Brine shrimp twice a 
day, flake mixed in once a day; we feed with squeeze bottles and 
just add the flake in the squeeze bottle.) 

< Dept. Neurobiology and Anatomy 
|       office: 1-801-585-1701 > < Univ. Utah Med. Center         
|          lab:1-801-585-1702 > < 50 North Medical Drive.        
|          fax:1-801-581-4233 > < Salt Lake City, UT  84132      
|  chi-bin.chien at hsc.utah.edu > 
<http://molbio.med.utah.edu/chien/chien.html>





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