[Zbrafish] obtaining eggs and sperm

Christian Lawrence via zbrafish%40net.bio.net (by pinaleno from gmail.com)
Fri May 11 15:53:19 EST 2007


I've never seen oviposition without males before.

That aside, assuming that you have one 5 gallon aquarium set up I would recommend doing the following in order to be able to strip viable eggs and sperm:

1.  Bump conductivity up to 500uS.

2. Do not add methylene blue to the water.

3.  Make sure your water in the tank/system  is buffered. You are buffering makeup water but depending on alkalinity and frequency and percentage of water change, your pH may be running unacceptably low. Is it always 7.6 in the tank?  If so, then good.  If not, or if you don't know then chances are you might have a pH problem.

4. Get some more females.  10-20 more will do.

5. Feed 3-4 small meals over course of day.

6. Facilitate natural mating so you know eggs are spawned.

7. 1 week later do big water change in morning.  That evening take females out and keep in static (filtered better if you can get it) holding tank(s) of fresh fish water  at densities of no more than 2-4 fish per liter.  If possible simply separate males from females within tank, but if not possible, then static tanks as described ok.

8. Next morning do IVFs when lights come on.


See if this helps with your problem.

Best,
Chris


                                                     
Christian Lawrence
Brigham & Women's Hospital
Karp Family Research Laboratories
One Blackfan Circle
Boston, MA 02115
Tel: 617.355.9041
Fax: 617.355.9064
    

-----Original Message-----
From: wratzan from uchc.edu
Date: 10 May 2007 15:05:49 
To:bionet-organisms-zebrafish from moderators.isc.org
Subject: [Zbrafish] obtaining eggs and sperm

Hello zebra fish community!

I am new to working with danio rerio and I am trying to conduct some
experiments which will involve in vitro fertilisation.  Today I
manually expressed eggs and sperm for the first time.  I was
successful at both, but the eggs were very small (250 micron diameter)
and did not fertilise.  Furthermore, a few of the females ovulated
groups of similarly immature oocytes in the holding tank on their
own.  Has anyone seen this before?  Do you know what could be the
reason for the immature oocytes?  What can I do to rectify this?

The only thing I could think of is that it is that the lights go on in
the room four hours before the aquarium light and that some ambient
room light is leaking into the top of the tank through the cut-outs
where the filtered water pours into the tank.  I started collecting
gametes just before the aquarium light was about to go on.

The water is at 200 uS conductivity (by adding sodium bicarb and sea
salts and HCl to pH 7.6, 1 drop of methelyne blue per gallon).  The
fish were purchased from Carolina Biological about two months ago, and
about ten males and ten females were mixed in the same 5 gallon tank
together ten days ago.  I feed them flake food and live brine shirmp
evey day, and they are on a 14 hours of light to 10 hours dark cycle.

Thanks in advance for any suggestions

-William Ratzan
University of Connecticut Health Center
(860) 679 2677
wratzan from uchc.edu

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