Requesting help keeping young fish alive in small

Richard Gordon gordonr at cc.UManitoba.CA
Tue Aug 10 12:45:55 EST 1999


Dear Barbara,
We've kept zebrafish embryos/fry alive for 2 weeks (at which time 
we let
them swim free) in methylcellulose. The trick seems to be the 
viscosity,
which impedes their motion, so their energy utilization is low, so 
their
yolk absorbs more slowly, so they can go much longer without 
eating.
Sounds like the time period might well overlap development of the
vestibular system, with which I'm not familiar. An advantage is that, 
on
earth, you could look at development of the vestibular system with the
embryo forced to have a specified orientation relative to gravity. See:

King, G. M., R. Gordon, K. Karmali & L. J. Biberman (1982). A new method
for the immobilisation of teleost embryos for time-lapse studies of
development. J. Exp. Zool. 220(2), 147-151.

I'll be in Davis Sept 3-4 for the Joel Keizer memorial symposium
(http://www.itd.ucdavis.edu/joelapalooza/), if you'd like to discuss it.
Would be glad to volunteer. Best, -Dick Gordon

>Dear Zebrafish community--
>
>    I am currently involved with a NASA project trying to send
>zebrafish
>embryos into space (on NASAs space shuttle) in order to study
>the effects
>of microgravity on the development of the vestibular system.
>
>    The problem we are having is that we need to keep embryos
>alive for 19
>days in a very small volume of liquid (35 mls).  Due to the
>constraints of
>the hardware, there is no way to have access to the aquaria to
>change
>water, etc. during flight.  The only possibility to add anything to the
>aquaria during flight is through an automatic mechanism (designed for
>injecting fixative, but which we will not be using for that purpose)
>which can inject 1 ml of liquid once during the flight.
>
>    What we do now:  Currently the fish are allowed to develop
>normally
>at
>28.5 degrees C until 10 hours post fertilization.  At that point we
>remove
>the chorions enzymatically using pronase, and transfer the
>embryos to
>10%
>Hanks solution containing PTU (to prevent melanin formation).
>>From this
>point on the fish are raised at 23 degrees C to slow down
>development.
>Fish are loaded into the small (35 ml volume) aquaria at 12, 36,
>and 60
>hours post-fertilization.  40 embryos are loaded in each aquarium.
>Paramecia are provided as a food source.  The aquaria are sealed,
>but
>provide access to air through teflon membranes.
>
>    The problem:  We get good survival (about 75% survival) and
>normal
>(though slowed) development of zebrafish under these conditions for
>aproximately 14 days.  After this there is a precipitous drop in
>survival,
>with all or nearly all fish dead by 19 days.
>
>    Solutions we have tried so far:  1) Lower number of fish in each
>aquarium.  This has not seemed to make
>a difference in survival-- even with as few as 2 fish per aquarium we get
>no significant survival at 19 days. This suggests the possibility that
>something else may be causing problems other than just the build- up of
>waste products.  2) Using fish Tris (Sigma) in the Hanks solution to
>better buffer pH and remove carbon dioxide also made no difference in
>survival.  3) Oxygenating the 10%Hanks at the beginning of the experiment
>by bubbling through 100% oxygen makes no difference.
>
>    We have noticed that there are very few paramecia left by 14
>days,
>so
>there is some possibility that the fish may be starving.  We have
>tried
>loading more paramecia with the fish at the begining of the
>experiment,
>but this actually seems to cause earlier death, presumably due to
>increased biomass causing more problems with waste products
>early in the
>experiment.  We are currently trying to use the fixation devise to
>inject
>more paramecia into the aquaria latter on in the experiment. We
>don't
>have
>results from this yet.
>
>    It has been suggested to us that adding a plant type to the
>zebrafish/paramecia ecosystem might help provide oxygen and
>remove
>wastes from the solution.  If anyone has any experience with using
>single- or
>few-celled plants in such a manner, advice would be appreciated.
>
>    Any other suggestions for maintaining zebrafish health under the
>conditions described above would also
>be greatly appreciated.
>
>    In addition, NASA would like to find 2-3 scientists willing to serve
>as advisors/peer reviewers for this project.  This would probably
>involve
>some e-mail and/or telecon exchanges, and the possibility of a trip
>(at
>NASAs expense, of course) to NASA's Ames Research Center at
>Moffet
>Field,
>California (Near Silicon Valley).  If anyone has any interest in
>volonteering for such a position, please let me know.
>
>Thanks very much.
>
>I would appreciate replies by e-mail.
>
>Barbara Chapman
>bxchapman at ucdavis.edu

Dr. Richard Gordon, Radiology, U. Manitoba, HSC, 820 Sherbrook Street,
Winnipeg R3A 1R9 Canada, Phone: (204) 789-3828, fax: (204) 787-2080/ Book
just out: The Hierarchical Genome & Differentiation Waves: Novel
Unification of Development, Genetics & Evolution:
http://www.wspc.com.sg/books/lifesci/2755.html, E-mail:
GordonR at cc.UManitoba.ca




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