[Zbrafish] Unfertilized eggs

Christian Lawrence clawrence at rics.bwh.harvard.edu
Fri Aug 11 20:19:40 EST 2006

(WARNING to all:  This is a LONG reply, but in my opinion, justifiably
Heather --

Once again, it is difficult to say for sure what the problem (or likely
problems) might be, but given the info you supplied, I can venture an
educated guess as to what might be going on with your fish.

Your problem, at least proximately, is most likely with your females. 
The fact that
the males from the wt population spawn and fertilize well when mated
against females from the mutant facility is not at all surprising -
males will almost always be fertile for longer and under more adverse
conditions than females will be.  At the most basic level, this is
sperm much more energetically cheap to produce than eggs are.

The fact is that females are much more high maintenance (no pun
intended).  Because eggs are more expensive to produce, it takes much
more out of them, especially under intense conditions, and consequently,
they break down sooner than males. This is exacerbated under suboptimal

Females are also much more difficult to maintain in top breeding
condition because of behavioral interactions among other females.  Work
done by Gabi Gerlach from the MBL in Woods Hole demonstrated that
females will quickly form dominance hiearchies when held in same social
situations with one another, probably via olfactory cues (pheromones).
These pheromones repress ovulation in subordinate females.  This
phenomenon is quite common in zebrafish facilities.  Take 4 or 5
females of similar reproductive condition and hold them together for a
few weeks in the same tank, and over time, you will see one or two
females dominate the others.  The dominant female or females will
continue to ovulate (evident by body shape), while subordinates will no
longer cycle (again this will be evident by body shape).  Hold them long
enough, and the subordinates will not only become senescent, but they
will typically start to get sick, because energy shunted away first from
ovulation, then somatic growth and finally immunity to deal with stress
dealing with aggression of dominants.

There are a few ways to get around this, including:
1. Mix and match individuals, particularly females, such that
individuals are not held in stagnant social situations for longer than
a week or two.
2. Do frequent, higher volume water changes before breeding to flush out
pheromones that are typically not metabolized by bacterial flora in your
3. Set crosses up in fresh (not from system) fish water.  This can be
difficult logistically in many setups, including AHABS (which I believe
you have), since most (AHAB, MBI, Schwarz, etc.)  are designed to take
water for crosses from system reservoirs, but if you have a reserve
tank of fish water, you may be able to use this for crosses.  Again,
this gets rid of pheromones and evens the playing field a bit.  Another
thing you can do if you don't have designated fresh water reserve is to
dilute system water used in crossing cages with RO.  50% will work
well, and this drop in conductivity usually promotes ovulation.

Your conductivity is a little on the low side, particularly if you're
running at 300uS.  While zfish are adapted to deal with low
conductivity, it is physiologically stressful for them to maintain at
this level, and at least one study has showed that breeding success
decreases with lower holding conductivities,  presumably for this
reason.  Bringing it up to 600+ ( we keep at 1000-1200 and others keep
at up to 2000) would probably help some.  This would also enhance the
effect of dropping it when setting up crosses (e.g. going from 1000 to
600 is better than 400 to 200).  The few studies done in wild
populations indicate that zfish are found at much higher conductivities
(2000 +), particularly during the dry season.

You and others may wonder why things seem to work fine for a long
periods of time at low conductivity levels, or other seemingly
suboptimal conditions (incompletely balanced diets, poor buffering,
etc., etc.) but this is likely the reason why fish in these situations
abruptly crash and burn at less than a year.  As things get tougher,
the window of time during which they can perform physiological
functions at high level (spawning 1-2x weekly) grows smaller.

A few other things to consider in closing..

The more outbred your wt populations are, the better.  The more closely
related they are, the less efficient breeding will be.  Natural
selection discourages inbreeding.  We can overcome this, certainly, in
culture, but the effects are still evident.  Always make sure you use
maximal number of founders when propagating next generation, and if
possible, carefully breed in individuals from outside sources to
enhance variation (i.e. import Abs from trusted outside source every
several generations and breed into your current Abs).  This is a tricky
problem that everyone deals with, especially when doing genetics.  But
it must be dealt with in most thoughtful manner possible.

It is best to have new generations coming up every 6-8 months, so that
once your prime stock starts to tail off, you can simply retire them
and move on to the next generation.

Mutant lines should be maintained by outcrossing.  This is particularly
the case when using viable homozygous pigment lines such as nacre or
albino.  If you?re generating from homozygous incrosses, your lines
will quickly go downhill.

Somewhat of an exhaustive post, I know, but this stuff is so much more
complex than we in the zfish biomedical culturing field typically make
it out to be, and taking all information regarding the biology and
natural history of the animal and applying this to husbandry standards
is necessary and important to increase efficiency and avoid problems
like the ones you're experiencing now.  The beauty and the curse of
this model is that because zfish are so tough and adaptable, you can
abuse them and still get results.  Over time, however, this strategy
doesn?t work, and so it makes sense to bring all info to bear when
designing management strategies for maximal long-term efficiency.

 Good luck, and of course feel free to question or refute any of the


Quoting Heather S McAllister <hmcallis at Princeton.EDU>:

> I took some of the suggestions I previously the, and it seems that
> our largest facility is back to normal.  The fish are laying great.
> These are all our mutants.  However, the room where we keep our
> wildtypes as well as the larvae have stopped laying almost
> altogether.  For instance, last night I set up 24 tanks and 5 laid,
> which is well below what we have previously experienced.  Two of
> these clutches were unfertilized, and one consisted of 12 eggs, and
> the other two were unhealthy.  This problem seems to be repeated on a
> daily basis.  The fish I set up have previously laid great clutches,
> even up to about a month ago.  Most were about a year old, though
> some were younger.  I set up WIKs, TLs, Nacres, and ABs.  The WIKs I
> didn't expect to lay that well.  I also set up 28 tanks in our
> quarantine room, and two laid, unfertilized clutches.  Again, they
> are pairs that previously laid well.  I am unsure the age of most,
> but at least 3 pairs were less than a year old.
> All of our rooms are supplied with R.O. water from the same system.
> We have an AHAB system, with 1L (between 1 and 4 fish), 2.75L
> (between 10 and 20 fish) and 9L (up to 50 fish), but the density of
> fish in the tanks are about the same in both rooms.  We monitor the
> water quality with a YSI system.  The pH is generally around 6.8 and
> the conductivity between 300 and 600.  The temperature in the main
> facility is about 28 degrees, while the wildtype room and the
> quarantine room are about 30 degrees (I know this is a high temp, but
> it has been consistently higher, so I am inclined to think that isn't
> the issue.)  I test the NH3, NO2 and NO3 once a week, and the nitrate
> is the only one that is ever elevated, and no more than 30 ppm.  As
> far as I know, the town hasn't changed the method of treating the
> water (but both rooms use the same water).  The light cycle has an
> alarm so that we will know if they are staying on all night (we
> previously had that problem), plus people are som
> etimes down there when the lights go out, so I am confident that is
> not the problem.  The only other difference between the two rooms is
> the lighting.  The main facility has lower lighting than the wildtype
> room, mainly because there are more racks arranged differently.  We
> feed Zeigler food in the morning and brine shrimp in the afternoon,
> but I think the breeding has improved since we switched to the
> Zeigler's.
> Another thing that is making this frustrating is that when wildtype
> males are taken from that WT room to be bred with a mutant female in
> the main room, the eggs are almost always fertilized.  When WT are
> incrossed, more often than not whatever clutches are laid are
> unfertilized.  They are set up about once a week.  We do a lot of
> injecting in our lab and this is really starting to impact the
> research.  Any suggestions would be greatly appreciated.
> I apologize for the length of this submission, but I hope that I have
> provided all the information one might need to know.  Thanks.
> Heather McAllister
> Research Specialist
> Burdine Lab
> Molecular Biology
> Princeton University
> Washington Rd.
> Princeton, NJ 08544
> 609-258-5782
> _______________________________________________
> Zbrafish mailing list
> Zbrafish at net.bio.net
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