[Zbrafish] Re: Unfertilized eggs

Wilson Clements wclements at ucsd.edu
Fri Aug 11 18:20:35 EST 2006


Dear Heather,

We have also confronted the problem of intermittent reluctance of our  
fish to mate.  I have several suggestions to try, although we have  
yet to find THE solution.  Here are some things that helped us.

1) I find I get more eggs and better fertilization when I set up  
multiple fish in a single breeding chamber.  For small breeding  
chambers, this usually means three females and two males.  I also do  
two females and two males.  Never more males than females.   
Especially if the problem seems to be lack of fertilization and not  
failure of the female to throw her eggs, I think this solution might  
help, since even if one male isn't doing his job, the other still  
might be able to step up.  In slightly bigger chambers you could try  
four and three, or five and four.  An added benefit is that you have  
to put up and take down less total chambers.

2) We get better lay rates when we set up females of one tank with  
males from a different tank and vice versa.  Of course you need to be  
able to sex them reliably in order to put them away correctly.  That  
this might be helpful to you is also suggested by the fact that  
you're getting pretty good clutches when you out-cross, but not so  
good when you in-cross.  We have also had pretty good success with  
increased fertility out of hybrid lines, such as WIK/AB* (sometimes  
called the "kick-ass line"; Griffin, K.J.P., personal  
communication).  If you're not planning on trying to do genetic  
mapping, trying to get some WIK/AB*s or another hybrid line going  
might be worthwhile.

3) I try to shield fish from activity in the room when I want them to  
mate.  In my old lab our cage inserts in the breeding chamber were  
not clear, but rather a translucent white plastic that allowed light  
in without letting the fish see what was going on in the room.  I  
think this kind of cage is ideal; unfortunately I can't find anyone  
that makes them.  As a substitute in my new lab, I place baffle  
screens or green plastic lid containers in front of and on top of the  
crossing chambers so that they are less able to see motion in the  
room. I think also a shower curtain or something would work.

I'm not sure if it matters, but we run our pH a little higher than  
you:  7.2-7.4.  I'm guessing that the main thing is not to have big  
fluctuations, and that the fish can deal with a static pH over quite  
a range.

Best,
Wilson
------------------------------------------------------------------------ 
--------------------
Wilson Clements, Ph.D.

wclements at ucsd.edu

Dept. of Biology
Section of Cell and Developmental Biology
University of California at San Diego
9500 Gilman Dr.
Natural Sciences Building 6324
La Jolla, CA 92093-0380

TEL    (858) 534-6955
LAB    (858) 822-4658
FAX    (858) 534-5457


On Aug 11, 2006, at 10:00 AM, zbrafish-request at oat.bio.indiana.edu  
wrote:

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>    1. Unfertilized eggs (Heather S McAllister)
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> ----------------------------------------------------------------------
>
> Message: 1
> Date: Fri, 11 Aug 2006 12:14:07 -0400
> From: Heather S McAllister <hmcallis at Princeton.EDU>
> Subject: [Zbrafish] Unfertilized eggs
> To: zbrafish at magpie.bio.indiana.edu
> Message-ID: <fb01939617276.44dc748f at Princeton.EDU>
> Content-Type: text/plain; charset=us-ascii
>
> 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
>
>
>
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