[Zbrafish] Re: Unfertilized eggs (Heather S McAllister)

[Tiago Filipe Baptista da Rosa Repolho]

Dear Heather,

My opinion is that you have several problems that you 
should solve in the Wt and Quanrantine Room:

- Feeding: you should try to change your feeding regime, 
i.e. giving diferent food items to fish and also to feed 
them less but more often (if it is the case of your 
feeding regime). If you want I can send you our feeding 
regime from our lab 
(http://www.bio.ua.pt/ZebraLab_UA_Webpage/Zebralab.htm).

- Concerning water parameters:
    - pH should be around 7 (check also carbonate hardness 
of the system to avoid pH "up and down"). It is better to 
have a system with a suboptimal pH than to have one always 
bouancing up and down (very dangerous to fish.
    - Ammonia and nitrite levels should be 0 (optimal). 
Remember that these are very toxic to fish (as an example 
high levels of nitrite cause what is called 
metahaemoglobin in fish whereas nitrite molecule binds to 
blood haemoglobin and does not allow O2 as well as CO2 to 
bind, resulting in anoxia as well as hipercapnia events 
and later death).
    - System water conductivity should have a narrow range 
(for example 450-550 microsiememens/cm) instead of 
300-600. If the conductivity has a broad range daily, fish 
will be stressed dur to osmotic pressure variation. In the 
case of Zebrafish (even though it is a registant fish) it 
can disrupt both the behaviour as well as reproductive 
capacity/performance.
    - RO water: Check if your RO purification unit has 
cationic as well as anionic resines because for an 
instance, if it is not equipped with those, phosphates can 
pass through the RO membrane and disrupt the system.
    - O2 levels: Always maintain them above 4ppm. Do not 
over saturate the water because then you might start 
having "bubble gas problems/disease" on your fish.
    - Check water flows on tanks.

- Environmental enrichement: Try to insert "underwater 
fake plants" on the tanks. Remember that these fish are 
originally from Rice fields and slow moving water streams, 
whereas plants exist.
- Fish density in the tanks. It depends on the stage of 
fish: Normally we have:
    - Adult: 2 adults per 1L tank
             5 adults per 1.9L tank
             10 Adults per 3.9L tank

If you have spare tanks on the system do not overcrowd 
them. Fish get stressed in higher densities.
It is like humans:
If I lived with 10 of my friends at my place instead of 
just me and my wife, it could be fun in the first day but 
after a few days I would start to be a little bit stressed 
as well as them, unless it was a huge house in a 
countryside farm.
I hope I could help,

Kind Regards,

Tiago

Tiago Filipe Baptista da Rosa Repolho
Departamento de Biologia
Universidade de Aveiro
Campus de Santiago
3810 - 193
Aveiro
Portugal
e-mail: trepolho at bio.ua.pt
web: http://www.bio.ua.pt/ZebraLab_UA_Webpage/Zebralab.htm



Em Fri, 11 Aug 2006 12:00:07 -0500 (EST)
  zbrafish-request at oat.bio.indiana.edu escreveu:
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>   1. Unfertilized eggs (Heather S McAllister)
> 
> 
> ----------------------------------------------------------------------
> 
> 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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> End of Zbrafish Digest, Vol 15, Issue 5
> ***************************************

><((((º>`·.¸><((((º>`·.¸><((((º>`·.¸><((((º>
Tiago Filipe Baptista da Rosa Repolho
Departamento de Biologia
Universidade de Aveiro
Campus de Santiago
3810 - 193
Aveiro
Portugal
e-mail: trepolho at bio.ua.pt
web: www.bio.ua.pt
><((((º>`·.¸><((((º>`·.¸><((((º>`·.¸><((((º>