Hello, the 1st thing that jumps to mind is your light cycle. If you dont have a consistent 14 on 10 off the fish wont give. Make sure your light cycle is properly functioning, often light cycles can be overriden by a switch leaving you with constant light. Are other fish in the lab producing?

Age and diet would be my 2nd and 3rd. 

There are other fish that live in the same cabinet that give eggs readily.  The first thing that we checked was the light cycle too. We feed our fish twice daily with zeigler adult complete diet and supplement with brine shrimp and spirulina three times per week. The fish for the majority are about a year and a half old.

Well this is a bit trickier then.

Well, you could try seperating the males and females in to two fish tanks, and in the spawning traps set them up with a divider the day before and remove the divider when the lights come on the next day.

I will just throw out a few suggestions to see if anything sticks:

Fill the spawning cages half-full with sys. H20 and tip up one end of the trap to create a gradient in the cage.

You always want to be +1 female ratio-wise in a mass spawn. 

If you leave the cross up an extra day change out the trap water. 

Dont spawn the same fish more than once every 7-10 days, you can use multiple tanks to sort these out. 

How is your nitrogen cycle? 

Do the fish appear to be in good health?

Make sure your fish are eating the dry food and just not holding out for artemia. 

Here is a link to our SOP on spawning, http://staff.washington.edu/dgw5079/ (spawn link on right side of the page), but I am sure you familar with the simple techniques descriped there. 

Hello Gina,

 Dave has given some good advice and ideas on things to look into and try. 

I would also ask how often these fish have been bred before this?  You are at the mark, being 1.5 years old, where these fish will start to decline.  Depending on the line and the background of the fish they may already have hit the wall and won't be very good.

Were they stagnant and not breeding for an extended period of time before this? 

Are they newer fish to this cabinet?  Even though you have fish breeding in there now if these fish are new they could still be adjusting to water quality etc?

Some some food for thought.

Thanks, Joe

We do not have any requirements for PPE on water quality testing at ABSL1 level. In general we follow the MSDS recommendations for our dosing system and water testing chemicals.

We also had no requirements for eyewash stations in our fish rooms according to our institute's environmental health and safety. 

Hey Doreen!

Interesting question.  Since leaving NIH and working with other facilities, I have not found one place that requires eye wash in the aquatic rooms at least not specifically for what could come from testing chemicals.

Many places will do water testing outside the rooms after taking samples.  this is either due to safety of not getting chemicals in the system or using an actual lab where they take the water sample.

As far as PPE in the rooms I have seen everything from nothing to just gloves to glove, booties, and lab coat.  Also places that require "fish room shoes" where they will wear dedicated shoes for the fish rooms that they leave on a rack in the vestibule area. 

In actuality this is another one of those things with aquatics that has no standard. 

Thanks - Joe

Hi David,

We didnt find a significant difference in survival between similar feeding regimes with rotifers or paramecia.  But we did with growth.  Specifically rapid growth on rotifers for a certain number of days in order have fish feed on Artemia.  

Yes, ease of culturing is also an important aspect of the diet you might choose to feed your larval fish.  Although rotifers and paramecia are very different to culture I have found both relatively easy.  The main exception with rotifers is their exceptional ability to gut load making them a much more suitable larval diet.   

One aspect I point out in the talk regarding rotifers is their ability to form a polyculture within each inoculated tank.  Especially when fed multiple times.  This increases density and in turn increases the rate of prey encounters.  

We did attempt to use challenged lines, specifically Casper.  However, they proved to be much more difficult to photograph in the larval stage vs. adult.  Either way, we do feed our rotifers to Casper and we have noticed rapid growth and an increase in survival with those fish.  

Thanks for your comments and interest in the study.  

Hi Bill,

I am happy you enjoyed the presentation.  

Concerning your question: we used the Panasonic DMC-LX3 with a tripod to take pictures of fish in petri dishes from above.  I tried to minimize the volume of water in each petri dish so all fish remain at approximately the same depth.  This makes for more accurate measurements because the camera focuses on only one plain.  Two shots were taken of each dish and the best image was used for measurements.

Using the Image Tool software, I manually calibrated a known measurement from each picture and then manually measured 10 fish chosen haphazardly.  The only criteria for a fish to be measured was that it had to be straight (no curvatures in the body or tail).  This means that there are only two clicks to measure the fish (from the anterior end to the posterior end).  As far as I know Image Tool does not have a way to measure by any automated process.  Although this would be a tremendous feature.  

http://ddsdx.uthscsa.edu/dig/itdesc.html  

Hope this answers your question.

Thanks again for attending. 

Katie,

We also feed dry powder from 5 dpf to about 10 or 15 dpf and then start feeding Artemia in combination with the dry powder.  The larvae start getting Artemia when they are visiually large enough to consume them, we inspect the tanks three or four times daily and make changes as needed.  You can easily see if the larvae have consumed the Artemia by the orange bulging stomachs.  We feed instar 1 nauplii  and our larvae certainly are not able to consume them at 5 dpf.  The species of Artemia could be a factor.  There are several different species and their size at the instar 1 stage can vary depending on species and region from which they came.  

I beleive healthy Artemia can survive a couple of hours in freshwater.  We run our conductivity at 625 uS and I have seen the Artemia survive for five or six hours.  The more you have in a small tank or container the faster they will die.  Adult zebrafish will eat dead Artemia but, larvae are much more reluctant to do so.  I would not leave Artemia set in a container dead throughout the day and then feed them to the fish in the evening as the dead Artemia will quickly create very favorable conitions for fungus and bacteria to grow.  If you save some for the evening feed I would try and keep them in saline water with air flow until they are ready to be used. We feed Artemia twice per day and harvest a fresh batch each time.  We also feed the larvae first to ensure they get the freshest Artemia possible as it takes at least 1.5 hours to feed the whole facility.  If the Artemia are freshly dead I would still use them but, if I did not know how long they have been dead or if there is any kind of foul odor to them I would not use them. The water often starts to turn orange if they have been dead for an extended period of time.

I hope this helps.

Jim Burris, Duke University

certainly we have seen that the fry need only a day or two of reasonable quality feeding before they can wean -  readily - onto artemia.   this is key because once they do this, their growth rapidly increases and their survival stabilizes.

it is possible to present artemia as the first item at day 5 or 6, but the feeding is very inefficient for a number of reasons.  gape size is an issue - the fry cannot consume the nauplii whole so must take swipes and pieces off the carapace of the animal.  to do this they must be able to position themselves in the water effectively and catch up to fast swimming nauplii.  the problem is that early swim up zfish larvae are not the strongest swimmers - it takes a few days after swim up (with concurrent feeding) before they are strong or fast enough.   in our hands it takes a full two days or more before they seem to wean on to artemia if presented with nothing else.  survival is ok, maybe 60-70%, but it's variable.  it is likely that they eat whatever else they can find in tanks; bacteria, slime molds, ciliates, etc.  but only after a number of days will everyone in the tank wean on. it's during those days, especially the latter part, where the survival drops.

what we showed in our experiment is the value of a smaller, slower live feed item to bridge that gap.  both paramecium and rotifers work in this regard, but rotifers performed better, presumably because we can get their densities higher (and hence increase encounter rates) in tanks and they are of a higher nutritional content because of their superior gut loading capacities.  we have also used a powdered, encapsulated diet to get them to artemia, but it was messier, more time consuming, and more expensive.  but it worked, comparing favorably to our results with paramecium and rotifers. 

in short, it seems that this gap is one to two days post swim up.  cover it with a live feed or a reasonably well designed dry formulated diet.  but get them to Artemia as soon as you can.

a few comments about Artemia --

- make sure you're feeding out the artemia as close to hatching as possible.  the longer they go past hatching (15-30 hours post set up depending on conditions), the larger they get, and, if you're not feeding them, the less nutritious they are.  either way, first feeding larvae don't have a chance.

- you can culture artemia at lower salinities- don't need to do it at 33ppt.  you can go as low as 5-10 ppt depending on cyst origin (you have to test them).  artemia cultured at lower salinities will 1) hatch faster, 2) have higher energy content after hatching than those cultured at higher salinities, and 3) will live longer in fresh water than those cultured at higher salinities.  we culture ours at 5-8ppt.

- most brine shrimp nauplii, even if cultured at 33 or 35 ppt, will survive in fresh water for hours.  the best way to feed out is harvest and immediately feed out after hatching, but if you are going to harvest once and stash for later, you can put them in fish water and throw them in the fridge and they will be ok for 8 hours or more. 

good luck