Survey/Current husbandry of zebrafish – Diana Baumann, Zoltan Varga

DIANA BAUMANN: Thanks very much, Steve. My name is Diana Baumann, I’m at the Stowers
Institute, which is a research institute for biomedical research, and there I have the
pleasure of running the aquatic facility, where we work with multiple invertebrates,
aquatic species, as well as vertebrates, which does include zebrafish. I was also on the organizing committee, and
Zoltan and I put together, with the committee, a survey that we sent out. We advertised it at the recent zebrafish meeting
in Madison and then also sent it out, too, throughout the zebrafish community through
multiple channels. What we’re doing here really speaks to Steve’s
point when he gave his introduction on the second objective of this workshop, which is
learning about our current status, where we are. We will see insofar, we’re not very far. That’s a good starting place to know where
you are to be able to build on it. So … number of questions that were sent
out. The first one was “At what life stages do
you use?” We had 63 respondents to our survey. Sixty-three, and so if you look at the total
here of 244, you can see that we had many, many groups who were using animals at all
the different stages, from the embryos, the larvae, through the young adults, and through
even to aging studies. This really speaks to the diversity of studies
and the complexity of research that is being done in zebrafish these days. We’ve moved well beyond the embryonic development
beginnings, which is still very solid, but I was really surprised, and if we just put
that in graph form, just how many varieties there were. Although, of course, the predominance, as
you can see from the top two bars, is still on the embryonic work, but as it was very
nicely brought up in the last comments, to get good embryos for good research, you need
good breeders, and good breeders come from good nutrition. And we don’t really know what’s in that
embryo and what’s in that yolk sack unless we start defining things. The next question that we asked were “What
diets you do provide to your fish?” And going back to the historical perspective
that Chris provided for us, we did see out of our 63 respondents, that Artemia came in
highest in that traditional beginning of the Artemia, the brine shrimp, combined with the
flake food. The majority of our facilities in our respondents
were using Artemia. GEMMA came in next as the top used dry commercially
available food, followed by Zeigler and then next live food would be the rotifers. Now many people, but not all, were using the
rotifers in a polyculture, and this is where you start your larvae out in what looks like
a green suit, where you have the larvae and the rotifers and rotifer feed all coexisting
together, which removes that manual labor. I suspect it has some other challenges of
its own, not to mention breeding rotifers in the first place. Flake food, still very popular, predominantly
TetraMin, but there were a number of other mentioned vendors, and I shouldn’t have
done, but I also threw in algae wafers, which were being used by a few facilities into the
flake food category to make that 17. Paramecia, the last of our live foods that
came up, coming in at 14 of our respondents, and so about 22 percent. Golden Pearls came up as a similar number
to paramecia of our respondents, that might well be because the master mix that’s published
on the (indiscernible) website is actually a mix of the Zeigler spirulina and Golden
Pearls, so I think that might raise the awareness of Golden Pearls, particularly in the U.S.
zebrafish environment. ZM is a diet out of the UK. There were some people that couldn’t get
a hold of this who wanted to be using it. Sparos, again, came up much higher in the
European survey that … there at Portugal, and they would also make small batch custom
feeds, which a lot of vendors aren’t able to do for us at this time. Spirulina, Argent Hatchfry Encapsulon for
your fry, and SDS—specialized diets services—they all came in with smaller levels of usage. And then there were ones, the very low frequency
ones, that just came up for one or two respondents and that included, krill, the Ridley NRD pellets,
Sera Micron, Hikari pellets, trout larvae food, tubifex worms, the ImmunoPro and caviar
that we saw more predominantly in the European survey, (indiscernible) starter, freeze-dried
bloodworms, mysis shrimp, which surprised me, I would’ve thought they were a bit large
for zebrafish, but there was one lab reported that, and then a number of self-made diets,
which I presume are some conglomeration of the previously-stated ones. Our next question was “why did you choose
the diets that you chose?” And the largest response was, “based on
experience.” And this particularly is looking at the two
things we mentioned so far: the growth rate and the fecundity/fertility, and you need
both. Because it’s no good having wonderfully,
100 percent fertile embryos if you’ve only got five, so you need to have a good number
of embryos with also good viability. Survival was also listed, but not nearly as
much as growth and the fecundity/fertility. Along with that also was just what’s worked
in the past. We’ve already heard that, you know, don’t
break something that’s already working. Recommendations from others was a large reason
why people were doing what they were doing. They’d either moved lab samples with them,
it’s what they were trained to do, a couple of people, Christian, referenced your 2012
aquaculture paper as a reason for doing what they were doing. Other literature searches were mentioned,
talking to colleagues, and also vendor recommendations, so the people that put in the housing systems
were advising on what foods to be used in their housing systems, so I was interested
to see that one come in as well. Next reason for using the food was cost, ease,
availability. Ease definitely plays into trying not to use
the live foods, because of the massive amounts of manual labor involved with that. Availability: Certainly, there were some diets
that some labs couldn’t use because of the location they’re currently in, and they
couldn’t get diets that are made in other countries, so that played in as well. Food composition: 13 groups out of 63 mentioned
food composition. Fairly low, and again, reflecting a lot of
what we’ve seen today, that it’s more important what we see as an output rather
than what we know is going in as an input. Those that did mention food composition, the
only component that was specifically mentioned really was protein being of concern by groups. Other: This is where we already see one of
the challenges that Christian mentioned coming into play, which was … for the food being
able to work with the equipment you use, particularly for those labs that are lucky enough to have
the automated feeders. Some foods work much better in the automated
feeders than others do (indiscernible), so that definitely came into play. Particle size based on the growth of the fish
was definitely a big issue in determining which foods (indiscernible) life stages. Also relating to equipment, there was one
group that talked about some foods leading to much more mold growth in the tank than
others, so looking at what that food does in your system, if you have overfeeding. Enrichment definitely came up here as a topic. There were number of groups that wanted to
stay with their live feed because of the enrichment aspect of the natural behavior. So those were some of the categories coming
in. There was one group … a lot of groups talked
about providing variety. There was one group, they said, “Well we
did a survey, and we talked to a lot of labs, and every individual lab said what they were
doing is the best. So we just took a combination of what they
were using and put them all together, that’s our mix.” So that was a novel approach. Our next question … oh, and six of the groups
that responded did talk about their own in-house trials to determine what would be the best
food input for their research fish. Next question was, “How often are your fish
fed per day?” You can see if I graph that out, twice per
day came out most common, but there was also, this is “other,” so combinations of above. So some groups—many, many groups, in fact—feed
different life stages different amounts of food, and the most common being that the juveniles
get more than the adults. If we look at what came up in the text responses
to this answer, we find that a number of facilities feed less on the weekends. It’s going back to the manual labor aspect
and having staff or students available on weekends to do that. So those facilities that normally feed twice
a day would go down to once a day on the weekends. There were other permutations, as well, such
as they if they fed two types of food, they’d put them both in the same feeding instead
of different times a day on the weekend, or they wouldn’t do any live foods on the weekend,
they’d only do dry foods on the weekend. So a number of variations there. But automated feeders … those with automated
feeders have the capacity to feed many more times in a day than those with manual labor
only, and facilities where they only have some racks with automated feeders and some
racks fed by manual labor, the racks with the automated feeders were receiving a lot
more feedings—not necessarily food, but a lot more feedings—than the racks that
were fed manually, so again, it comes to determining which fish you have on which racks and needs
for those studies. Stage of life: Definitely, there were a lot
more groups that were feeding the juveniles and the high-growth-rate groups, larvae and
juveniles, than were feeding the adults. There were also a number of groups that fed
their breeders more than their mates, and that’s …we heard that in mice at the (indiscernible),
and so a common thing was that adults would get fed one or two times a day, juveniles
would get fed three of four times a day, and then those groups feeding their breeders would
give their breeders one extra feeding a day. There was one lab that talked about the transition
of going from the growth feedings that lab fed four times a day down to the adult two
times a day, and they actually did a two-month transition, 4x, 3x, 2x. Most people I think just hit a certain date
post-fertilization and just do the switch from 4x to 2x or 3x to 2x. The next question that we asked was, “How
much are your fish being fed?” Large variance. Answers varying from no standard to controlled
measured amounts per fish and per number of fish in the tank, because that is also going
to affect it based on how many fish they’re interacting with, and their levels of activity
and behavior will determine that consumption as well. So, the highest predominance of answers was
“whatever they consume within a few minutes,” but there were also combinations of said results,
as well. A lot of the comments came up relating to
stuff we’ve already heard, so if you have rotifers being raised in a polyculture environment,
they have that ability to feed continuously. The juveniles get more, the breeders get more,
and those then those that do that actually gave a specific number said that it was between
1 and 4 percent body weight was where they were aiming at for the amount in a single
feed. We finished off, again, by looking at interest
in participation in intra-laboratory research, and this slide was looking at the desire to
determine the nutritional requirements. And the majority—again putting it in graph
form—the majority was strongly in favor. We’ve got over half of our respondents there,
and if you include the “agree,” “agree with support,” and “maybe,” we’re
looking at 87 percent of our respondents were interested, but the majority of them would
require support, whether that was in funds or free test diets to test. But there’s a lot of interest there. And tying in with that, our final question
was looking at whether they’d be interested in developing and testing a basic defined
diet with zebrafish. Again, very high amount of “yes.” If you look at the top three bars, that gives
us a 92 percent of our respondents were interested. Again, the majority would need support in
someway or another, but it does show there is a tremendous amount of interest in our
community, not just in having a defined diet, but in helping develop that defined diet. Whilst I agree that this is a self-selected
subset that responded, of those that did respond, we have a lot of people behind us here and
wanting to look at this. With that, I’ll hand it over to Zoltan to
give us some updates. ZOLTAN VARGA: Thanks for having me today. You know, it actually plays off really well,
because I’m really curious now to see, after I put together my slides, where we fit in. I have no clue, right? I really would like to give this presentation
with the thought in the background that we’re just another (indiscernible). We’re one of the many. And we have no food. Or maybe we have a little, but not much. Our mission is … we are genetic resource
center, we have wild-type transgenic mutant zebrafish. Our purpose is to acquire these, maintain
these, and redistribute these so researchers don’t have to do that, right? Instead of spending their research dollars
on researching international shipping documents, we do that for them, and the money they save
goes to research. In a nutshell, that’s our mission. Now, because our day-to-day operations are
so centered on customer service and client service, it’s really, really hard for us
to devote consistent time into researching new things. Yet we’re in the peculiar situation, that
because of our health services, we also consult with a lot of researchers and research facilities
about the fish health and how they run the fish facilities, and that includes (indiscernible). We may not be the best advisor, but that’s
why we’re here to learn. The first thing I want to talk about is our
food. As Diana already pointed out, what we have
is a mix of foods. The idea behind this is that for each life
stage we recognize the gape of the larvae, the gape of the juveniles, and that of the
adults changes, so there’s a limit of what they actually ingest without too much effort. From the get go, we have the larval mix …does
this work? No, okay. So we have a larval mix, which focuses on
the smallest food sizes, but it is also a mix of different food sizes, and it includes
rotifer diet, which is mixed in the in Zeigler diet in equal parts. The juvenile mix is, again, different particle
sizes of Zeigler diet and Golden Pearls, and the master mix is a mix of adults’ Zeigler
diet, Spirulina, and Golden Pearls. In the olden days before I was at the ZIRC,
a veterinarian looked at what information was available from (indiscernible), and then
decided to use the information that was available to put together something that looked like
a complete diet. May not be, but that was the idea. What we do is we take these bags, we mix them
and break them up into portions that we can feed within a week or two, and then we pack
and seal and store them in the freezer. We also feed Paramecia and then Artemia and
Paramecia are mainly for the larvae and also Artemia for the larvae and for adults. We know that Artemia is essentially like sausage
and bacon for the zebra fish: They have very little nutritional value, so we (indiscernible)
enrichment, at least that’s what we tell our (indiscernible) because fish go crazy
when they see something that’s moving, and so they obviously, you know, have the hunting
instinct, so we keep feeding that as a complement the other diets. The goal of mixing the dry feeds is to compensate
for potential deficiencies and, based on the information that actually was available from
suppliers, we tried to make a complete list of vitamins, trace elements, 14-amino acids,
fats, and pigments. And, of course, we want to support all the
different life stages, and it’s especially important the transition between various life
stages—for example, between larvae to juveniles. So that’s one thing. Change gears here. This is the information that was available
in 2005, for example, on the Zeigler diet, and this is, on the right side, it’s what
is available today. And because, now, they list much, much more
ingredients, we could probably say … we could get away only with Zeigler because they
probably have a complete diet, but do we really know? Also, there are some things we suspect might
not belong in the fish food, like poultry byproduct, (indiscernible). I’m not sure if that is what we should be
feeding them. Now, how do we feed? We have several different feeding strategies. We have little spoons. These are 3D printed spoons, and you go to
(indiscernible) and download the pad, and do that on your own 3D printer. You can adjust the size of the spoon, and
what we see here is actually a part of a feeding test that we started recently in spite of
all our shortcomings. UNIDENTIFIED FEMALE: Use your mouse; click
on Next. Use your mouse; click on Next. ZOLTAN VARGA: Then we have the (indiscernible),
that’s how we call it. You can see the central parts have different
size holes, and then this can be pushed through the feeding hole and dispense a certain amount
of food. So the volume of the spoons and the size of
the hole helps us control the amount that we put into each tank. We try to make it such that per tank, we try
to dispense a certain amount of food per fish in the tank. We also aim for about 3 percent body weight
measured at zebrafish that are 6 months old. Most recently, we started going high tech
using Arduino and Raspberry Pi technology, such as here this little motherboard, and
we 3D printed another food gun, which uses an auger to dispense food, and the food would
be dropping down from this fountain tube into the auger and then would dispense here at
the front. This is computer controlled; Raspberry Pis
are mini computers, and this is the thing in action, actually. There’s a little movie that I can show for
those who are interested. And then the goal is to have actually on our
bar codes for each tank a little extra QR code, which indicates nothing but the number
of fish in there, and the food gun reads the bar code and dispenses for the appropriate
number of fish that we (indiscernible). UNIDENTIFIED MALE: Does that work? Is that theory? ZOLTAN VARGA: It works in principal, (laughter)
but we have to do more on it. It’s not pretty; let’s put it that way
around. It’s not pretty, but it works. Recently, we did find a little time that we
could commit to testing foods. What I told you about these food mixes go
back almost 15 years now. Yes, you can put us into the camp, “What
ain’t broken, don’t try to fix it,” but in all realistic … we have to do something
about the progress that has been made and that is available in terms of food. What we have here is now six test groups of
food side-by-side in our facility, and we’re testing three doses each day. For example, the red label food would be one
type of food, and we feed it once, twice, or three times per day, and we do that six
times, parallel. And then we want to learn about the growth
curve that these fish experience, the weight curve, we want to look at fecundity and breeding,
and then we also want to look at general health of zebrafish, which includes adipose tissue
and/or resistance to certain pathogens. This is how it looks like in real life. We have these tanks nicely labeled, and the
food and the spoons are labeled accordingly, and that removes all bias. In principal, we shouldn’t know what food
it is, except I do, unfortunately. The results for the 3-month feedings looks
like this. So we have the six different food types—there
is a red label in there—and this is our stopping point. We started feeding when the fish were 1 month
old. They were 13.6 milligrams then, and since
then they have gained mass. This is only the mass of the fish, and this
is for one time feeding per day, twice a day, and three times a day. You can see that the different diets failed
in different ways, and we actually learned something from them. The most interesting part is that the physical
characteristics of the food really make a big difference. For example, in our tanks, as soon as we drop
the food from the spoon into the tank, the water that drops into the tank and the air
that bubbles from the tank creates a vortex and pulls the food backwards and into the
side, much as Chris described, but some feed stayed in the water column longer, and others
dropped to the bottom like a stone. When that happens, the fish will follow it
from the bottom, but not extensively. However, they will go crazy about food that
stays in the water column, and so feeds that are very successful, like the yellow and the
blue, they are feeds that float well and stay in the water column longer. Our own diet, which is the red, that’s the
Zeigler mix with Artemia, and the just Zeigler flakes back here, is actually not performing
that well as these other more modern feeds that have been developed recently. This is how we learn and how we adapt our
feeding strategies and our feeding program, and hopefully it will be something that can
contribute to the community. UNIDENTIFIED MALE: Zoltan, I’m sorry, just,
on that chart, is that male and female weight? ZOLTAN VARGA: We have scored males and female
separately, but this is now just a summary. Males and females across the board. UNIDENTIFIED MALE: What was the orange and
blue food? (laughter) Keeping it secret? ZOLTAN VARGA: I want to keep it secret, because
this is the first time point … well I can tell you. It’s SPAROS and it’s Otohime. AUDIENCE MEMBER: So, GEMMA didn’t make it
to the top round, huh? ZOLTAN VARGA: Didn’t make it because it
is a heavier food, and the fish don’t feed as well on it as on the lighter flowing feeds. So GEMMA is actually the pink … here. GEMMA is the pink. Our Zeigler flake only is yellow. Zeigler with Artemia is coming in under that,
so what we learn from that is Artemia actually dilutes the nutritional value of whatever
we put in the tank. Then we have Otohime and SPAROS zebra feed
on top. Then we have an experimental food from a good
friend that is very heavy and sinks to the bottom like stone, but would normally suggest
that it should grow the larvae really well. So physical characteristics of the food seem
to be important. UNIDENTIFIED MALE: What are the times of feeding? ZOLTAN VARGA: Say again? UNIDENTIFIED MALE: What are the times of feeding? Three times? ZOLTAN VARGA: The first feeding is 9 a.m.,
second feeding is noon, and then the last feeding we try to spread out as far into the
evening as we can, anywhere between 3 to 5, 6p.m. UNIDENTIFIED MALE: Thanks. UNIDENTIFIED MALE: Questions again? (applause) We had one or two questions, and
then we need to move to content. UNIDENTIFIED MALE: I’m a zebrafish researcher. I have a question about feeding zebrafish
and the food. When you’re feeding, the food goes into
the water; is the assumption that you’re feeding the amount that the zebrafish will
eat in a reasonable amount of time? Because my simple understanding is if the
food sticks around for a long time and doesn’t get eaten, somebody said it’s going to cause
a sludge. And then wouldn’t it ultimately dissolve,
and the nutrients then be … they’re swimming through everything now. How do … what assumptions are made when
you’re feeding? ZOLTAN VARGA: There’s two responses here. When we started feeding the larvae, in the
first week, we saw the sludge, and then it broke down and went away. So what happens there is when we feed with
the spoon, we feed the same 80 milligrams per feeding to the larvae and to the 1-year-old
fish. What it does is, it feeds the larvae 12 percent
or 10 percent of their body weight and the old ones about 1.5 percent of their body weight,
and it averages out to 3 around 6 months of development. Clearly, we’re all feeding (indiscernible). All of these things are going into the design
of this. UNIDENTIFIED FEMALE: One of the biggest challenges
with overfeeding on the assumption, “Hey, there’s just food in there, they can self-dine,”
is that because it goes into a recirculating system, it then severely impacts your water
quality, your filter changes, and hence more manual labor, and other aspects of the recirculating
system, so overfeeding is usually a recipe of disaster. ZOLTAN VARGA: Here, again we might be different. We were just discussing this: Our facility
was built at a time when these water systems weren’t very efficient. We have a huge amount of water volume, and
our pollutant equilibrium is so stable. You can … I don’t know. You can do a lot of it before we see a pH
variation. Other systems are much smaller in the volume,
so when you feed, you will see, for example, sludge build up right away in the (indiscernible)
much, much more than the (indiscernible). UNIDENTIFIED MALE: One more? UNIDENTIFIED MALE: I was just going to ask,
are you checking for health parameters, as well? Because optimizing the growth hasn’t been
mentioned and not necessarily good for the fish in the long term. ZOLTAN VARGA: We have a staff veterinarian,
and part of our services is a health service, a diagnostic health service, and when the
fish are 9 months old, she will look at adipose tissue development, at general infection rates,
and those kinds of things. Pretty much part of our routine, not the adipose
tissue, but the infection rates pretty much part of our routines health monitoring in
the facility.

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