Diet and life stage: what we need to know Louis D’Abramo

Diet and life stage: what we need to know Louis D’Abramo


LOUIS D’ABRAMO: Well, I appreciate being
invited here, and I had a luxury of listening to all the talks today, so I kind of looked
at my presentation and maybe made some changes as I’m going along, but I do want to approach
this more as a…philosophical presentation in a sense. I don’t have many slides or anything like
that. I started working on standard reference diets
back in the ’80s. Kind of proposing them for crustacea and after
working with John Castell and Doug Conklin, I really didn’t get anywhere. We were looking at the work that had been
done by AIN and all that, and we just couldn’t get people interested in it, and I hope that’s
one of the…we’re going to have one of those problems here I think in the future,
but I think we’ve got a lot of people here, a lot more in terms of helping one another
to make that come to fruition. Then I did some work on what I would call
a defined reference diet for Macrobrachium, which is a freshwater shrimp, the larval stages. It’s 11 larval stages, and I completely
replaced Artemia; in fact, it was better than Artemia. Then I started talking to Dr. Watts about
that, and then we started talking about a standard reference diet that might be working
with zebrafish. So, we’ve been working together probably
since about 2000 on reference diets for zebrafish. I’m going to start this presentation off
by…in a little bit differently, because I’m the last speaker. So, that I know that we can engender some
discussion, so I’m going to give you some of my biases, in a sense, and that can hopefully,
like I say, engender some discussion. Is there a need for special life stage–defined
standard reference diets? I’m going to be emphatic. Yes! Do we need to know all the nutritional requirements
of zebrafish to produce exceptional standard reference diets? No. Do potential standard reference diets for
zebrafish exist? Yes. Are they really essential? Again, my opinion, Yes. So, I’m going to go into a few things here;
some of it is going to be repetitious in a sense of I’m the last speaker, and a lot
of people have covered all these things, but I will try to talk specifically about life
stages and what we know from some of the traditional fish nutrition or the fish nutrition that
dealt with aquaculture organisms. So, the critical need here is to manage the
nutritional physiology of the different life stages. I look at them as distinct physiological packages
in a sense, and in order to…we have to understand the different physiology of these life stages
to provide greater confidence, comparative value, and efficient progress in biomedical
research. People have shown this before—life stage,
life cycle stages of zebrafish—and as you can see I’m concentrating on three parts
there—life history, life stages—larval, juvenile, adult. In some cases, we’ve heard from people that
are using exclusively formulated diets, commercially available diets, for the larval stages or
combination of live food and formulated diets, then moving into formulated diets specifically
for juvenile culture, and then on to adult culture. Some people using combinations of formulated
diets. So, you’ve all seen that. Oh, sorry. Now, just to show you a relationship here,
a growth-age relationship, I think when you look at growth, growth is a fundamental physiological
process, and it’s simple here to see you’re moving from larva to adult, that there are
changes in the growth rates as it relates to age, and I’m trying to demonstrate that
on the left side with length, but it also is true with weight. So we’ve also seen research that was done
many years ago where dietary protein source influences body size and the composition of
juvenile zebrafish. So most of this is related to protein. Protein deposition—that’s what growth
is all about. So looking at dietary protein requirements—and
a lot of this information comes from aquaculture, and I’ve kind of generalized here—we’re
looking at again different stages. First feeding larvae to brood stock, and you
can see how changes in protein requirements—and again this is a range, because we’re looking
at probably omnivores, herbivores, carnivores—and you can see how things change, and a lot of
it has to do…and a lot of it is shown in terms of growth rate. You’re first feeding larvae or your fastest
growing—a lot of the protein is being used for deposition; some of that protein is being
used as an energy source. So … and then you go down to the fingerlings,
and you get a reduced amount of protein that is in the diet; brood stock, going back up,
and that is related to moving into a reproductive stage, along with requirements for lipid. Dr. Hardy talked about all the…all the knowledge
that we have about zebrafish nutritional requirements—it’s basically nothing—and he put zebrafish down
at the bottom of—what do you call it—the triangle pyramid, and you can see that it’s
true. There’s really no information on nutritional
requirements of zebrafish, and you compare it to trout, where we’re moving into sustainable-type
feeds, where all the nutritional requirements are known. Something that I found that was published
in 2016 gives us a good idea that this research that was done—although I have some problems
with using fishmeal as an exclusive protein source to determine protein requirements,
but be that as it may—it comes in probably at the level that I showed you in the previous
slide in terms of requirements for juveniles. So what I’m trying to say here is that the
existing nutritional information that has been generated for other species of fish,
I think, has application as a foundation that we can use to develop a defined diet. So, again, the support for foundational application
of existing knowledge—that’s in relationship to my answer to my question, I should say—about
whether we can have these defined standard research diets without knowing anything specifically
about the nutritional requirements of zebrafish. So nutritional requirements change during
the stages of life cycle. I can tell you that this does happen; it’s
obvious in some of the work that has been shown here today. How do we meet those requirements? Again, these are things that were mentioned
by various people in their presentations: physical, size, shape, form—whether you’re
using inert or live food; consumption and palatability—whether the food is chemically
attractive, whether it is palatable; and nutrient sources—is it coming from live food, or
is it coming from formulated diets? Those are how the requirements are going to
be met. You’ve got to have the right size and shape,
you’ve got to get them interested in it, it’s got to be palatable, and it’s got
to have the proper nutrients. Here’s an experiment that was done in Dr.
Watts’ lab; this was done back in I think about 2003. This was trying to illustrate to people of
all the variety of diets that were being used that some of the problems that you were confronted
with. You’ve got zebrafish that are 28 days postfertilization,
having been previously fed a live food—rotifers. You had five commercially available diets
at that time, and you had two experimental laboratory diets that were kind of based upon
the diet that I had used with freshwater prawn, freshwater shrimp, and so, we wanted to take
a look at those compared to these five commercially available diets. They were fed… ad libitum and for 9 weeks. A lot of data here. You can see the two experimental diets—A
and B—have been highlighted, and you can see some of the other diets that have been
used in the past. I think we’ve moved farther ahead. There are a lot of other diets that are being
used that are much better that are not listed here, but that’s not the point. The point is if you take a look at weights
for females throughout the diets, you have—after 9 weeks, you’re dealing with 68 milligrams
to 279 milligrams. For males, 72 to 181 milligrams. If you take a look at the experimental diets,
you’ll see experimental diet A had the largest in terms of average weight males; experimental
B had the largest experimental weight for females. So, the experimental diets are working very
well. Ok, you’re getting very, very good growth
rates. That may not what—that may not be what you
want, but in this case, it’s saying that what you have developed here is equivalent
with what is available in these commercially available diets that are undefined. Okay. I got it. So, how is that control achieved, in terms
of, let’s say, protein quality as it relates to developmental stage and—ooh, one minute,
that should be another one before that—How do those commercial diets differ? Obviously, if they were flakes or pellets,
they differed in terms of shape and size and, therefore, had an effect on consumption. Energy nutrient content—the protein content
in those diets varied from 31 to 60 percent and the lipid from 5 to 34 percent. So again, the differences that you’re seeing,
I think, are related to nutrient content, but I think there’s a lot of other things. You can even say the microbiome that has been
created by these differences in protein and lipid in the diet certainly had an effect
ultimately on the growth of these fish. The energy content, the nutrient source, the
quality itself—how good are they, are they carrying any kind of toxicants or things like
that?—and the nutrient availability. Once it is consumed, how well is it digested,
given the complement of enzymes that are available in that particular life stage of the zebrafish,
and how well are they assimilated? So all of these influence physiological processes—and
when I talk about physiological processes, I’m talking about distinct physiological
packages that are associated with the different life stages. So, my conclusion is nutritional control is
essential. Now, how is that control achieved? Well, just to give you an example of just
the protein—protein source for the different developmental stages and the quality—you
have to have an readily available source; that source has to be consistent in terms
of its composition. I like to have at least two different sources. It has to be very highly digestible, you’ve
got to have that balanced amino acid profile, and it can’t be subject to vagaries in nutrient
composition, something that was talked about over and over again in previous presentations
here today. Again, this information comes from a lot of
published information on aquaculture species, but again, I think it has direct application
to the development of a defined standard reference diet for zebrafish. Take a look at larval fish. Phospholipids—very, very important—and
you might say, don’t they synthesize phospholipids? Yes, they do, but apparently, they don’t
synthesize them at a rate that is sufficient to meet their needs because of their physiological
state, because of how fast they are growing. We saw mentioned today a lot long-chain omega-3
or n-3 polyunsaturated fatty acids—very, very important in early larval culture—particularly
for marine fish. High protein. I tried to demonstrate that in that relationship
between growth and age. Obviously, larvae are putting down a lot as
a percent of their body weight as they grow, and that is all due to protein deposition. Also, lipid energy sources. Again, we talked about carbohydrate a little
bit, and we see that a lot of species of fish do not handle the carbohydrates very well,
and a lot of that has to do with the proper enzymes for efficient digestibility. If you move to the juvenile and adult—again,
phospholipids seem to be something that are required. You can call them as a supplement almost,
because we know again that this particular life stage also synthesizes phospholipids,
but apparently not at a sufficient rate. Also, again, predominant lipid energy sources
in the diet, and if we move to the brooder, we’re talking about now high-lipid, high-protein
diets, we often talk about protein-energy ratios and what might be the best or optimum
protein-energy ratio to have in the diet. Your n-3 long-chain polyunsaturated fatty
acids also become very, very important, particularly in terms of the production of eggs; vitamin
C requirements increase; vitamin E requirements increase; B6 pyridoxine also increase. We also need, and I forgot to put it, but
somebody mentioned it today—carotenoids in the diet. Make sure the carotenoids are there, particularly
canthaxanthin or…canthaxanthin, I should say, is primarily the one, or astaxanthin. So, you can see the differences, and a lot
of it has to do again with where they are in development and that they are distinct
physiological packages. … I thought I was going to come up here
and be the only one that didn’t have a problem. Okay, so steps toward standardized diets. Standardized—what I call reference diets—for
aquatic biomedical research models, and again, I think we can get around having to develop
a diet that we can use specifically to try to increase our knowledge of nutritional requirements. That should continue, but I think we can jump
over that step based upon all the information that has been provided to us through aquaculture
research. So use that existing knowledge to develop
that standard reference diet for the key developmental stages. And I see this already: Limit live feeds as
much as possible. I’m one of those people that think that
we have to move away from live feeds and start feeding as soon as possible. Now we run into problems, too, because I can
think of a larval diet, and you’re saying I’m just I’m kind of promoting the idea
that you have to have a lot of lipid in the diet. Well think about producing the small-size
feed that they need that has a lot of lipid, so there’s manufacturing problems here,
too, that we have to consider. Provide the formulated diets through a commercial
manufacturer for testing, and I would think again—as I listen to everyone again—the
idea here is to kind of engender some discussion; I may be biased, but I think we should try
to develop a diet specifically for the juvenile stage, and see how that works first among
the people who want to try it. They can go ahead with their feeding management
practice—whatever they want to do—but this is the reference diet we want you to
look at, and you can compare it in terms of what you’re doing now—whether it has to
do with a one diet, a combination of diets, a live and a formulated diet—and then establish,
based upon that, hopefully the fundamental acceptance and use of standard reference diets
by the scientific community. I couldn’t do it about 30 years ago. I hope that we can do it, because I think
it’s essential for the kind of progress that we’re going to have to need. This is a very unique—I learned more today
about how unique this particular organism is as a biomedical model. It’s just great to look at it. So here, I’ll give you just one more thing
to think about. When I said something to the effect “Do
I think that a standard reference diet exists already?” This is something that is being used in Steve’s
lab, and again you can take a look at some of the things. The first three obviously are protein sources. Why are they chosen? They’re chosen because they don’t change
in terms of their composition. They were chosen because they are mostly protein. They were chosen because they are highly digestible—all
the things that you would want to need, and then you’ve got some carbohydrate sources
in there: wheat starch and dextrin, your oils, your lipids. You can see that right here it’s about 6
percent with the safflower oil and the menhaden, which is a combination of your n-6, n-3 fatty
acids, α-cellulose is…it’s not a…it’s a insoluble source of fiber; diatomaceous
earth is just a filler; soy lecithin, that’s your phospholipid; alginate is your binder. You’ve got your—what we talked about before—your
vitamin and mineral mixes and things, and canthaxanthin being your pigment source—so
basically something like this—and this is something that has gone through multiple generations—Something
like this I think we can look at as a potential foundation for a standard reference diet in
terms of our advocacy for that, and I would say that instead of trying to develop diets
for the three different life stages that I’ve outlined, maybe go with the juveniles and
see what happens there, and let people stay with their particular feeding practices to
start with, and see how this diet performs and if we can get people interested in it. I’m going to stop there and, hopefully,
I kind of engendered some discussion about my biases that I had before I came and the
ones that I developed while I was here. {Applause}
UNIDENTIFIED MALE SPEAKER: I imagine a lot of the points that Dr. D’ABramo brought
up will be subject to discussion in the longer session. Does anyone have maybe one question now, on
anything specific, and uh… LOUIS D’ABRAMO: Yes. UNIDENTIFIED MALE SPEAKER: I thought you showed
data you said they kept animals, the fish, on ad libitum diets for 9 weeks. LOUIS D’ABRAMO: Mm-hmm. UNIDENTIFIED MALE SPEAKER: We heard earlier
today that someone said that keeping the food in the water all constantly 24/7 was a bad
thing. You couldn’t do that long term. So how did you get around that, or is that,
or am I misunderstanding something about how… LOUIS D’ABRAMO: I wasn’t advocating that. The question is about feeding them for 9 weeks
ad libitum, and what other people were saying is, that’s being something detrimental. Right? Is that what you’re saying? UNIDENTIFIED MALE SPEAKER: Or hard to maintain,
because it’s dirty for the water, dirty for the tank, or whatnot… Is that something you were able to get around
with in your methodology, or… UNIDENTIFIED MALE SPEAKER: Ad libitum, at
that point in time, was primarily defined by being a unit of food and allowing them
to clean up as much as they can within a defined period of time. That was defined as ad libitum at that point
in time. You wouldn’t define that as ad libitum now. So that’s what he’s referring. LOUIS D’ABRAMO: Yeah. UNIDENTIFIED MALE SPEAKER: He’s not referring
to actual 24/7… LOUIS D’ABRAMO: No no no, ok, no, no. UNIDENTIFIED MALE SPEAKER: {indiscernible}
You don’t feed that way anymore. LOUIS D’ABRAMO: Remember that was 2000…actually,
the work was probably done in 2002 and published in 2003, so we’re talking like 15 years
ago. {indiscernible}
UNIDENTIFIED MALE SPEAKER: So, we have a 15-minute break scheduled, but we’re just a tad behind,
and so maybe a 9-minute break, so start at 3:50? {indiscernible, 21:16-21:20} Let’s
thank the speakers again. LOUIS D’ABRAMO: Ok, Thank you.

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