127
7
Genetics
Rex Dunham
CHAPTER MENU 7.9 Xenogenesis, 146
7.1 Introduction, 127 7.10 Genetic Engineering, 146
7.2 Basic Genetics, 128 7.11 Gene Editing, 151
7.3 Epigenetics, 130 7.12 Combining Genetic Enhancement Programs, 152
7.4 Domestication and Strain Evaluation, 130 7.13 Genotype‐environment Interactions, 152
7.5 Selection, 131 7.14 Future Developments, 153
7.6 Inbreeding and Maintenance of Genetic Quality, 136 7.15 Summary, 154
7.7 Crossbreeding and Hybridisation, 136 References, 154
7.8 Chromosomal Techniques, 140
7.1 Introduction et al., 2012; Gjedrem and Robinson, 2014). This is true for
genetic stocks used for new aquaculture species, which
Genetic intervention has been used to enhance animal are essentially wild. However, these reports of lack of
and plant agriculture production for centuries and has application of genetic enhancement programs only con-
intensified during the last two centuries. Aquaculture sider selection as genetic improvement. Obviously, there
genetics has tremendous potential for enhancing aqua- are alternative genetic enhancement programs, and after
culture production and is now being applied to aquatic 40–50 years of genetics and breeding research, coupled
organisms to improve production traits. Modern research with genetic biotechnology, established commercial spe-
on Aquaculture Genetics began sporadically 80 years ago cies such as carps, catfish, salmonids, tilapias and oysters
and became common place in the 1970s. During the last are essentially all genetically improved. The best available
three decades, research in this area has steadily grown, genotypes may have performance levels of up to 10‐fold
and now research on traditional selective breeding, that of poor performing wild genotypes, and the rate of
genetic biotechnology, transgenics and genomics is quite progress and genetic gain certainly rivals that or exceeds
active. Modern techniques, such as marker‐assisted that for terrestrial livestock. Regardless of the species and
selection, are being researched, and there is broad appli- the genetic gain made as of today, much greater genetic
cation of genetic enhancement in aquaculture, including progress can continue to be accomplished.
selection, multiple‐trait selection, marker‐assisted selec-
tion, interspecific hybridisation, polyploidy, genetic mon- Effective programs have goals and plans, and this is
osexing and recently genetic engineering also true for genetic enhancement programs. Goals are
the important traits of economic importance that we
Twenty years ago, most marine fish production want to improve, and the extent to which we consider it
remained equivalent to the use of undomesticated ances- feasible to improve these traits. Genetic enhancement
tral cattle and chickens in ancient terrestrial agriculture; programs are then the plans that we use to accomplish
however, selection programs are now common place for these goals and objectives. The primary purpose of this
some of the major species. Still, some reports indicate that chapter is to review these genetic enhancement pro-
genetic improvement programs in the aquacultuare indus- grams to show how effective they have been with the
try are not widespread, and only 10% of commercial aqua- focus on production traits of finfish, molluscs and
culture populations are genetically improved (Gjedrem crustaceans.
Aquaculture: Farming Aquatic Animals and Plants, Third Edition. Edited by John S. Lucas, Paul C. Southgate and Craig S. Tucker.
© 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.
128 Aquaculture 7.2.2 Qualitative Traits
7.2 Basic Genetics Qualitative traits are phenotypes that are expressed in an
all or nothing fashion. For example, albino or normal col-
7.2.1 Gene Action ouration is usually a result of gene expression from a sin-
There are two basic types of gene action, dominance/ gle or only a few loci. Colouration and deformities are
recessive and additive. examples of qualitative traits.
In the case of dominance, only one copy of the domi- Qualitative traits such as changes in colour, finnage,
nant allele (A) in a diploid organism is needed for expres- scale pattern or deformities can be desirable or detri-
sion of the associated trait. In the case of the recessive mental in aquaculture. Obviously, qualitative traits are
allele (a), two copies of the allele are needed for the important and the primary basis for the ornamental
recessive phenotype to be observed: aquaculture industry. Deformities can be valuable in the
●● In a completely dominant system (Table 7.1a), a ornamental trade, but are usually undesirable in the food
trade industries. If these qualitative traits are a result of
large unit of phenotypic change occurs when going dominant gene action, they can be easily eliminated as all
from the homozygous recessive genotype (aa) to homozygous dominant and heterozygous (carrier indi-
the heterozygous genotype (Aa) and no unit of viduals) phenotypes are obvious, and those individuals
change when going to the homozygous dominant can be immediately selected against, resulting in a popu-
genotype (AA). lation with none of the dominant detrimental allele. An
●● For additive gene action, the alleles act in an additive example of this is the saddleback mutation in tilapia. On
fashion with similar units of change when compar- the other hand, it is extremely difficult to eliminate a del-
ing the different genotypes (aa → Aa → AA) or as eterious recessive allele from a population, since hete-
in mathematical addition, the stronger allele will rozygous carriers cannot be identified by simple visual
make a greater contribution to the phenotype observation (Table 1a). If the trait was of high economic
(Table 7.1e). importance or damage, the heterozygous carriers could
●● In overdominance, the heterozygote is superior or out- be eliminated, by mating them with individuals of known
side the range of the phenotypes of the two parental genotype and examining the phenotypic ratios in the
homozygotes (Table 7.1c). progeny: progeny testing. Since fish are highly fecund
Further, in the case of alleles at more than one locus, and produce large numbers of progeny, progeny testing
the alleles at one locus can affect the expression of alleles could eliminate the deleterious allele from the popula-
at another locus. This type of gene interaction is termed tion in a single generation.
epistasis.
Table 7.1 Basic types of gene expression.
(a) Complete dominance Genotypes Phenotypes Unit of phenotypic
(b) Incomplete dominance change between
(c) Overdominance bb red genotypes
(d) Co‐dominance Bb black ↕ large
(e) Additive BB black ↕none
bb white
Bb dark grey ↕large
BB black ↕small
ss malaria prone
Ss healthy (best) ↕large
SS sickle cell (sick) ↕large
AA black
Aa black and white large equal units of change
aa white
rr white large equal
Rr pink units of change
RR red
When deformities are observed, fish culturists often Genetics 129
assume that the deformities have a genetic basis and that
they are likely to be increasing because of inbreeding in Figure 7.2 Line scale pattern in common carp. Genes for scale
the population. However, these assumptions are often pattern in common carp have epistatic interaction, pleiotropy and
false. Many deformities observed are environmentally semi‐lethality. Source: George Chernilevsky. Reproduced under
induced and are often related to low egg quality or poor the terms of the Creative Commons Attribution Share Alike
water quality in the hatchery. license, CC BY‐SA 3.0.
There are different types of dominance (Table 7.1), as effects and this applies to the alleles responsible for
discussed earlier. In the case of complete dominance, the saddleback in tilapia.
trait is fully expressed in the heterozygous and homozy- 3) Heterosis is the increased or decreased function of
gous dominant genotypes. Normal colouration (in con- any biological quality in hybrid offspring as heterosis
trast to albinism, which is homozygous recessive) and can be positive or negative. In the case of genetic
saddle back are examples of complete dominance in fish. enhancement of performance traits in animals the
terms heterosis and hybrid vigour are interchangea-
The heterozygous genotype allows a major, but not ble. However, we suggest for fish genetic enhance-
complete unit of change in the phenotype in incomplete ment this be standardised to heterosis as in population
dominance. The homozygous dominant genotype is nec- genetics hybrid vigour refers to reproductive fitness
essary to make the complete maximum shift in the phe- and occurs when F1 produce more living offspring
notype. In the case of overdominance, the phenotype of than the parental genotypes.
the heterozygous genotype is outside the range of the 7.2.3 Phenotypic Variation
two homozygous genotypes (Table 7.1c). The phenotype Individual phenotypes, the appearance, characteristics
associated with each allele is observed in the case of co‐ and performance of individuals, are a result of three
dominance (Figure 7.1). main components:
●● the environment;
1) Epistasis is the interaction of genes at different loci. ●● the genotype;
Alleles at one locus can affect the expression of alleles ●● the interaction between the genotype and the
at another locus. Epistasis is the basis of some colour environment.
types in fish. For instance, it is the explanation for Thus, phenotypic variation (Vp) is a result of genetic vari-
some red and black colour variants in tilapia and scale ation (Vg), environmental variation (Ve) and variation
pattern in the common carp is also influenced by epi- due to the genotype – environment interaction (Vge):
static gene action (Figures 7.2 and 16.5).
Vp Vg Ve Vge.
2) Pleiotropy is when one gene affects more than one Genetic variation in populations has several potential
trait. The alleles and loci that affect scale pattern in components as well:
common carp are an excellent example of this phe- ●● additive genetic variation;
nomenon. These genes not only affect scale pattern, ●● dominance genetic variation;
but also growth, survival, other meristic traits, toler- ●● variation due to epistasis interaction;
ance of low DO, haemoglobin and haematocrit and ●● variation due to maternal heterosis.
the ability to regenerate fins. Some mutations can also The type of genetic variation has a bearing on the success
have semi‐lethal or lethal effects, resulting in reduced of the genetic enhancement program being attempted.
viability or death. Again, certain alleles affecting scale
pattern in common carp have semi‐lethal or lethal
homozygous homozygous heterozygous
Parental Parental F1 genotype
type 1 type 2
Figure 7.1 Co‐dominance – both alleles are expressed equally.
DNA or protein banding patterns illustrate the concept.
130 Aquaculture Percent improvementstrains of fish usually have better performance in aqua-
culture settings than wild strains of fish. Strain variation
For selection to be successful, a trait must have signifi- is also important, since strain affects other genetic
cant heritability and the ratio of additive genetic varia- enhancement approaches, such as intraspecific cross-
tion to total phenotypic variation (narrow sense breeding, interspecific hybridisation, triploidy, sex con-
heritability) must be high. However, absolute quantities trol and genetic engineering.
of additive genetic variation and phenotypic variation
are of equal or greater importance. Dominance, epistasis When wild fish are moved to aquaculture or hatchery
and overdominance are the genetic basis of heterosis: the environments, they are exposed to a new set of selective
relative performance of crossbreeds and hybrids com- pressures that will change gene frequencies. Thus, an
pared to their parents. Thus, significant dominance‐ organism better suited for the aquaculture environment
related variation must exist for crossbreeding and begins to develop. This process, termed domestication,
hybridisation programs to be successful. occurs even without directed selection by the fish cultur-
ist. Domestication effects can be observed in some fish
7.3 Epigenetics within as few as one to two generations after removal
from the natural environment (Dunham, 2011).
The expression of genes can be greatly affected by envi-
ronmental factors. Thus, qualitative and quantitative In channel catfish (Ictalurus punctatus) an increased
genetic variation can be even more complicated than growth rate of 3 to 6% per generation was observed due
once believed. Epigenetics relates to changes in gene to domestication selection (Figure 7.3), and the oldest
expression without alteration of the DNA sequence. The domesticated strain of channel catfish (107 yr), the
environment alters gene expression by ‘silencing’ genes Kansas strain, has one of the fastest growth rates of all
or ‘waking them up’, hence epigenetic modification, strains of channel catfish.
which is associated with the epigenome, the overall pat-
tern of activation of the genome. The influence of epige- Domesticated common carp in Hungary showed better
netics was once thought to primarily affect embryonic growth and resistance to Aeromonas hydrophila than
stages of development, but it is now known that these wild strains. Although most domesticated strains usually
epigenetic influences can accumulate over time and perform better in the aquaculture environment than wild
actually be more prevalent in adult stages. Although strains, there have been some putative exceptions, such
brought about by the environment, epigenetic changes as wild Nile tilapia, Oreochromis niloticus and rohu,
can be transmitted to one or more generations. Thus, Labeo rohita, which appeared to grow better in the aqua-
how a fish is cared for, fed, exposed to disease and other culture environment. However, the explanation for this
environmental factors can have long lasting effects in the anomaly appears to be related to a lack of maintenance of
individual that can be passed on to its progeny and even genetic quality and genetic degradation in the domesti-
further generations. cated strains compared to these wild fish. Poor perfor-
mance of some domestic tilapia is related to poor founding
There are multiple mechanisms for epigenetic gene (parental) lines, random genetic drift, inbreeding and
silencing. The main epigenetic mechanisms for altering
gene expression include DNA methylation, chromatin 60
modifications (histones) and non‐coding RNAs. Kansas select
Methylation is the attachment of methyl groups to DNA Kansas random
sequences in a gene, specifically to cytosine when it is
adjacent to guanine. The functioning of the epigenome is 40
also related to histones, proteins that control access of
genes for transcription, for which the abundance can 20
again be environmentally influenced.
7.4 Domestication and Strain 0
Evaluation 01234
Generations
Use of established domestic strains that are best
performing is the first step in a genetic improvement Figure 7.3 Percent improvement for Kansas select channel catfish
program and the mechanism to make the most rapid compared to Kansas random channel catfish after first, second,
initial progress in genetic improvement. Domestic third and fourth generation of selection for increased body
weight. Source: Reproduced with permission from Rex Dunham.
Table 7.2 Strain variation in Nile tilapia (Oreochromis niloticus). Genetics 131
Comparison of Egypt, Ivory Coast and Ghana strains for some
traits that are important for aquaculture. effective and requires the least resources. However, since
no pedigree information is available, individual selection
Trait Best performing strains will eventually lead to inbreeding and inbreeding depres-
sion of performance. However, in relatively small popu-
Growth rate Egypt, Ivory Coast lations, 20 pairs breeding per generation, this inbreeding
Reproduction Ghana will not occur until 5–6 generations have elapsed. In
Cold tolerance Egypt highly, fecund species such as Chinese and Indian carps
Seinabilitya Egypt, Ghana for which only a few spawns are required to meet hatch-
ery needs, mass selection will lead to rapid inbreeding
a vulnerability to be seined in harvesting. depression.
introgression with slower growing species, such as O. Family selection should lead to more rapid genetic
mossambicus, and slower growing strains such as Nile gain. It allows genetic gain for lower heritability traits
tilapia from Ghana (Table 7.2). and prevents inbreeding since pedigrees must be main-
tained. Family selection also allows selection of both
Domestication of farmed shrimp was relatively slow sexes for sex‐limited traits and selection for traits that
compared to that of finfish because of: require sacrifice of the fish such as carcass yield. The pri-
●● the use of wild broodstock and postlarvae; mary disadvantage to family selection is the extra
●● a lack of understanding of shrimp reproductive biol- resources and record keeping required to conduct this
program. Combined selection includes individual selec-
ogy for domestication of the species; tion within families, and theoretically, results in the most
●● endemic disease challenges; rapid genetic improvement via selection.
●● laws restricting movements of shrimp and disease‐free
Research on selection in fish for relevant aquaculture
certification; traits began in the 1920s (Embody and Hayford, 1925),
●● the relatively recent nature of shrimp aquaculture. but very little selection was conducted prior to 1970.
As is the case with fish, domesticated shrimp are more Unfortunately, during this period, several potentially
cost‐effective than wild strains for aquaculture applica- high impact experiments did not include adequate
tion, but the reproductive performance of domesticated genetic controls to prove genetic gain. From 1970 to the
Penaeus monodon and brown tiger shrimp, P. esculentus, present, research on selection and traditional selective
are similar to wild brood stock. There are likely to be breeding has continued to grow rapidly (Dunham, 2011;
environmental causes for this similarity rather than Gjedrem et al., 2012; Gjedrem and Robinson, 2014)
genetic. despite the excitement about and increased funding in
the area of molecular genetics and genomics. In general,
Strains of fish show large amounts of variability for the response to selection for growth rate in aquatic spe-
many different traits. There are strains of channel catfish cies is very good compared to that with terrestrial farm
and rainbow trout strains that differ in growth rate, dis- animals and these programs have been highly successful
ease resistance, body conformation, dressed carcass %, (Table 7.3). Fish, shrimp and bivalve molluscs often have
vulnerability to angling and seining, age of maturity, time higher genetic variance compared to farmed land ani-
of spawning, fecundity and egg size. Okamoto et al. mals: genetic variation for growth rate is 7–10% in
(1993) reported that an infectious pancreatic necrosis farmed terrestrial animals and 20–35% in fish, shrimp
virus (IPNV)‐resistant strain of rainbow trout showed and bivalves. Fecundity is also higher in aquaculture
4.3% mortality compared with 96.1% in a highly sensitive species compared to warm‐blooded agriculture animals
strain. Other strains of some marine fish species vary for allowing for higher selection intensity for aquaculture
upper‐temperature tolerance, including loss of swim- production improvement, and a few hundred heritability
ming equilibrium and disassociated caudal fin. estimates have been obtained for several traits of cultured
fish and shellfish (Tave, 1993).
7.5 Selection
Selection for increased body weight has a high proba-
7.5.1 Selective Breeding bility of success in the vast majority of aquatic organisms
Individual or mass, family, combined and index selection and in the vast majority of strains within a species. Six
are alternative selection programs with various advan- generations of selection increased body weight by 30% in
tages and disadvantages. Individual selection is simple, rainbow trout. An increase of 7% was achieved within a
single generation in Atlantic salmon (Salmo salar) and
an increased growth rate of 50% was achieved with 10
generations in coho salmon (Oncorhynchus kisutch).
Body weight was improved in channel catfish, by 12–20%
132 Aquaculture
Table 7.3 Examples of improvements from selective breeding in aquaculture species.
Species Parameter Number of generations % improvement
of selection
Rainbow trout Body weight 30
Coho salmon Growth rate 6 50
Atlantic salmon Growth rate 10 7
Channel catfish Growth rate 1 55
Brook trout Resistance to bacterial furunculosis 4 2% to 69% survival
Rainbow trout Resistance to Flavobacterium psychrophilum 3 32
Common carp Body weight 1 5 per
Vietnamese 6 generation
GIFT Nile tilapia Body weight 11–13 per
8–14 generation
8–9
Bivalve molluscs Growth rate 1 4.4
White‐leg shrimp Growth rate 1 12.4% survival
White‐leg shrimp Resistance to Taura virus 1
with one to two generations of genetic selection, and the for disease resistance than for body weight, and often no
best line grew twice as fast as typical non‐selected strains. selection response is found for some strains while others
After three generations, the growth rate of channel cat- will show significant enhancement of disease resistance as
fish in ponds was improved by 20–30% and this was fur- a result of selection. In the case of salmonids, selection for
ther increased to 55% after four generations of selection disease resistance has been particularly successful
in a Kansas strain of channel catfish (Figure 7.3). (Embody and Hayford, 1925). Three generations of selec-
tion for resistance to endemic bacterial furunculosis in
Selection for body weight has also been successful in brook trout (Salvelinus fontinalis) improved survival from
marine species. Selection for body weight was success- 2% to 69%. Resistance to furunculosis in brown trout
ful in gilthead seabream (Sparus aurata). Selection (Figure 7.4) and brook trout has been improved via selec-
improved growth of the marine shrimp, Marsupenaeus tion. One generation of selection increased resistance to
japonicas. Improvement in the growth, survival and total Flavobacterium psychrophilum (bacterial cold‐water dis-
yields were obtained in two selected lines (10‐15 % ease) in rainbow trout (Oncorhynchus mykiss) by an abso-
increase in mean yields). lute 32% and a relative 105%. Selection response for
resistance to viral and bacterial diseases in salmonids has
Selection is an effective genetic enhancement program sometimes been as much as 18–19%.
to improve growth rates in bivalves and crustaceans.
One generation of mass selection for growth rate in Selection for increased disease resistance and survival
Pacific oysters increased growth rate by 8%, and mass has also been successful in crustaceans and bivalves. A
selection of adult oysters gave a strong response to selec- response for one generation of selection of 4.4% for
tion for growth rate in Crassostrea virginica. In other growth rate and 12.4% for survival was obtained in the
experiments, a 10–20% gain in growth rate of oysters was white‐leg shrimp, Litopenaeus vannamei, when exposed
achieved after one generation of selection. A genetic gain to Taura syndrome virus. More dramatically, resistance
of 9% increased growth rate in Sydney rock oysters to Taura syndrome virus had an absolute 30% improve-
(Saccostrea lomerate) was achieved in a generation and ment and a relative improvement of 100% with two gen-
in the Chilean oyster (Ostrea chilensis). A 9% per genera- erations of selection in L. vannamei. Additionally, growth
tion of selection for growth rate has been estimated for and pond survival were improved 5–6% per generation.
the hard‐shell clam or quahaug (Mercenaria merce-
naria). Selection for increased body weight has been Heritability1, additive genetic variation and selection
successful for a variety of marine shrimp, freshwater response can vary among strains for body weight, and
prawns and crayfish (Gjedrem et al., 2012). Rate of genetic
improvement in bivalves and crustaceans appears to be 1 Heritability is an estimate of how much variation in a phenotypic
similar to that of finfish. trait within a population is due to genetic variation among individuals
in that population. It most commonly refers to narrow sense
Selection has been effective for improving disease resist- heritability which is the proportion of phenotypic variation due to
ance, but not as consistently as selection for body weight. additive genetic variation.
Strain variation for selection response is more prevalent
Figure 7.4 Brown trout, Salmo trutta. Source: USFWS. Photograph Genetics 133
by Eric Engbretson http://www.underwaterfishphotos.com
growth improvement after 7 generations of selection.
tilapia and common carp (Cyprinus carpio) are several of During the first two generations of selection, similar
the more prominent examples of this phenomenon. Body responses to selection in Nile tilapia grown in low input
weight of common carp initially appeared unresponsive environments as were found in the first two generations
to selection as five generations of selection for increased in the GIFT population, which was selected in a variety of
body weight resulted in no genetic gain, and five genera- environments. The 11% genetic gain per generation in
tions of family selection resulted in modest gains of about GIFT tilapia was better than that obtained in most other
5–10%. However, in a Czechoslovakian strain of common species of fish, which typically average 5–7 % per genera-
carp heritabilities for body weight were estimated at tion as demonstrated for salmonids following approxi-
0.15–0.49. Vietnamese common carp had a heritability of mately 10 generations of selection. However, other
0.3 for growth rate, and six generations of selection exceptional examples exist such as that for channel cat-
increased body weight by 5% per generation. fish, which had an increased body weight of 14 % per gen-
eration over four generations, and the 13–14 % increase
Mass selection has improved body weight in the per generation observed in some cases for salmon.
Mozambique tilapia (Oreochromis mossambicus), red
tilapia, Nile tilapia (O. niloticus), O. shiranus and, blue Response to selection can differ depending on the
tilapia (O. aureus). However, selection for increased body direction of selection. Body weight of common carp in
weight in red tilapia has been variable. Even greater vari- Israel was not improved over five generations but could
ability to the selection response has been observed in Nile be decreased in the same strain selected for small body
tilapia: from no response in some strains, 1–7% gain per size. Virtually identical results for Nile tilapia have also
generation in others and as much as 11% per generation been reported. In general, it is easier to select to make
in the Philippines for the GIFT strain. The lack of response traits smaller rather than larger, which, of course, would
in some strains may reflect a narrow genetic base in the rarely have aquaculture significance. There are excep-
founder stock or sole use of mass selection in cases where tions to the above observations above as common carp in
additive genetic variation was low. Some selection pro- the Czech Republic responded to selection for increased
grams for Nile tilapia were moderately successful, 14% body weight, but not for decreased body weight.
body weight increase over two generations for a synthetic
Egyptian strain. Selection for increased growth in GIFT Body conformation can be dramatically changed via
Nile tilapia was much more productive, with 77% to 123% selection. Heritability for body depth is quite high in
common carp. Recently, a significant heritability was
found for deformities in the Atlantic cod, Gadus morhua.
The implication is that deformity rate could be reduced
through selection.
Reproductive traits tend to have high selection
responses. Spawning date can be shifted in coho salmon,
Oncorhynchus kisutch, by about 14 days with four gen-
erations of selection. Age at sexual maturation, fecun-
dity and even sex ratio are heritable traits in fish. Strong
genotype‐environment interactions for sex determina-
tion can exist for fish. Heritability for gender and sex
ratio can be dramatically different at different hatching
and rearing temperatures, allowing for selection for
highly skewed sex ratios or monosex populations.
Long‐term selection appears feasible in fish. Nile tila-
pia body weight was doubled through seven generations
of selection. The 8th to 14th generations of selection for
body weight in GIFT Nile tilapia resulted in a response
per generation of 13 % even after this length of time.
7.5.2 Correlated Responses to Selection
and Indirect Selection
When selection is conducted upon one trait, positive,
negative or no correlated responses to selection can
occur for other traits depending upon the nature of
genetic correlations among traits (Table 7.4). Additionally,
in mass selection programs there is the potential for
134 Aquaculture
Table 7.4 Examples of correlated responses to selection.
Species Trait selected Positive Correlated traits Negative
No correlation low DO tolerance
Channel Increased body weight dress‐out %a body composition
catfish feed consumption seinabilityc
Growth rate FCEb
Atlantic Growth rate feed consumption dress‐out %
salmon Muscle lipid content FCE fillet %
European bacterial cold‐water disease resistance FCE body weight
whitefish thermal growth coefficients
Rainbow
trout
Rainbow
trout
a (body weight without head, viscera and skin) × 100 /total body weight.
b FCE (food conversion efficiency) (%) = 100/FCR.
c vulnerability to be seined in harvesting.
decrease in performance in some traits, long‐term, 4th generation selected for growth rate. The wild fish
because of the accumulation of inbreeding. had eight percent lower retention of both energy and
protein. There are strong genetic correlations between
Genetic correlations, if positive, among traits allow for growth rate and feed conversion in European whitefish,
the possibility of indirect selection, selecting a second Coregonus lavaretus, and channel catfish, and the
trait to improve the primary trait of interest. Under cer- nature of the heritabilities and genetic correlations
tain conditions indirect selection can be more effective indicate that indirect selection for feed conversion by
than direct selection. Additionally, indirect selection has selecting for growth rate would more effectively
some of the advantages of family selection as it can allow improve feed conversion than direct selection for feed
selection of sex‐limited traits and lethal traits. conversion efficiency.
Although selection for body weight has generally been The relationship between body weight and carcass
associated with positive correlated responses such as traits is not consistent from one species to another. The
increased survival and disease resistance, in some cases nature of the heritabilities and genetic correlations
long‐term selection results in decreased bacterial resist- among body weight, visceral fat, muscular fat, muscular
ance either due to changes in genetic correlations or due moisture and muscular ash in gilthead seabream, Sparus
to inbreeding depression. Increased fecundity, fry sur- auratus, would allow development of a selection index to
vival and disease resistance were correlated with selec- improve growth, fat content, texture and carcass yield
tion for increased body weight in channel catfish after simultaneously. Dress‐out and fillet % also had positive
one generation of selection for body weight. Three and heritabilities in gilthead seabream. However, % body
four more generations of selection resulted in increased weight and % fillet had a negative genetic correlation
dress‐out percentage2, decreased tolerance of low DO indicating that it might be difficult to simultaneously
and no change in body composition or seinability3. select for both traits. In the case of sea bass, Dicentrarchus
Progeny from select channel catfish had greater feed labrax, body weight, % viscera, % visceral fat, % fillet and
consumption, more efficient feed conversion and greater % head weight all had significant heritability. Body
disease resistance than controls. weight had a positive correlation with each of these traits
except a negative correlation to % head weight, indicat-
Atlantic salmon show a positive correlated response ing selection for increased body weight would also
in feed conversion when selected for growth rate. increase % fillet, but result in fish with a higher % visceral
Wild salmon had a 17% higher intake of energy and fat. Body weight, % fat, relative head length, relative body
protein per kg of growth compared with fish from the height, relative body width, % processed body and fillet
yields had moderate to high heritabilities in common
2 (body weight without head, viscera and skin) × 100 /total body carp in the Czech Republic. Body weight was highly
weight.
3 Proportion caught when pond is seined for harvesting.
correlated with % fat. Relative head length had strong Genetics 135
negative correlation with % fat, % dress‐out and % fillet.
Thus, indirect selection for reduced relative head length selection for body weight and fillet percentage improved
should increase % fillet, and % dress out, but also increase both traits in Nile tilapia.
% fat. Selection for increase body weight would also
result in a fattier common carp. 7.5.4 Marker‐assisted Selection
and Genomic Selection
Muscle lipid content in rainbow trout responded to bi‐
directional selection. Selection for muscle lipid content Aquaculture genomics has generated an explosion of
did not impact dress‐out % or fillet %. information during the past 22 years. Framework linkage
maps have been constructed with large numbers of
A variety of genetic relationships exist among growth markers, particularly type I markers of known genes,
and survival traits. Selecting for resistance to bacterial have been generated for a number of aquaculture spe-
cold‐water disease in rainbow trout did not affect cies. Normalised cDNA libraries for EST analysis and
body weight or thermal growth coefficients. Resistance functional analysis have been constructed. Functional
to furunculosis, infectious salmon anaemia and genomics has advanced rapidly and the knowledge of
infectious pancreatic necrosis all had relatively high gene expression responsible for growth, disease resist-
heritabilities in Atlantic salmon. Additionally, the ance, reproduction, sex ratio and response to cold tem-
genetic correlations among these traits were all zero, perature and other traits has been greatly expanded for
which should allow simultaneous selection for all three aquaculture species utilising tools such as EST (expressed
traits without any negative correlated responses to sequence tag) analysis and microarrays. Radiation hybrid
selection. panels in tilapia and analysis of BAC (bacterial artificial
chromosome) libraries in catfish has greatly advanced
Heritability for upper thermal tolerance is significant the area of physical mapping of fish genomes. For some
in rainbow trout. Genetic correlation between this trait aquaculture species such as catfish and salmon, the
and body weight was essentially zero, thus no corre- majority of the genes have been isolated and cloned.
lated responses on the corresponding trait would be Catfish have about 27 000 genes of which currently
expected when selecting for either of these traits. around 50% are of known function. Sequencing of fish
Melanin deposits of Atlantic salmon were negatively genomes is well advanced for some aquaculture species
correlated with pericarditis, pericarditis was not corre- and is nearing completion in some cases. This progress
lated with body weight; however, pericardial fat was in the past 22 years is quite remarkable.
correlated with body weight. Shell closing strength has
a high heritability in Japanese pearl oysters, Pinctada Quantitative trait loci, QTL, analysis generates the
fucata. This trait is correlated with high summer sur- data to allow marker‐assisted selection, MAS; selection
vival as the ability to close the shell tightly appears to be based on genetic markers associated with a quantitative
a major survival trait. trait. A great deal of QTL mapping and analysis has been
conducted for aquatic organisms, but very little MAS
7.5.3 Multiple‐trait Selection because of the added expense and need for both greater
resources and record keeping compared to traditional
More than one trait can be selected at a time. Various selection. Marker‐assisted selection programs have been
techniques can be applied, but the most common and successfully evaluated in various animal and plant sys-
effective is multiple‐trait selection using a selection tems. Much theoretical research has been conducted
index. Each trait is weighted with regression coeffi- which indicates that marker‐assisted selection has the
cients that are generated from the heritability, genetic potential to greatly accelerate genetic improvement in
correlation, phenotypic correlation and economic breeding programs. Initial experiments with corn, toma-
value. Little multiple‐trait selection has been con- toes, barley, pigs and dairy cattle have all given positive
ducted in aquaculture because of the extra effort and results indicating that the utilisation of DNA and protein
record keeping required. However, this technique has markers has the potential to accelerate genetic improve-
great potential in aquatic organisms compared to ter- ment in various crops or terrestrial animals.
restrial animals as the high fecundity of fish, crusta-
ceans and bivalves allow more intense multiple‐trait However, marker‐assisted selection is not always the
selection. most efficient or cost‐effective method. These initial
experiments indicate that when heritability for a trait is
Multiple‐trait selection was not successful in white‐leg high, marker‐assisted selection does not provide any
shrimp (Litopenaeus vannamei) for growth and resist- faster rate of genetic gain than traditional selection.
ance to Taura virus syndrome as these two traits were However, when heritability is low, the rate of genetic gain
negatively correlated (Argue et al., 2002). Simultaneous obtained from marker‐assisted selection can be substan-
tially higher than that for traditional selection. Another
136 Aquaculture
disadvantage of MAS is that the genetic markers are usu- have prevented rapid generation of GWAS results in
ally strain specific, preventing the use of the background aquatic organisms followed by genomic selection.
research across strains and populations. Genomic selection is increasingly being used in livestock
selective breeding and genetic enhancement. Initial
QTL markers for growth, feed‐conversion efficiency, experiments indicate that genome selection should pro-
tolerance of bacterial disease, spawning time, embryonic duce more rapid genetic gain for bacterial cold‐water
developmental rates and cold tolerance have been identi- disease resistance than traditional selection in rainbow
fied in channel catfish, rainbow trout and tilapias. trout (Vallejo et al., 2016).
Putative linked markers to the traits of feed‐conversion
efficiency and growth rate have been identified for chan- 7.6 Inbreeding and Maintenance
nel catfish (Dunham, 2011). In trout and salmon, a can- of Genetic Quality
didate DNA marker linked to infectious haematopoietic
necrosis (IHN) disease resistance has also been identi- Even if genetic enhancement is not a goal, loss of genetic
fied and for IPN disease in Atlantic salmon. A single IPN quality and avoiding reduction in performance will always
QTL found on LG1 accounts for most of the variation in be a goal. It is as important to prevent production losses
IPN resistance and a highly resistant line can be devel- due to inbreeding as it is to increase production from
oped by selecting this marker. QTL maps have been genetic enhancement. This is especially true for species
developed that have multiple markers for bacterial dis- with high fecundity, such as carps, where few brood stock
ease resistance in Japanese flounder as well as for body at a single hatchery are necessary to meet demands for fry
weight, total length and a variety of body conformation and brood stock replacement. The detrimental effects of
traits in channel catfish and body weight in Asia seabass, inbreeding, the mating of related individuals, are
Lates calcarifer. A SNP for RuvB‐like protein in Giant well documented and can result in decreases of 30% or
Tiger shrimp is associated with fast growth rate. greater in growth production, survival and reproduction
(Dunham, 2011) once the inbreeding coefficients reach
Marker‐assisted selection has not been broadly applied 0.125 and 0.250 (percent increase in loci homozygous due
in fish. However, MAS lead to the development of a line to the mating of relatives). For many traits, as the inbreed-
of lymphocystis disease resistant Japanese flounder, ing increases, the extent of the inbreeding depression also
Paralichthys olivaceus. These fish were widely applied on increases. Inbreeding depression is easily correctible.
farms and demonstrated high levels of disease resistance When an inbred individual is mated to an unrelated indi-
and survival. MAS may be a mechanism to improve the vidual, the inbreeding coefficient of the progeny returns
efficiency of monosex male production in Nile tilapia. A to 0.0 and the effects of inbreeding on the performance of
microsatellite marker has been found on linkage group the progeny are also eliminated and inbreeding depres-
23 that is associated with sex determination. MAS sion is also zero. This was demonstrated in channel cat-
improved feed‐conversion efficiency by 11% for aquacul- fish. In most aquaculture businesses inbreeding will not
ture species, while traditional selection improved feed‐ become a problem because brood stock populations are
conversion efficiency by 4.3%. The growth rate of relatively large. If 50 breeding pairs are randomly mated
rainbow trout was increased by 26% based on selection per generation, the accumulated inbreeding should not
for an mtDNA marker, but this method was strain‐ result in inbreeding depression for 25–50 generations.
specific because the relative performance of fish with the Formulas for calculating inbreeding and determining the
specific haplotype was consistent across males within impact of random genetic drift are thoroughly discussed
strains but not across strains. In contrast, six generations in Tave (1993).
of traditional selection were required to increase body
weight by 30% in rainbow trout. In rainbow trout, 25% of 7.7 Crossbreeding and
Hybridisation
progeny showed a high degree of upper‐temperature tol-
erance after MAS for heat tolerance. 7.7.1 Intraspecific Crossbreeding
The opposite of inbreeding, crossbreeding is the mating
Genomic selection is on the cusp of revolutionising of unrelated individuals. Intraspecific crossbreeding
(crossing of different strains, lines, breeds or races) has
selective breeding, and, theoretically, new schemes based the potential to increase growth rate and other traits, but
on whole genome selection may enhance rate of genetic heterosis (differences between offspring and parents)
gain even for traits with high heritability. In this case,
genome wide association studies, GWAS, generate the
data on the impact of every single nucleotide polymor-
phism, SNP, on performance of all traits. These studies
are just beginning in aquatic organisms. The SNPs can
be strain specific, but many are species wide. The new-
ness of the technology, the expense, the need for high‐
powered bioinformatics analysis and record keeping
may not be obtained in every case. However, intraspecific Genetics 137
crossbreeding is a relatively effective genetic enhance-
ment program to improve growth rate, and tends to be higher growth rate was found in crossbreeds than the
highly effective for improving survival‐related traits and parent strains. Crossbreeds of different strains of
reproductive performance. This excellent genetic European catfish, Silurus glanis, have outstanding adapt-
enhancement program is probably underutilised because: ability under warmwater holding conditions and mixed
diet feeding regimes. Crossbreeding can also improve
●● it does not have international advocates like selection; performance in crustaceans and resulted in heterosis for
●● requires some effort to identify strains that combine growth rate, but not survival in Chinese white shrimp
(Fennropenaeus chinensis).
well; and then
●● requires maintenance of two or more strains without Crossbreeding often improves survival traits. Strains of
cold‐resistant carp, Ropsha carp, for cold zones in north-
mixing and genetic contamination. ern Russia have been developed by crossing local carps
and Siberian wild carps from the River Amur. Wild strains
Approximately 55% and 22% of channel catfish and rain- of common carp are less susceptible to koi herpes virus (=
bow trout crossbreeds evaluated, respectively, elicited carp interstitial nephritis and gill necrosis virus), whereas,
improved growth rate (Dunham, 2011). Chum salmon domestic strains tend to be vulnerable. Two domestic
crossbreeds, however, had no heterotic increases in strains, two domestic × wild crossbreeds and one domes-
growth rate (Dunham, 2011). Common carp cross- tic × domestic crossbreed were compared for viral resist-
breeds generally express low levels of heterosis and only ance. In the laboratory, the most resistant genotype was
about 5% of the crossed carp that were evaluated had one of the domestic × wild crossbreeds and one of the
enhanced growth rates. However, those that showed pure strains was the least resistant. The remaining genetic
positive heterosis are quite important and at one time groups were intermediate in viral resistance. When the
were the basis for carp aquaculture in Israel, Vietnam, challenges were repeated in ponds, the results were the
China and Hungary. same except the other domestic × wild crossbreed had
excellent resistance in ponds, although its performance
In the case of channel catfish, reciprocal crosses did had been intermediate in the laboratory.
not perform the same and there appeared to be a mater-
nal effect on combining ability. Crossbreeding of the walking catfish, Clarias macro-
cephalus, improves resistance to Aeromonas hydrophila
The crossing of common carp lines in Hungary demon- infections. Crossbreeding improved phagocytosis activ-
strates the frequency of success in long‐term crossbreed- ity in African catfish, Clarias gariepinus, but did not
ing for this species. During a 35‐yr period, more than 140 enhance body weight, total length, the specific immune
crosses were tested. Three were chosen for culture, based response to A. hydrophila, phagocytic index or male
on ca. 20% improvement in growth rate and other quali- reproductive performance. In the case of channel catfish,
tative features, compared to parent and control carp lines. reciprocal crosses did not perform the same and there
Approximately, 80% of common carp production in appeared to be a maternal effect on combining ability.
Hungary is generated from these Szarvas crossbreeds. In
Israel, the crossbreeding of the common carp strain, Domestication has a strong influence on the success of
DOR‐70 and the Croatian line, Nawice, resulted in fast crossbreeding programs. Domestic × domestic crosses
growth and was widely utilised on Israeli farms. The are more likely to result in heterosis than wild × domestic
Czech Republic also utilises improved growing cross- and wild × wild crosses for several traits. Domestic ×
breeds, South Bohemian × Northern mirror carp and domestic channel catfish (Table 7.5) and rainbow trout
Hungarian 15 × Northern mirror (Figures 7.2 and 16.5). are more likely to show heterotic growth rates than
In Vietnam, crossbreeding of eight local varieties of com- domestic × wild crossbreeds.
mon carp, along with Hungary, Ukraine, Indonesia and
Czech strains resulted in significant heterosis in the F1 Table 7.5 Number of channel catfish (Ictalurus punctatus)
progeny. The Vietnamese × Hungarian common carp from domestic (D) by domestic (D × D) crosses and domestic ×
crossbreed was particularly popular, due to fast growth wild (W) (D × W) crosses showing positive, negative or no
and high survival rates under different production condi- heterosis (hybrid vigour) for growth rate.
tions. Double crosses among Vietnamese, Hungarian and
Indonesian strains have subsequently been used for carp Heterosis
selection and crossbreeding ceased throughout Vietnam
because farmers had difficulty maintaining pure parental Cross Positive None Negative
lines for the crossbreeding.
D×D 4 0 1
Heterosis for growth rate, body shape, fillet yield and % D×W 2 3 1
visceral body fat has been observed in Nile tilapia. In the
case of the silver barb, Barbodes gonionotus, 23–35%
138 Aquaculture can occur and impede fry output in channel catfish and
Nile tilapia, and this appears to be more strongly influ-
Again, crossbreeding does not always result in genetic enced by the female than the male.
improvement. No heterosis was observed for reciprocal
crossbreeds between a domestic and wild strain of 7.7.2 Interspecific Hybridisation
Chinook salmon, Oncorhynchus tshawytscha, for
growth survival, saltwater growth, saltwater tolerance, In principle and in genetic basis, interspecific hybridisa-
stress response and recovery and fecundity. Crossbreeds tion is similar to intraspecific crossbreeding. This has
between wild and domestic Atlantic salmon were inter- been a popular breeding program as over the years fisher-
mediate in performance for body weight, condition ies biologists have repeatedly tried to combine the best
factor and sexual maturation. When wild strains of traits of more than one species, mostly with little success.
European sea bass, Dicentrarchus labrax, were crossed, Interspecific hybridisation rarely results in heterosis.
large strain differences were obtained for survival, However, interspecific hybridisation sometimes resulted
growth, shape, sex ratio, muscular fat content, visceral in fish with increased growth rate (Figure 7.5), manipu-
yield and spinal deformities, but not fillet yield. There lated sex ratios, sterilised animals, improved flesh quality,
was no heterosis among these wild strains and no GXE increased disease resistance, improved tolerance of envi-
interactions. This helps illustrate the fact that heterosis ronmental extremes, and other altered traits.
is less likely to be obtained from wild strains than for
domestic strains. Although interspecific hybridisation rarely results in
an F1 suitable for aquaculture application, there are a few
Apparently, domestication also affects the success of important exceptions to this rule. The channel catfish
crossbreeding in crustaceans as well. When two wild female × blue catfish, Ictalurus furcatus, male is the only
strains of giant freshwater prawn, Macrobrachium rosen- hybrid of nearly 50 North American catfish hybrids
bergii, were crossed with a domestic strain (Bangkok), examined that shows superiority for growth rate, growth
large additive strain effects were observed for growth, uniformity, disease resistance, tolerance of low DO lev-
but no heterosis. Wild strains were involved and little els, dress‐out% and harvestability. This is by far the best
heterosis observed. The domestic strain was the fastest genotype for ictalurid farming. This is now the best
growing of the strains. The most rapidly growing prawns example of commercialisation of interspecific hybrids as
were some of the crossbreeds. Reciprocal crossbreeds 70% of US catfish production is now hybrids. Also, this is
did not have the same performance. the best example of overall genetic enhancement in
aquaculture.
One crossbreeding practice is to develop inbred lines
to use in crossbreeding programs to obtain heterosis. Although they do not show heterosis for such a
The existing data indicate that it is unlikely to obtain broad spectrum of traits, crosses of the silver
true genetic gain using this strategy. The crossbreeding carp (Hypophthalmichthys molitrix) and bighead carp
of inbred lines of Pacific oysters, Crassostrea gigas, (Aristichthys nobilis), black crappie (Pomoxis nigromacu-
often resulted in heterosis for growth and survival. latus) and P. annularis and African catfish hybrids
Additionally, crossing of inbred lines of Pacific oysters (Clarias gariepinus, Heterobranchus longifilis and H.
reduces summer mortality. Similarly, the crossing of bisorsalis) all show faster growth than parent species. In
two inbred lines of cockle, Fulvia mutica, resulted in marine fish, the family Sparidae, hybrids of P. major and
increased shell length and whole body weight, but inter- common dentex, Dentex dentex, also grow faster than
mediate survival. It is likely that this is not true genetic parental genotypes.
enhancement. It has been demonstrated that inbreeding
reduces growth in channel catfish. Crosses of the inbred In the case of shellfish, various hybrids between the
lines did indeed grow faster than the parental inbred Thai oyster species (Crassostrea belcheri, C. lugubris and
lines. However, the performance of the crossbreeds only Saccostrea cucullata) were compared, but no heterosis
negated the inbreeding depression and was equivalent was observed. However, heterotic pearl production has
to that of the original population, so no true genetic gain been achieved in China using interspecific hybridisation.
was obtained as performance returned to the original The hybrid between the freshwater pearl mussels,
baseline. Production of gynogenetic female lines and Hyriopsis schlegel ♀ and H. cumingii ♂, increased pearl
gynogenetic sex‐reversed inbred male lines from com- size by 23%, pearl output by 32% and the frequency of
mon carp with the best combining ability was an impor- large pearls by 3.7 times.
tant part of the Hungarian crossbreeding programs. A
higher heterosis was expected from crossing inbred Heterosis for a single trait is not necessarily essential
lines, but the growth rate of F1 crossbreeds was only for an F1 hybrid or cross to have increased value com-
10% higher than controls. pared to the parents. The composite performance may
make the F1 the culture genotype of choice. The ‘sun-
One small potential impediment to crossbreeding pro- shine’ bass between white bass, Morone chrysops and
grams is seed production. Strain mating incompatibility,
Frequency (%) 40 Genetics 139
BC
●● tambaqui (Colossoma macropomum) and the pacus,
30 N = 127 Piaractus brachypoma and P. mesopotamicus;
20 ●● green sunfish (Lepomis cyanellus) crossed with blue-
gill (L. macrochirus);
10
●● gilthead seabream (Sparus aurata) with red seabream
0 (Pagrus major).
40
One of the best examples of commercial application of
F1 interspecific hybridisation is that for walking catfish. At
30 N = 59 one time the primary catfish cultured in Thailand was
the hybrid between African (Clarias gariepinus) and
20 Thai (C. macrocephalus) catfish. Although it does not
grow as fast as pure African catfish, it grows faster than
10 the Thai walking catfish and its yellow flesh is acceptable
to Thai consumers in contrast to the red flesh of the
0 African walking catfish. However, consumer and social
40 attitudes influence breeding goals. The younger genera-
tion of Thai accept red flesh, making the African walking
WC catfish the preferred culture genotype. Another example
30 N = 47 of a good ‘compromise’ hybrid is the rohu (Labeo rohita)
x catla (Catla catla) hybrid which grows almost as fast as
20 pure catla, but has the small head of the rohu considered
desirable in Indian aquaculture. Catla catla × Labeo
10 fimbriatus (fringe‐lipped peninsula carp) hybrids have
the small heads of L. fimbriatus, plus the deep body and
0 growth rate of catla.
5 10 15 20 25 30 35
Total length (cm) Another potential benefit of interspecific hybridisa-
tion is that some species combinations result in progeny
Figure 7.5 Length – frequency distribution of age1+ (1989 class) of with skewed sex ratios or monosex progeny. Monosex
black crappies (BC) (Pomoxis nigromaculata) and white crappies (WC) populations of fish are desirable when growth differ-
(P. annularis) and hybrids (F1) collected in autumn (fall) 1990. The ences between the sexes, sex‐specific products such as
dotted lines indicate the minimum size for fishing. Source: Travnichek caviar are wanted, reproduction needs to be controlled
et al. 1996. Reproduced with permission from Taylor & Francis. or when other exploitable sexual dimorphism exists.
Hybridisation in tilapias or centrarchids often result in
striped bass, M. saxatilis, grows faster, with better over- near monosex hybrids. Hybridisation between the Nile
all culture characteristics for growth, good osmoregula- tilapia and the blue tilapia, Oreochromis aureus, results
tion, high thermal tolerance, resistance to stress and in predominantly male offspring. Tilapia matings, which
certain diseases, high survival under intense culture, produce mainly male offspring, include Nile tilapia ×
ability to use soybean protein in feed, handling tolerance O. urolepis honorum or O. macrochir, and O. mossambicus
and angling vulnerability than either parent species. × O. urolepis honorum. Conversely, the hybrid between
Other examples of crosses that have resulted in improved striped bass and yellow bass (M. mississipiensis) pro-
overall performance in experimental aquaculture condi- duces 100% female individuals.
tions include:
●● common carp with rohu; Theoretically, the production of sterile hybrids can
●● mrigal (Cirrhinus irrhosis) and catla (Catla catla); reduce unwanted reproduction or improve growth rate
by energy diversion from gametogenesis or reduction
in sexual behaviour. Karyotype analysis is believed to be
a general predictor of potential hybrid fertility. Hybrids
of Indian major carps are generally fertile because
they share similar chromosome numbers (2 N = 50).
However, when they are mated with common carp
(2 N = 102), the hybrids have what is equivalent to a 3 N
chromosome number and they are sterile. A natural
triploid also occurs when crossing between grass carp,
Ctenopharyngodon idellus and bighead carp. Grass
140 Aquaculture Table 7.6 Cumulative mortality from cold exposure
for Oreochromis aureus (AA), red backcross O. aureus (AR),
carp are commonly utilised for aquatic macrophyte Oreochromis niloticus (NN) and red backcross O. niloticus (RN)
control in the US, but there is concern about them illustrating the correlated performance of the red backcross and its
establishing in the natural environment, resulting in associated backcross parent species.
potential impact on desirable vegetation in the ecosys-
tem. The grass carp – bighead carp is not a viable option Cumulative mortality (%)
for weed control as, although this triploid hybrid has
reduced fertility, some progeny maintain diploidy and Time Genotypes AA AR NN RN
could be fertile. An exception to the chromosome num-
ber‐fertility rule includes some crosses of sturgeon spe- 1 0 0 7 17
cies with different chromosome numbers that produce 2 0 0 60 80
fertile F1 offspring. 3 7 10 100 100
4 27 37 100 100
Hybridisation is a good program to improve disease 5 100 100 100 100
resistance in fish such as is the case for coho salmon,
(Oncorhynchus kisutch) hybrids, which are considered Hybridisation among marine species, and among
resistant to several salmonid viruses. However, overall marine and freshwater spawning species, has not shown
viability was poor. Viability increased when hybridisa- much promise for developing improved fish for aquacul-
tion was followed by creating triploids. In some cases, ture application. Reciprocal hybrids between Sparus
the salmon hybrids showed the outstanding viral resist- aurata and Pagrus major developed vestigial gonads at
ance, but very poor growth. Triploid Pacific salmon two to three years and were sterile, and no growth or sur-
hybrids sometimes show earlier seawater acclimation. vival superiority was observed compared to the parent
Similarly, increased tolerance of various environmental species until sexual maturity. Hybridisation between
factors may also be inherited by F1 hybrids when one European sea bass (Dicentrarchus labrax) females and
parent species has a wide or specific physiological toler- striped bass (Morone saxatilis) resulted in viable fry.
ance. Several tilapia hybrids display enhanced salinity Only triploid fry survived to 6 mo of age and at 8 mo the
tolerance. Florida red‐strain hybrids (O. mossambicus × survivors showed poor growth compared to diploid D.
O. urolepis hornorum) can reproduce in salinities as high labrax. Such hybrids would only be of commercial value
as 19‰, which is not necessarily a good trait when con- where reproductive confinement is needed for ecological
sidering the potential environmental impact. reasons and a highly desirable flesh quality was obtained.
Just as was the case for intraspecific reciprocal F1 cross- 7.8 Chromosomal Techniques
breeds, reciprocal F1 interspecific hybrids usually show
different phenotypes and performance. Reciprocal hybrids 7.8.1 Gynogenesis, Androgenesis
of O. niloticus (N) × O. mossambicus (M) demonstrate dif- and Cloning
ferent salinity tolerances. Genetic maternal effects were Gynogenesis, and androgenesis are techniques to
evident as the hybrid with the O. niloticus mother had a produce rapid inbreeding and cloned populations.
higher survival rate after salinity challenges at 20‰ than Gynogenetic individuals (‘gynogens’) produced during
pure O. niloticus, but lower survival rates than those of the meiosis (‘meiotic gynogens’) are by definition ‘inbred’,
reciprocal hybrid. At 30‰ salinity, a direct transfer killed since all genetic information is maternal. ‘Meiotic gyno-
all tilapia with O. niloticus maternal ancestry. Growth gens’ are not homozygous, since cross‐overs and recom-
rates of N × M hybrids were comparable to those of Nile bination during oogenesis produce different gene
tilapia, while those of the M × N hybrids and O. mossam- combinations on the chromosomes of the ovum nucleus
bicus were comparable, but lower, than the first two and nucleus of the second polar body, which is expelled
groups, an additional example of maternal genetic effects. during meiosis. The rate of inbreeding through gynogen-
esis is roughly equivalent to one generation of full‐sib
Backcrosses, MN × N, also showed the highest salinity mating. Meiotic gynogens are totally homozygous, with
tolerance (comparable to that of O. mossambicus), but no identical genes on each pair of chromosomes. They are
significant differences in salinity tolerances were found more likely to die during embryonic development due to
in the remaining backcross (N × NM, NM × N, N × MN) the higher frequency of deleterious genotypes found in
or pure O. niloticus; thus some type of maternal effect 100% homozygous individuals.
from the maternal nuclear genome, cytoplasm or mito-
chondrial genome continued to be transmitted to the
backcross generation. Carcass yield of the backcross
hybrids, however, tended to be higher than those of the
parent species. Interspecific backcrossing has also been
used to successfully introgress genes for cold tolerance
and colour among closely related tilapia (Table 7.6).
Androgenesis, or all‐male inheritance, is more difficult Genetics 141
to accomplish than gynogenesis, since diploidy can only
be induced in androgens at first cell division, a difficult variation decreases, environmentally induced variation
time to manipulate the embryo. Also androgens are increases and at a more rapid rate than in heterozygous
totally homozygous, so a large percentage with deleteri- populations.
ous genotypes will probably die.
7.8.2 Polyploidy
Gynogenesis and androgenesis can be used to eluci- In normal development of fish, the diploid egg nucleus
date sex‐determining factors in fish. If the male is the undergoes a mitotic division after a sperm penetrates
homogametic sex when androgens are produced, the through the outer membrane to fertilise the egg. One of
androgens will be 100% ZZ (all‐male). If the male is the two 2 N nuclei resulting from this mitosis is extruded
the heterogametic sex, XX and YY androgens will be from the egg as the first polar body. The 2 N nucleus of
produced, resulting in both sexes. the egg then undergoes a meiotic division and one of the
resulting haploid (N) nuclei is extruded as the second
Fully inbred clonal lines have been produced in polar body. The egg now contains two haploid nuclei:
zebrafish, ayu, common carp, Nile tilapia and rainbow one from the egg and one from the sperm. These fuse to
trout using both gynogenesis and androgenesis. produce a diploid nucleus in a zygote, which then under-
Technology has not yet been shown to directly target goes an initial division into two cells as the first step of
and clone an individual fish. However, two successive embryonic development.
generations of mitotic gynogenesis or androgenesis
results in a clonal, although randomly generated popu- Polyploids, gynogens and androgens are produced by
lation. These individuals within the clonal population disrupting the above processes at various stages through
should have identical genotypes throughout their shocks (Figure 7.6). Various chemical, temperature and
entire genome. Since they will be homozygous for pressure shocks are used shortly after fertilisation to
sex = determining genes, sex reversal must be used to p roduce triploidy and shortly before first cell division
perpetuate these populations. The performance of indi- to produce tetraploidy (Figure 7.6). The timing of the
viduals within such clones is highly variable. Individuals d isruption is critical and that, together with the most
with extreme homozygosity apparently lose the ability effective shock varies according to the species. Meiosis
to respond to environmental variables in a consistent, in shellfish is different from fish. In this case, triploids
stable manner and even micro‐environmental differ- can be produced by blocking either the first or second
ences affect performance among individuals. As genetic meiotic division.
Figure 7.6 Stages of egg nucleus Triploid Tetraploid Gynogenetic Androgenetic
development when shocks are applied to UV UV
produce triploids, tetraploids,
gynogenetics and androgenetics. Source: EN
Tave 1990. Reproduced with permission ES
from the World Aquaculture Society.
SPB S S
h h
o
o c
k
c
k
2N N S
NN h
S o
h c
2N 2N o k
c
3N k 2N
4N 2N
142 Aquaculture Triploid performance can be influenced by strain and
family effects. Diploid Arctic charr (Salvelinus alpines)
Polyploidy was thoroughly evaluated in fish and grew faster than triploids. However, both ploidy level
bivalve molluscs, especially during the period 1970–2000 and family affected growth, and family predicted the
(Dunham, 2011). Triploid evaluation usually emphasises performance of triploids. Triploids from fast growing
the traits of growth, sterility and flesh quality. Triploid families grew more rapidly than diploids from slow
organisms are generally sterile. Females produce less growing families.
sex hormones and, although triploid males may develop
secondary sexual characteristics and show spawning Triploidy can have adverse effects on low DO toler-
behaviour, they are generally unable to reproduce. ance. Triploid channel catfish and triploid catfish hybrids
Triploidy can also be used to restore viability to nonvia- had decreased tolerance of low DO. Similar results have
ble interspecific hybrids. been obtained with salmonids.
Usually, triploidy will not improve growth rates in finfish Triploidy generally results in the prolongation of good
until after sexual maturation, which is beyond market size flesh quality. The flesh of triploid rainbow trout females
for most species. However, there are exceptions: channel was superior to that of diploid females because post‐
catfish triploids grown in tanks were larger than diploids at maturation changes were prevented. Combining mono-
about 9 mo of age (90 g), which is shortly after the first sex breeding and triploidy can produce fish with both
emergence of sexual dimorphism in body weight. This is superior growth rate and flesh quality. The triploid chan-
not advantageous commercially as triploid channel catfish nel catfish had 6% greater carcass yield at three years of
and triploid catfish hybrids did not grow as rapidly as dip- age, which was well past the time of sexual maturity and
loids in commercial environments, such as earthen ponds, market size. However, carcass percentages and resist-
and they had decreased tolerance of low DO. Triploid sal- ance to haemorrhagic septicaemia (caused by Aeromonas
monid hybrids show similar or slower growth than diploid hydrophila) did not differ between triploid or diploid
hybrids, but again may grow faster than controls once they Thai walking catfish.
reach maturity. Triploid chinook salmon (Oncorhynchus
tshawytscha) were less aggressive during feeding than dip- Triploidy can be very beneficial when applied to
loid fish, but grew at the same rate as diploids. bivalve culture. Triploid induction in oysters, such as
Crassostrea gigas, increases their size and flesh quality
In the case of common carp, most one year‐old trip- (Dunham, 2011). In Sydney rock oysters, Saccostrea cuc-
loids had undeveloped gonads and were sterile. The trip- ullata (Figure 7.7), triploidy increased the flesh content
loids grew slower than their diploid siblings under all of the oyster relative to diploid siblings (Kesarcodi‐
conditions investigated. The potential for culture of trip- Watson et al., 2001) (Figure 7.8). Triploid oysters do not
loid common carp appears questionable; however, results produce large gonads, increasing marketability and flesh
from India indicate that triploid common carp had a quality. This technique may or may not result in com-
higher dress‐out % than diploid controls at least partially plete genetic sterilisation in oysters, as some triploids are
compensating for the slower growth.
Figure 7.7 Sydney rock oysters, Saccostrea
cucullata. Croajingolong National Park,
Victoria, Australia. Source: Stevage 2007.
Reproduced under the terms of the
Creative Commons Attribution Share Alike
license, CC BY‐SA 3.0, via Wikimedia
Commons
70 Genetics 143
Total body energy (KJ/oyster) 60 of low DO in finfish as well as having implications for
crustaceans. The triploid shrimp grew faster during sex-
50 ual maturation, but not before this time. Polyploidy is
not commercially feasible for all species because the
40 reproductive biology of some species places limitations
on artificial propagation technology needed for triploid
30 induction. For instance, mouth brooding of many tilapia,
low numbers of eggs per batch and asynchronous spawn-
20 70 80 90 PLOIDY ing prevents or would greatly impede commercial pro-
10 Shell length (mm) 3n duction of triploid tilapia.
2n
0 Tetraploidy is extremely difficult to accomplish in fin-
60 100 fish. Most tetraploid individuals die as embryos. In the
rare cases where a few tetraploids hatched, they were
Figure 7.8 Relationship between total body energy, which to a weak, slow growing and had low survival, but were fer-
large extent is soft body tissue, and shell length in adult and tile. Tetraploids are viable for bivalve molluscs. In this
triploid oysters of Saccostrea cucullata. Each data point is a value case, they are a valuable tool for crossing with diploids to
for a single oyster. Source: Kesarcodi‐Watson et al. 2001. make triploid populations (see above).
Reproduced with permission from Elsevier.
In summary, triploidy is usually not effective for
able to reverse a portion of their cells back to the diploid increasing growth rate, but is very effective for sterilisa-
state, creating potentially fertile mosaics (Dunham, tion and increasing flesh quality. However, triploidy can
2011). Growth performance of sibling triploid and dip- be effective for increasing growth past sexual maturation
loid oysters was correlated, but not their ability to repro- and, in general, is effective for increasing size and growth
duce. With regard to summer mortality, performance of in molluscs and shrimp.
triploid Pacific oysters was much more erratic than that
of diploids. 7.8.3 Sex Reversal and Breeding
A variety of strategies and schemes utilising sex reversal
Benefits of triploidy are not as straightforward in other and breeding, progeny testing, gynogenesis and andro-
mollusc species. Growth and survival were no different genesis can lead to the development of predominantly, or
for the blacklip abalone (Haliotis rubra) up to 30 months completely, male or female populations (genetically and
of age; however, the triploids had a more elongated shell phenotypically) and populations with unique sex chro-
and greater foot muscles than diploids. Triploids had mosome combinations. The goals of this strategy are to
higher feed consumption than diploids, but diploids had take advantage of sexually dimorphic characteristics such
superior feed‐conversion efficiency. Triploids of greenlip as growth and flesh quality, control reproduction or pre-
abalone (Haliotis laevigata) had heavy mortality com- vent establishment of exotic species. All‐female popula-
pared to diploids in several life stages. Diploids also grew tions have been successfully developed for salmonids
faster than triploids, although the triploid abalone (Figure 7.9), cyprinids and tilapias using the scheme pre-
yielded up to 30% greater meat weight compared to same sented in Figure 7.10. Populations of YY males have been
length diploid abalone during the spring‐summer matu- established for Nile tilapia on a commercial scale and on
ration periods at 36 and 48 mo. Diploid abalone pro- an experimental scale for channel catfish (Dunham, 2011),
duced equivalent meat weights to triploid abalone and the procedure is illustrated in Figure 7.11. Genetic
between the maturation periods. Fatty acid composition production of monosex populations has the advantage of
of the meat was the same for triploids and diploids. In reduced hormone use compared to direct sex reversal
the majority of examples of various species of molluscs, using hormones, which of course has environmental and
triploidy was beneficial (Dunham, 2011). regulatory implications.
Triploidy is the only technique that can guarantee that Sex‐determining mechanisms were reviewed by
marine shrimp populations are skewed towards the (Devlin and Nagahama 2002, Dunham, 2011). While
faster growing sex, the female. Triploidy is also used to many commercially cultured families show the usual
prevent the theft of elite stocks/germplasm. XX/XY sex determination mechanism (carps, salmo-
nids), where XX are females and XY are males, others
In the Chinese white shrimp (Fenneropenaeus chinen- may be sequential hermaphrodites (change sex as they
sis) triploids had a reduced number of haemocytes. This mature), such as gilthead seabream and groupers, or
may be a key to explain the trend of reduced tolerance have temperature‐controlled sex determination in addi-
tion to an XX/XY mechanism such as in Nile tilapia and
144 Aquaculture
Percentage of ova types 100 All female are XY can be turned into phenotypic females by use of
Triploid sex hormones and can then be used as breeders. The sex
ratio of progeny from the mating of XY female and XY
80 Mixed sex male channel catfish was 2.8 males: 1 female, indicating
that most, if not all, the YY individuals are viable. All‐
60 male progeny are beneficial for catfish culture, since they
grow 10‐30% faster than females. YY males are also via-
40 ble in salmonids, Nile tilapia and goldfish. The channel
catfish YY system has stalled, however, because YY
20 females have severe reproductive problems, and large‐
scale progeny testing is not economically feasible to
1986 1988 1990 1992 1994 1996 1998 identify YY males. A combination of sex reversal and
Year breeding to produce all‐female XX rainbow trout is now
the basis for stocking most of the culture industry in the
Figure 7.9 Percentage of all‐female, triploid and mixed‐sex United Kingdom, as is the case for the chinook salmon
rainbow trout, Oncorhynchus mykiss, utilised in and from 1986 to industry in Canada. All‐female populations are desira-
1998, illustrating the increasing and almost exclusive adoption ble, in this case, because males undergo maturation at a
of the all‐female production technology Source: D. Penman. small size, and have poorer flesh quality. Monosex chi-
Reproduced with permission from Dr Penman. Data from nook (O. tshchawystcha), and coho crossed with chinook
Marine Scotland. have also been produced.
hirame. Different mechanisms may also be found in YY male Nile tilapia were as viable and fertile as XY
closely related species: males, and capable of siring 96% male offspring. YY geno-
●● the Nile tilapia has the XX/XY system with the female types can be feminised to mass produce YY males with
YY × YY matings, thus eliminating the need for time‐
being homogametic, XX, and the male XY; consuming progeny testing to discriminate XY and YY
●● the blue tilapia has a WZ/ZZ system with the male male genotypes. This has enabled the production of
YY males and then all‐male progeny, XY, after crossing
being homogametic, ZZ, and the female,WZ. with normal XX females. These normal all‐males derived
Similar differences in closely related species are likely to from the YY males are sold commercially as ‘genetically
exist for centrarchids, ictlaurids and perhaps others. male tilapia’ [GMT®] to distinguish them from sex‐
Additionally, sex determination has polygenic influences reversed male tilapia, The YY male technology provides
in some species. an effective solution to culture problems with early sexual
maturation, unwanted reproduction and overpopulation.
Sex ratios of Nile tilapia at 36 °C become a quantitative
trait. Three generations of selection for maleness Sex ratios vary widely between spawnings of Nile tila-
resulted in 93% male progeny, whereas selection for pia, but at the population level, they maintain a normal
femaleness resulted in a sex ratio of 1:1. Heritability was distribution of around 1:1 males to females. Sex ratios
high for maleness. The response to selection for female- vary among strains of Nile tilapia and greater heterogene-
ness was the result of a lower heritability coupled with ity was found in the sex ratios of families collected from a
maternal effects. mix of strains, some of which were introgressed with the
Mozambique tilapia (O. mossambicus). YY males crossed
Sex reversal and breeding has allowed production of with XX females produce 95–100% males and there were
YY channel catfish males that can be mated to normal no females among 285 progeny from the mating of a
XX females to produce all‐male XY progeny. Males that
Take a sample of fry and sex reverse to all males with methyltestosterone, Figure 7.10 Scheme for producing all‐female XX
resulting in XX and XY males populations of fish. Source: Reproduced with
permission from Rex Dunham.
Progeny test with normal XX females: XX × XY yields 1:1 sex ratio
: XX × XX gives all females
Sex reverse a sample of XX fry to males with methyltestosterone for future
broodstocka
aIn a perpetual XX population, all of the progeny are XX female—thus if you do
not sex reverse some of these to males, eventually all of your XX females will
die of old age and there are none left to mate with XX males and the XX
population dies out.
Genetics 145
Figure 7.11 Scheme for producing all YY male Take a sample of fry and sex reverse to females with beta-
populations of fish. Source: Reproduced with estradiol XX females and XY females
permission from Rex Dunham.
Progeny test with XY male:
XX female × XY male yields 1:1 sex ratio
XY female × XY male yields 1 XX: 2 XY: 1YY = 1 female : 3 males
Take the male progeny XY and YY and progeny test with and XY
female from the mother’s generation:
XY male × XY female yields sex ratio of 1 female : 3 males
YY male × XY female yields a sex ratio of 1 XY : 1 YY = all males
Take sample of these fry and sex reverse to female with beta-
estradiol YY and XY females
Progeny test with XY males:
XX female × XY male yields a sex ratio of 1 female : 3 males
YY female × XY male yields a genotype ratio of 1 XY: 1 YY = all males
Cross YY males with YY females to generate all YY progeny
Sex reverse a sample of these YY males to females with beta-
estradiol for future brood stocka
a In a perpetual YY population, all of the progeny are YY males—thus if you do
not sex reverse some of these to females, eventually all of your YY males will
die of old age and there are none left to mate with YY females and the YY
population dies out.
single YY male crossed to 10 separate females indicating feminisation of YY genotypes and existing hatchery
the potential to select for lines that can produce 100% systems without any special facilities or labour require-
males. In fact, three generations of gynogenetic Nile tila- ments. YY male technology has been widely dissemi-
pia have been produced, and males from this line were nated in the Philippines since 1995, Thailand since 1997
used for mating with gynogenetic Nile tilapia females, and, to a lesser extent, in a number of other countries
resulting in consistent production of 100% males. including Vietnam, China, Fiji and the USA. In the
Philippines and Thailand, broodstock are distributed
YY‐GMT® technology has commercial application, from breeding centres to accredited hatcheries to
maintain quality control and proprietary control. The
since YY Nile tilapia, unlike channel catfish, can be sex‐
reversed to produce functional females. The progeny of availability of further improved GMT®, along with
the YY‐GMT® males increase yields by up to 58% com- increasing resistance to use of hormones in aquaculture
should allow this technology to impact tilapia culture
pared to mixed‐sex tilapia of the same strain, as well as on a global scale.
greater yields than sex‐reversed male tilapia. In addi-
In Israel, all‐female common carp populations have
tion, YY‐GMT® fish have more uniform harvest size, been established using sex‐reversed XX gynogenetic
females crossed to sex‐reversed XX males. All‐female
greater survival and better food conversion ratios. offspring were released to commercial farms and resulted
Although the development process is time‐consuming
and labour‐intensive, once developed the production of
monosex males can be maintained through occasional
146 Aquaculture male germ‐line stem cells, which can regenerate by sper-
matogonial dedifferentiation.
in 10–15% yield improvement over existing commercial
stocks. Gynogenesis and sex reversal have also success- Both primordial germ cells (PGCs) and spermatogonia
fully induced in temperate basses (Morone spp.) to pro- A (SSCs) have been transplanted from a donor species to
duce monosex populations to avoid limitations on a related host species with the recipient species produc-
introductions to areas where this species is exotic. ing sperm and eggs (originating from testicular PGCs or
SSCs) of the donor species. This procedure can be suc-
Monosex female Java barbs (Barbonymus gonionotus) cessful utilising both cryopreserved or fresh donor cells
are another example of sex manipulation and progeny opening the possibility of many potential applications. In
testing being adapted to a commercial scale over a rela- the case of the salmonid xenogens, testicular develop-
tively short period (8 yr) in Thailand and Bangladesh. ment was normal. A 30–70% success rate was achieved
Gynogenetic Java barbs were all‐female and were hor- among injected host embryos. The xenogenic individu-
monally masculinised to produce XX male. Most of the als did have reduced fecundity (possible age effects), but
breeding of the resultant neomales produced all, or the F1 offspring were normal in genotype, performance
nearly all, female progeny. These gave greater yields in and appearance.
pond culture than mixed‐sex batches and, perhaps sur-
prisingly, had higher survival rates than the mixed‐sex Alternative methods also have the potential to allow
fish. This increased survival was likely to be due to the xenogenesis and autogenesis via PGC transplantation. A
all‐female fry being large enough to better utilise zoo- single PGC from pearl danio (Danio albolineatus) was
plankton when stocked into ponds. Monosex female fin- transplanted into the blastula of a zebrafish whose native
gerlings from neomale broodstock are now supplied on a PGC production had been knocked out by an antisense
commercial scale in Thailand. morpholinos oligonucleotide against dead end. The
donated PGC formed a single testis that produced pearl
7.9 Xenogenesis danio sperm. Xenogenic pearl danio males were sex‐
reversed to femaleness and mated with untreated males to
A xenogen is an organism comprised of elements typi- produce normal, fertile pearl danio offspring. Similarly,
cally foreign to its species, and xenogenesis is a method the zebrafish host was able to develop goldfish (Carassius
of reproduction in which successive generations differ auratus) and loach (Misgurnus anguillicaudatus) testis
from each other. This technology was first accomplished that produced donor sperm from the injection of a single
on a small scale with salmonids by Japanese scientists donor PGC. Normally, a few dozen PGCs are needed to
and has many potential genetic applications. Since those form gonads containing germ cells. This study showed
initial experiments, xenogenesis has been demonstrated that one PGC and perhaps a single SSC are capable of pro-
in a variety of species including zebrafish, ictalurid cat- ducing a single testis. The next step is to determine if xen-
fish, Nile tilapia, pejeerey and some marine species. This ogenesis can be applied on a large scale. Xenogenic triploid
procedure is a powerful tool for genomics research, gene channel catfish harbouring diploid sperm from blue cat-
knockout applications, cloning and reviving extinct lines fish (Ictalurus furcatus) were mated with normal channel
and species. catfish females resulting in 100% channel catfish female ×
blue catfish hybrids. This opens the possibility of more
Xenogenesis is a new biotechnological tool that allows naturally and easily producing hybrids as well as a mecha-
one to interspecifically transfer embryonic stem (ES) nism to more easily produce difficult to spawn species.
cells. Totipotent spermatogonia from the testes of dip-
loid rainbow trout were intraperitoneally transplanted The potential for innovative applications of xenogene-
into newly hatched sterile triploid masu salmon sis is expanding as the host‐donor combinations have
(Oncorhynchus masou) resulting in xenogenic individu- been found to be quite plastic. Stem cells can be success-
als. Upon maturity, these triploid salmon produced only fully transplanted across families of fish and across
donor‐derived, pure, rainbow trout offspring. orders of fish.
Testes contain five major types of cells, spermatogonia 7.10 Genetic Engineering
A, committed spermatogonia B, spermatids, mature
sperm cells and somatic cells. Type A spermatogonia 7.10.1 Current State of Genetic Engineering
cells possess stem cell potential. These cells divide dur- in Aquaculture
ing spermatogenesis producing isogenic germ cells com- Transgenic fish have been developed that have improved
mitted to meiosis. Spermatogonia A from fish are stem growth, colour, disease resistance, survival in cold and
cell‐like, and when transplanted into developing rainbow body composition, and that can produce pharmaceutical
trout (Oncorhynchus mykiss) embryos, these embryos
produced sperm or eggs derived from the transplanted
cells. Similar results were previously found for Drosophila
Figure 7.12 Microinjecting DNA into salmon eggs. Genetics 147
Source: R. Devlin. Reproduced with permission from R. Devlin.
also approved the consumption of GH transgenic salmon,
AquaAdvantage Salmon. The first 4 tonne were placed in
the Canadian market, and were all quickly sold. This is
the first government approved consumption of any trans-
genic animal. Expansion into the US appears eminent in
the near future as AquaBounty Technologies Inc. has
invested in a large, indoor recirculating system in the
state of Indiana.
Transgenic carp and tilapia with growth hormone
genes are near commercialisation in some countries.
Commercialisation of these was once thought to have
taken place in some countries such as China and Cuba;
however, no official documents are available to confirm
this and government representatives indicate that
approvals are still pending.
To expedite increased commercialisation and mini-
mise environmental risk, transgenic sterilisation needs
to be developed. When transgenic fish research was ini-
tiated, a large percentage of the work was actually con-
ducted on commercial aquaculture species, but an
increasing amount of research is conducted with model
species. Potential positive impact of transgenic fish
appears likely in many areas.
proteins. Transgenes elicit pleiotropic effects, some 7.10.2 Growth Hormone (GH) Transgenics
positive and a few negative, but most of the negative Norman Maclean and S. Talawar of Southampton
effects appear to lower fitness traits which is positive for University, UK, were the first researchers to inject
biological containment. Transgenic fish appear to pose cloned genes (Figure 7.13) into fish (rainbow trout) eggs
little environmental risk, but this research is not fully (Maclean and Talawar, 1984). This was followed by
conclusive. Zuoyan Zhu at the Institute of Hydrobiology in China
reporting production of a transgenic goldfish (Zhu et al.,
The first application has actually been in the orna- 1985). Thirty or so years later, transgenic fish applica-
mental fish industry rather than in general aquaculture. tion sits on the cutting edge and is making its first major
GloFish (Figure 7.12), a transgenic fish of the zebrafish impact.
(Danio rerio) containing fluorescent protein genes, GFP,
YFP and RFP, are now commercialised. These fish were Figure 7.13 GloFish® fluorescent fish: genetically modified black
actually an output of experiments to develop transgenic tetras (Gymnocorymbus ternetzi). Source: K. Głąb 2011. Reproduced
fish for environmental monitoring, but were an obvious under the terms of the Creative Commons license, CC BY‐SA 3.0.
choice to create a marketing niche in the ornamental
fish trade.
In November 2014, the Canadian government approved
the sale of triploid growth hormone (GH) salmon
embryos for export to countries that would allow the cul-
ture of transgenic fish (Fisheries and Oceans Canada,
2013). History was made when the flesh of these triploid
transgenic salmon produced by AquaBounty Technologies
Inc. was approved by Food and Drug Agency of the USA
for consumption of these fish in the USA (U.S. Food and
Drug Administration, 2015). This is the first known
approval of the consumption of transgenic animal meat
in the world. Several groups are still trying to block the
sale of these fish, but consumer acceptance of transgenic
meat is the next potential obstacle to the successful com-
mercial application of transgenic fish. Now Canada has
148 Aquaculture
Figure 7.14 Transgenic AquAdvantage
Atlantic salmon vs non‐transgenic sibling.
Source: AquaBounty Technologies.
Reproduced with permission from
AquaBounty Technologies.
The greatest amount of work has focused on transfer Transgenically produced biomedical compounds
of GH genes. Due to the lack of available fish gene should be safe from human pathogens, should eventually
sequences, transgenic fish research in the mid‐1980s be less expensive and more widely available.
employed existing mammalian GH gene constructs, and
promoters from viral, bacterial and mammalian sources. Fish have potential advantages as bioreactors com-
In the early 1990s most GH research then switched to pared to mammals. These advantages include a short
using fish GH constructs. Growth (size and rate) generation interval, low cost of maintenance of the ani-
enhancement has ranged from 0% up to an incredible mals, easy maintenance, large numbers of individuals,
300% under some conditions (Figure 7.14). All fish GH high density culture and mammalian viruses and prions
constructs introduced into salmonids elevated circulat- are not found in fish. Several examples are now available
ing GH levels by 40‐fold, and in some cases, induced demonstrating the potential of fish as bioreactors for
5–11 fold increases in weight after one year of growth. medical products as well as compounds that can be used
Precocious smoltification (physiological adaptation from in fish spawning.
fresh water to sea water) also occurred.
CMV‐human coagulation factor VII was produced in
7.10.3 Body Composition transgenic zebrafish, African walking catfish and Nile
Transgenic alteration of the nutritional characteristics tilapia eggs. Clotting activity was detected indicating
of fish could be beneficial for consumers, and it is now proper post‐translational modifications. Proteins could
possible to directly alter body composition via transgen- be collected in eggs, serum or possibly different proteins
esis. Zebrafish transfected with B‐actin‐salmon desatu- in different tissues for other types of genes.
rase genes had enhanced levels of omega‐3 fatty acids,
docosahexaenoic acid (DHA) and eicosapentaenoic Transgenic Nile tilapia secreted human insulin in
acid (EPA), in their flesh. Similar results were obtained Brockmann Bodies. Islet tissue was used for xenotrans-
when the same transgenes were transferred to common plantation and successfully transferred to diabetic nude
carp and channel catfish, and the expression of these mice reversing the effects of diabetes.
transgenes was verified.
Single chain goldfish luteinising hormone(LH) gene
7.10.4 Bioreactors was injected into rainbow trout eggs. At 4‐days of age
Transgenic mammals such as cows, goats, sheep, and goldfish LH was isolated from the eggs and the recombi-
rabbits have been used as biological factories to produce nant LH injected into goldfish. Testosterone levels were
pharmaceutical compounds and biomedical proteins elevated in male goldfish after the injections proving bio-
such as clotting factors and blood thinners. Such tech- logical activity.
nology is especially important in the modern world since
human extracted products have the potential to be con- 7.10.5 Disease Resistance
taminated with HIV, hepatitis viruses and other human Transgenic fish with enhanced disease resistance would
pathogens. These products can also be quite expensive. increase profitability, production, efficiency and the wel-
fare of the cultured fish. Genetic gain is also possible
through traditional selective breeding, but it appears
that the rate of genetic improvement and the consistency
of genetic improvement may be greater with the trans-
genic approach for disease resistance. Selective breeding
Genetics 149
Table 7.7 Enhanced resistance to bacterial disease demonstrated by transgenic channel catfish containing cecropin genes in a natural
epizootic and an artificial tank challenge.
Transgene Disease challenge Environment Survival % Control
Transgenic 27
Preprocecropin B Flavobacterium Pond 100
Cecropin B columnare Tank 41 15
Edwardsiella
ictauri
Source: Adapted from Dunham et al. 2002 with permission from Springer.
may also have the drawback that the disease organisms potentially because of differences in genetic back-
may well respond to selective forces as well, negating ground, variable insertions sites, copy number, epistasis
some of the selection response in the fish. and other factors. This necessitates coupling selection
with gene transfer to obtain maximum genetic gain
Expression of viral‐coat protein genes or antisense of from the gene transfer.
viral early genes has improved viral resistance in rainbow
trout. Shrimp have been genetically engineered with Grass carp (Ctenopharyngodon idellus) have been
antisense Taura syndrome virus‐coat protein gene result- transfected with carp B actin – human lactoferrin gene
ing in a doubling of the resistance to this disease. resulting in P1 individuals that were more resistant to
Aeromonas, showed enhanced phagocytosis and more
Bacterial disease resistance may be easier to geneti- viral resistance than controls. Japanese flounder keratin
cally engineer than for diseases caused by other classifi- promoter linked to both the hen egg white (HEW)
cation of pathogens and is more well‐studied. Bacterial lysozyme gene and green fluorescence protein (GFP)
disease resistance may be improved up to 3 to 4‐fold gene, and transferred to zebrafish resulted in F2
through gene transfer. One approach that has been uti- t ransgenic zebrafish with lytic activity of protein extracts
lised is the transfer of antibacterial peptide genes. from the liver 1.75 times higher than in the wild type
zebrafish. In a challenge experiment, 65% of the F2 trans-
Insertion of the lytic peptide, cecropin B construct, genic fish survived an infection of Flavobacterium
driven by the CMV promoter, enhanced resistance to columnare and 60% survived an infection of Edwardsiella
bacterial diseases such as columnaris and enteric septi- tarda (likely pisicida), whereas 100% of the control fish
caemia of catfish 2 to 4‐fold in channel catfish, Ictalurus were killed by both pathogens.
punctatus (Table 7.7). A greater percentage (100%) of
transgenic individuals containing preprocecropin B con- 7.10.6 Cold Tolerance
struct survived than non‐transgenic controls (27.3%) There have been attempts to improve cold resistance in
during an episootic of Flavobacterium columnare in an fish. Early research involved the transfer of the antifreeze
earthen pond. In this case, the transgene appears to have protein gene of the winter flounder. The primary pur-
imparted complete resistance. pose of this research was to produce salmon that could
be farmed under Arctic conditions, but expression levels
Also, a greater percentage (40.7%) of transgenic indi- obtained have been inadequate for increasing cold
viduals containing cecropin B construct survived than tolerance of salmon. However, preliminary results with
non‐transgenic controls (14.8%) when challenged with goldfish show some promise for increasing survival
Edwardsiella ictaluri, causative agent of enteric septicae- within the normal cold temperature range. Recent
mia of catfish, ESC, in tanks. There were no pleiotropic research indicates that GH transgenic channel catfish
effects, and growth rate of the transgenic and non‐trans- have increased survival at −0.5 °C compared to non‐
genic siblings was not different. However, cecropin transgenic full‐siblings,
transgenic rainbow trout have shown increased bacterial
and viral disease resistance. 7.10.7 Pleiotropic Effects
The insertion of a transgene, a single gene, can affect more
Transfer of cecropin genes to Japanese ricefish than one trait, the pleiotropic effect. If the breeder is
(Oryzias latipes) resulted in increased resistance to fortunate, these pleiotropic effects will impact other traits
Pseudomonas fluorescens and Vibrio anguillarum,
which killed about 40% of the control fish in both cases.
However, only 0‐10% of the F2 transgenic fish were
killed by P. fluorescens and about 10‐30% killed by
V. anguillarum. Family variation was observed, and
family variation can be extreme for transgenic fish
150 Aquaculture common carp. F2 progeny inheriting this transgene
had higher survival than controls when exposed to a
in a positive way. Transfer of growth hormone genes has series of stressors and pathogens such as low DO,
been documented to affect body composition, body shape, anchor worms, Lernia, Aeromonas and dropsy. GH
feed‐conversion efficiency, disease resistance, reproduc- transgenic common carp had higher lysozyme activity
tion, tolerance of low DO concentrations, carcass yield, in the serum compared to age‐matched non‐transgenic
swimming ability and even predator avoidance. control fish. The serum bactericidal activity in the
transgenics was 20% higher than in the controls. Values
Increased growth rate in transgenic individuals may for leukocrit and phagocytic percent of macrophages
reflect increased food consumption, FCE4, or both. Fast in head kidney were higher in transgenics than con-
growing common carp containing the rtGH gene were trols, but the phagocytic indices and relative spleen
found to have better FCE than controls. Additional weights in the transgenics and the controls were not
transgenic common carp families demonstrated different. GH transgene expression apparently not only
increased, decreased or unchanged food consumption, stimulated growth, but also the non‐specific immune
but had improved FCE. rtGH transgenic common carp functions of common carp.
had more protein, less fat and less moisture than non‐
transgenic full‐siblings (about a 10% change). Growth Conversely, GH transgenic salmon were more sensi-
hormone promotes synthesis of protein over fat, thus the tive to Vibrio compared to controls so GH gene transfer
protein/lipid ratio is higher in transgenic fish with ele- does not always confer increased disease resistance.
vated growth hormone. Survival among GH salmon families was sometimes
improved, sometimes decreased and sometimes
Increased protein levels in the muscle of transgenic unchanged relative to controls. These differences in
common carp also increased levels of amino acids, how- salmon could be related to alterations in expression for a
ever, amino acid ratios and fatty acid ratios are virtually myriad of disease‐related genes in relation to the altered
identical in non‐transgenic and transgenic common carp. growth hormone expression. Growth hormone appears
Fecundity or precocious sexual development appear to to have pleiotropic effects and causes a cascade of events
be unaffected by insertion of rtGH in common carp; in a large number of biochemical pathways:
however, transgenic male tilapia have shown decreased
sperm production. Body shape of common carp was also ●● haem oxygenase;
changed by insertion of rtGH genes. Transgenic individ- ●● Acyl‐coA binding protein;
uals have relatively larger heads, and deeper and wider ●● NADH dehydrogenase;
bodies and caudal areas compared to controls. These ●● mannose binding lectin‐associated serine protease;
morphological changes do not affect condition factor, ●● haemopexin‐like protein;
but do improve the dressing percentage. ●● leucyte‐derived chemotaxin2 (LECT2);
●● and many other genes had enhanced expression in
Endocrine stimulation can lead to excessive, deleteri-
ous, deposition of cartilage in some GH transgenic hepatic tissue of immature transgenic salmon.
salmon with hyper‐growth rates (30X compared to con-
trols), analogous to mammalian acromegaly. The effect While the following had decreased expression compared
can be severe enough to impair viability. This is not a to non‐transgenic controls:
great problem as these genotypes are eliminated by natu- ●● complement C3‐1;
ral selection, and lines showing 10X growth rates do not ●● lectin;
show this anomaly and have good viability. ●● rabin;
●● alcohol dehydrogenase;
To date, two types of transgenic processes have been ●● Tc1‐like transposase; and
successful for improving disease resistance, blocking ●● pentraxin genes.
viruses with antisense and overexpressing antibacterial Gene expression pattern changed when transgenic
compounds from distant taxa. This is the extent of salmon approached maturation with the following hav-
reported research using transgenesis to directly improve ing enhanced expression:
disease resistance. However, transfer of other genes can ●● haemopexin‐like protein;
indirectly effect disease resistance through pleiotropy, ●● haem oxygenase;
both in a positive and negative way. If the pleiotropic ●● inter alpha‐trypsin inhibitor;
effects are known, they could be intentionally manipu- ●● LECT;
lated for genetic gain in a manner analogous to indirect ●● GTP cyclohydrolase; and
selection. ●● I feedback regulatory protein (GFRP).
Survival is an important commercial trait and the
insertion of the rtGH gene altered the survival of
4 FCE (food conversion efficiency)(%) = 100/FCR
While the following showed decreased expression: Genetics 151
●● Bikunin; GH gene transfer alters respiration and metabolism in
●● Lectin; many ways that could affect the ability to overcome dis-
●● Apolipoprotein; and eases either in an enhanced or detrimental manner. The
●● Pentraxin. results from common carp indicate GH transfer could be
used as an indirect method to transgenically enhance
Lectin was found to be highly suppressed in all F2 and disease resistance. The salmon situation may be different
immature F3 salmon. Serum lysozyme activity of the because of their different life history, the fact that they
innate immunity system was decreased in both genera- are a cold rather than warmwater fish and that their GH
tions of GH transgenic fish. GH transgenic amago enhancement is much more dramatic. The extent of plei-
salmon (Oncorhynchus masou macrostomus) had altered otropic effects is likely to be a product of the magnitude
hepatic gene expression relating to iron‐metabolism and of the change in the primary target trait and the associ-
innate immunity. ated expression strength of the transgene.
GH gene effects respiration, which in turn could have 7.10.8 Transgenic Sterilisation
a multitude of intertwined relationships with, and effects The ultimate means of preventing environmental or eco-
on, growth, low DO tolerance, disease resistance, stam- logical impact of transgenic, domestic hybrid or exotic
ina and predator avoidance. When subjected to low DO, fish are transgenic sterilisation or gene editing, which will
0.4 ppm, mean absolute survival was the same for trans- be discussed in the next section. In this case, escaped fish
genic rtGH and control common carp. However, when are incapable of breeding or their progeny are incapable
mean survival time was calculated, the transgenic indi- of breeding, resulting in absolute reproductive confine-
viduals had longer mean survival time than the non‐ ment and the prevention of introgression of transgenes
transgenic full‐siblings. Ventilation rate could also be a into wild populations and any potential associated
possible explanation for the slightly better tolerance of impacts. Several potential transgenic approaches have
low DO showed by the transgenic common carp. been evaluated including antisense, shRNAi and overex-
Transgenic channel catfish with the same rtGH con- pression of cDNAs. Some systems have shown promise,
struct as the common carp had a lower ventilation rate but they require fertile individuals at some point in the
when subjected to low DO compared to controls. process. In this case, long‐term environmental risk can-
not be eliminated. If repressible systems are used and all
Pleiotropy of GH gene for DO tolerance characteristics individuals in the population are homozygous for the
varies from one species to another. GH tilapia have a 58% sterilisation construct at all life stages, environmental risk
higher metabolism than controls, compensate for oxygen can only be short‐term in the worst‐case scenario.
consumption and have the same maximum swim speed
as non‐transgenics. GH tilapia tolerate hypoxia equally as Tet‐off systems of transgenic sterilisation have shown
well as controls despite higher demand for oxygen. great promise as repressible systems. The primary prob-
lem with these systems is that the transgene contains
GH transgenic salmon have an increased need for DO, small viral sequences. This does not pose any food safety
however, after 4 days of starvation. GH individuals had or biological risk; however, public perception and nega-
the same oxygen uptake as controls. After feeding, GH tive advertising could prevent the marketing of such
transgenics had 40–70% increased oxygen demand even aquatic organisms. Tet‐off like systems have been
when controls consumed equivalent amounts of feed. recently developed, which contain no viral sequences.
Adult transgenics salmon had higher oxygen demand, One potential drawback of transgenic sterilisation are
poorer swimming ability and longer recovery time com- potential negative pleiotropic affects as knocking out
pared to ocean‐ranched salmon. reproduction can affect other traits similar to what might
be observed with triploidy. For example, knock out of
GH transgenesis can have significant metabolic costs. some primordial germ cell genes resulted in channel cat-
Cardiac function was enhanced by GH transgenesis, but fish with no gonads, and these fish had a 25% reduction
there was no universal upregulation of cardiorespiratory in growth rate.
physiology in post‐smolt (adult) GH transgenic salmon.
Differences in arterial oxygen transport such as cardiac 7.11 Gene Editing
output and blood oxygen carrying capacity are impor-
tant for aerobic capacity; however, diffusion‐limited pro- Gene editing is the targeted deletion of specific base
cesses may be bottlenecks that would need to be sequences with the most common goal being the muta-
enhanced to achieve substantial improvements in meta- tion and disabling of a specific gene. The genes are
bolic and swimming performance. These diffusion‐
related limiting factors associated with gill function and
morphology may explain differences in results from one
study to another.
152 Aquaculture is now the technology of choice. CRISPR/Cas9 utilises
more potential recognition sites and can be engineered
disabled without the introduction of exogenous DNA. to target any sequence in the genome. Cas9 can be used
Technology for targeted gene editing is advancing rap- with any guide RNA. Mutations in multiple genes can be
idly with almost weekly innovations being reported. generated simultaneously in a single step by introducing
Gene editing can also be used in conjunction with genetic a cocktail of sgRNAs.
engineering for targeted gene insertion in known areas
of the genome, to substitute single base pairs to create In the case of fish, the myostatin gene of the yellow cat-
alleles and to correct defective genes. Gene editing has fish (Pelteobagrus fulvidraco) has been edited. Rainbow
been used to create polled cattle and double muscled cat- trout master sex‐determining gene was knocked out
tle without the need to introgress these mutant alleles using ZFNs resulting in the conversion of males to females
through crossbreeding. including ovarian development. Luteinising hormone
gene was knocked out in channel catfish to achieve trans-
The initial gene editing technology involved the use of genic sterilisation using ZFNs. Small indels were identi-
zinc finger nucleases (ZFNs), a major breakthrough fied in addition to substitution deletion complex, resulting
allowing targeted gene editing. ZFNs are fusion proteins in fish that were not able to successfully spawn. Knockout
of a modular DNA‐binding domain yolked to a FokI of follicle‐stimulating hormone receptor in zebrafish
endonuclease monomer. When a pair of ZFNs binds to using TALENs resulted in failed follicle activation and sex
their target in the correct orientation, FokI monomers reversal to males. Myostatin gene knockout in Japanese
can dimerise and introduce a double‐stranded DNA ricefish (Oryzias latipes) using TALENs resulted in 25%
break (Kim et al., 1996), which can be repaired by nonho- larger body weight compared to controls. As expected,
mologous end‐joining (NHEJ). The result is a small knockout of tyrosinase utilising CRISPR/Cas9 results in
insertion or deletion (indels), which causes a frame‐shift, albinism in salmon.
disabling encoded proteins in 67% of the indels. ZFN
knockout can be limited by available target sites. 7.12 Combining Genetic
Enhancement Programs
Transcription activator‐like effector nucleases (TALENs)
were the first major improvement in the gene editing tech- The best genotypes for aquaculture applications in
nology and are nucleases that join FokI endonuclease with the future will be developed by using a combination of
the modular DNA‐binding domain of TALEs. TALENs traditional selective breeding, the new biotechnologies
were an improvement on both complexity and transforma- and molecular/genomic approaches. Initial experiments
tion rates. TALENs are more easily manufactured with indicate good potential for this combined approach, with
straightforward design and assembly strategies, less examples using:
expensive and can result in high knockout rates (2–75%, ●● mass selection and crossbreeding;
often > 50%) in primary cells and embryos of livestock and ●● genetic engineering and selection;
fish. Multiple deletions (knockout alleles) can be generated ●● genetic engineering and crossbreeding; and
in a founder individual with upwards to six alleles found in ●● sex reversal and polyploidy;
a single founder. Gene editing can also lead to off‐target all working more effectively in combination than alone
effects, unintended mutation of genes that are not the tar- to improve traits such as growth, sterility and flesh
get, leading to cytotoxicity, the death of mutated cells or quality.
other negative effects on performance of the mutated indi-
viduals. Fewer off‐target effects and lower cytotoxicity 7.13 Genotype‐environment
occur with TALENs compared to ZFNs. Interactions
Clustered regularly‐interspaced short palindromic The best genotype for one set of environmental circum-
repeats CRISPR)/CRISPR‐associated protein (Cas9) sys- stances is not necessarily the best genotype for a
tem in eubacteria and archaea target foreign DNA. second set of environmental circumstances. Genotype‐
Endonuclease Cas9 with CRISPR RNA (crRNA) and environment interactions occur either when the value
transacting RNA (tracrRNA) can cleave of foreign DNA, of the genotypes change in rank or the relative value of
and a single guide RNA (sgRNA) chimera mimicking the two genotypes substantially change in relation to each
crRNA:tracrRNA complex in conjunction with Cas9 can other when the environment changes (Dunham, 2011)
produce site specific DNA double‐stranded breaks in
fish. A modified Cas9 system optimised with zebrafish
codons appears to be another quantum improvement
compared to TALENs both in increased simplicity as
well as the apparent production of homozygous/biallelic
founder individuals that possess the mutation in almost
every cell. CRISPR/Cas9 is much more efficient, effective
and less expensive compared to ZFNs and TALENs and
Figure 7.15 (a) Genotype–environment (a) Genetics 153
interaction occurs when the rank of (b)
genotypes changes when the PerformanceGenotype A
environment changes. (b) Genotype– of genotype Genotype B
environment interaction: rank of PerformanceGenotype B
genotypes does not change, but the of genotypeEnvironment Genotype A
magnitude of the performance difference
changes when the environment changes. Environment
Source: Reproduced with permission from
Rex Dunham.
(Figure 7.15 a,b). Genetically improved animals that differences cause genotype‐environment interactions
work well in a research environment may not necessarily among strains and crossbreeds of oysters. Similarly,
be the best performers under commercial conditions. In temperature and salinity differences can result in geno-
general, genotype‐environment interactions increase for type‐environment interactions in genetic evaluations
aquacultured animals with increasing genetic distance of finfish.
and increasing environmental differences, especially
associated with species such as carps or tilapias that can 7.14 Future Developments
be cultured simply and low on the food chain or with
complete artificial feeds. Genetic improvement of aquaculture species is an ongo-
ing process. As current demands increase and wild
The best genotype for catfish aquaculture in the USA is stocks are overexploited, more management tools will be
the channel catfish female × blue catfish male interspe- required to increase aquaculture production. Genetic
cific hybrid. When grown in aquaria or small cages, this enhancement is an increasingly important component of
hybrid performs and grows poorly compared to channel the management and, if used properly, has strong poten-
catfish. However, in ponds this hybrid has much greater tial to enhance aquaculture production, efficiency and
production than channel catfish. If researchers had not sustainability. Although considered old‐fashioned the
done genetic evaluations in multiple environments, the benefits of traditional selective breeding are far from
value of this fish would never have been discovered. being completely tapped.
Heritability for body weight was higher for the shrimp, Transgenic salmon were recently approved for public
Litopenaeus vannamei, when grown at high densities consumption. This may allow dramatic increases in
compared to low densities. Thus, choice of the environ- genetic enhancement of aquacultured organisms if there
ment and the subsequent genotype‐environment inter- is public acceptance of transgenic fish meat in the mar-
action could have impact on the success of selection ketplace. Genomic information that has been generated
programs. has not yet been utilised for genetic enhancement to any
appreciable extent. However, there have been some
Genotype‐environment interactions occur in F2 trans- examples of successful marker‐assisted selection.
genic zebrafish harbouring Japanese flounder keratin Genomic selection is on the cusp of becoming a reality
promoter‐hen egg‐white (HEW) lyoszyme transgene and is making dramatic impacts in livestock genetic
when challenged with either Flavobacterium columnare improvement. The cost of this technology coupled with
or Edwardsiella tarda (likely pisicida), under varying the current lower economic power of the aquaculture
challenge conditions. Genotype‐environment (nutri- sector compared to the livestock sector, and the fact that
tion) interactions were also observed when transgenic such a large number of species are used in aquaculture
European seabass (Dicentrarchus labrax) were fed on are likely to slow the adoption of genomic selection for
fishmeal vs. plant‐based diets. This also affects the aquatic organisms. A greater number of aquaculture
results of desaturase transgenesis to increase n‐3 fatty students and young scientists need training in both
acids. If the diet does not contain the proper precursors, quantitative and molecular genetics to take advantage of
the effect of the transgene will not be observed. these emerging genetic enhancement programs. The
recent emergence of highly effective gene editing tools
In the case of shellfish, no genotype–environment is ready to revolutionise genetic improvement in a
interactions were observed for total body weight, foot
colour and epipodium pattern in families of greenlip
abalone (Haliotis laevigata) grown in varying flow rates.
However, geographic location and associated temperature
154 Aquaculture in sterility, fast early growth and better flesh quality in
triploid shellfish compared to diploids.
non‐transgenic manner. However, regulatory agencies ●● Combining two or more genetic enhancement pro-
are contemplating the extent of their involvement in grams yields the greatest potential of genetic improve-
oversight of this technology and its products. In concept ment, especially for the overall phenotype.
it is no different from generating mutants by irradiation ●● Genotype‐environment interactions are relatively
and chemical mutagenesis, which was commercialised common in aquaculture.It is critical to measure geno-
with plants, which is not regulated, but not in animals. type‐environment interactions when the goal of
Genetic enhancement in aquatic organisms continues research is to genetically improve aquatic organisms
to develop rapidly, and the food production, efficiency for on‐farm application.
and potential positive environmental impacts using ●● Genetic engineering has been shown to dramatically
genetic enhancement appears increasingly promising change performance of aquaculture species and the
for the future. first government approval, i.e., for growth hormone
gene transgenic salmon, for consumption has opened
7.15 Summary the door for the first commercialisation of transgenic
aquatic organisms.
●● There is broad application of genetic enhancement in ●● Xenogenesis, a method of reproduction in which suc-
aquaculture, including selection, multiple‐trait selec- cessive generations differ from each other, is a stem
tion, marker‐assisted selection, interspecific hybridi- cell technology allowing a host species to produce
sation, polyploidy, genetic monosexing and recently, gametes from a donor species. This allows several new
genetic engineering; such that large impacts have been genetic methods for both aquaculture enhancement
made in genetic enhancement particularly for well‐ and germplasm conservation.
established aquatic species. ●● Gene editing is a new technology that has been suc-
cessfully implemented to produce phenotypic change
●● Traditional selection can double growth rate after sev- in fish, allowing the knockout of genes, insertion of
eral generations of selection, and often can improve genes and the creation of alleles. It has great potential
disease resistance and reproductive traits. for future genetic manipulation.
●● Polyploidy has made greater impact on shellfish
genetic improvement than on finfish: triploidy results
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(2001). Physiological energetics of diploid and triploid
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available at http://www.dfo‐mpo.gc.ca/csas‐sccs/ Y. and Sano, T. (1993). Resistance of a rainbow trout
Publications/ScR‐RS/2013/2013_023‐eng.pdf (viewed strain to infectious pancreatic necrosis. Aquaculture,
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selective breeding for aquatic species: a review. 2nd edn. Van Norstrand Reinhold, New York.
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157
8
Nutrition and Feeds
Lou D’Abramo
CHAPTER MENU 8.6 Formulation, Manufacture and Digestibility of Feeds, 171
8.1 Introduction, 157 8 .7 Nutrition Management Strategies, 178
8.2 Energy Consumption and Partitioning (Bioenergetics), 158 8.8 Feed Management, 180
8.3 The Relationship between Growth and Food 8.9 Emerging Research Areas, 181
8 .10 Summary, 181
Consumption, 159
8.4 Requirements and Metabolic Functions of Nutrients, 160 References, 182
8.5 Digestion and Assimilation of Food, 170
8.1 Introduction worthy contributor to meeting the consumptive global
demand for animal protein. Within the context of the
The predicted demand for animal protein in 2050 derived future value and need for both intensive and resourceful
from an estimated global population of 9.1 billion repre- animal agricultural production systems (NRC, 2015),
sents 470 million tonnes (t) of animal meat production, aquaculture is poised to offer the most efficient source of
an increase of 200 million t from that in 2009 (FAO, sustainable management practices, as defined by envi-
2009). A large proportion of the animal protein demand ronmental, economic and sociological components.
will emanate from developing countries, stimulated by
relocation to cities and a corresponding increase in per A principal component of meeting the environmental
capita income. Meeting this surge in demand in meat and economic sustainability criteria for freshwater, estu-
consumption will probably fall to increases in cultured arine, and marine aquaculture is knowledge of nutrient
freshwater and marine seafood production (aquaculture) requirements combined with effective production and
as capture fishery landings have essentially remained management of feeds and feeding practices. In the
unchanged for more than 20 years (Figure 1.5). During majority of aquaculture enterprises, the cost of feed is
the period 1994‐2004, the annual increase of aquaculture the primary operational (variable) cost. Hence, knowl-
animal production exceeded that of terrestrial animal edge of the nutrition of the farmed organisms, including
food production by 5% (Duarte et al., 2009). Over the nutrient requirements, digestibility of feed ingredients,
past fifty years, growth in the supply of fish has exceeded and when and how much to feed is critical to success. As
world population growth and continues to gain impor- production systems continue to invoke sustainable man-
tance as a source of protein and other essential nutrients agement practices, knowledge and understanding of the
(FAO, 2014). Aquaculture continues to make an ever‐ nutritional requirements and nutritional physiology of
increasing proportional contribution to meeting animal farmed aquatic species are critical to producing environ-
protein demand, having achieved over 50% of the total mentally and economically sustainable feeds, which are
(by volume) of aquatic food products. Due to limited either nutritionally complete or supplemental, depend-
land and freshwater resources, the greater increases in ing on the culture system. Recognising this need, the
production may be realised through the marine environ- National Research Council (NRC) published specific
ment (only a small percentage of possible areas is being research recommendations and complementary research
used) whereby aquaculture will continue to be a note- priorities in nutrition to meet required levels of animal
protein production and global food security brought on
Aquaculture: Farming Aquatic Animals and Plants, Third Edition. Edited by John S. Lucas, Paul C. Southgate and Craig S. Tucker.
© 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.
158 Aquaculture for the performance of physiological function and for
growth. The source of energy is the ingested food (IE)
by increases in global population (NRC, 2015). This and the units of energy are expressed as joules (J) or tra-
source of guidance is based on the presumption that ditionally as calories. Energy that is potentially available
freshwater, marine and estuarine production systems to an organism via the consumption of food is found in
will need to become more intensive, and, in most cases, three macronutrients: protein, carbohydrates, and lipids
requiring nutritionally complete feeds. which contain 23 kJ, 17 kJ and 38 kJ, respectively, /g
ingested. These are termed physiological fuel values. After
The level of knowledge of the nutrition of aquatic ani- the food (energy) is consumed, some is lost in the form of
mals is considerably lower than that of terrestrial species faecal energy (FE), because organisms lack the ability to
such as beef cattle, swine, or poultry. However, during the digest all of the macronutrients that are consumed. Some
past three decades, knowledge of the nutrition of fish and of the remaining energy, called metabolisable energy
crustacean species has notably increased as evidenced by (ME), is used to perform three energy‐requiring physio-
the number of research publications and scientific jour- logical activities. These activities include:
nals that specifically address or highlight aquatic animal ●● basal metabolism;
production. A large portion of the knowledge of aquatic ●● heat increment (HE); and
animal nutrition is primarily founded in species of fish, ●● voluntary activity (VE).
but information about crustaceans, molluscs, and echi- Basal metabolism, calculated as basal metabolic rate
noderms is gradually increasing. Over the past 50 years, (BMR), is the energy used for the maintenance of basic
two notable attempts toward synthesis of the existing physiological activities. Heat increment (HE) is energy
knowledge of nutrient requirements of fish and shrimp consumed in excess of BMR to fulfil needs for the diges-
have been published (NRC, 1993, 2011). Two other pub- tion and molecular transformation of the macronutri-
lications have provided a compendium of knowledge ents consumed. Specific dynamic action (SDA) is another
about nutrient requirements, feed formulation, and feed- term for HE that is used in diagrams of the partitioning
ing of fish (Webster and Lim, 2002) and crustaceans of energy. Some of the energy consumed can ultimately
(D’Abramo et al., 1997). be lost in the form of excretions from gills (ZE) the body
surface (SE), such as the energy used to produce a pro-
8.2 Energy Consumption tective coat of mucus, or urine (UE) that is produced.
and Partitioning (Bioenergetics) Any of the consumptive energy that remains can be used
for voluntary activity such as swimming, water column
The allocation of energy moving through an organism migrations and predation.
(Figure 8.1) is often described through a bioenergetics
diagram that shows the flow of energy as losses with Whatever amount of energy that remains after loss to
some ultimately remaining for the allocation of growth. voluntary activity is commonly termed recovered energy
In an aquaculture enterprise, achieving the lowest cost (RE) that is applied to the production of added tis-
per unit of protein produced is accomplished through sue (growth) or the development of sexual organs and
the allocation of the greatest amount of consumptive
energy to growth. Energy is not a nutrient but is essential
Food Intake Digestible Energy Metabolisable Energy Recovered Energy (RE)
(IE) (DE) (ME) -Tissue
Gross Energy Faecal Energy Gill Excretions -Sexual Products
(FE) (ZE)
Heat Production (HE)
Urine (UE) -Basal Metabolism
Body Surface Excretions (SE)
-Speci c Dynamic Action
-Voluntary Activity
Figure 8.1 Bioenergetic diagram of the paths of the loss and utilization of energy derived from the consumption of dietary
macronutrients.
associated gametes. If the goal is growth, then, the RE Nutrition and Feeds 159
would ideally be converted into tissue protein. Under this
scenario, energy needs, other than those of RE would ide- achieve ‘optimum’ growth under the existing culture con-
ally be served through the metabolism of carbohydrates ditions. The higher energy requirements for BMR and for
and lipids to serve as the sources of energy for BMR, the increase in feeding activity induced by an increase in
SDA, and voluntary activity to maximise therefore, the environmental temperature will generally be offset by
channelling of protein energy to growth. This concept, increases in IE. Oxygen is necessary for the oxidation and
based on the efficient use of protein consumed for growth energy released from the macronutrients consumed
rather than as an energy source, is often termed ‘protein (Figure 8.2). Thus, the aquatic organism’s ability to trans-
sparing’. As an analogy, the ecological literature uses the form dietary macronutrients to energy is influenced by the
term ‘ecological efficiency’ to define RE/IE, the amount availability of oxygen as influenced by the concentration in
of energy that is transferred from one trophic level to the water and the uptake ability of the aquatic organism.
next higher trophic level divided by the amount of energy
consumed × 100. Ecological efficiencies are generally 8.3 The Relationship between
around 10% but can range from 6% to 37%. Growth and Food Consumption
The total energy consumed by an aquatic organism per Three principal factors, temperature, organism size and
unit of time can vary based on its size, the energy content ration influence the growth of fish. The ration, i.e., the
of the food and temperature of the water. The amount of amount of food consumed, is generally expressed as per-
this energy ultimately available for growth is, in turn, cent of body weight per unit of time and is the sole ‘driv-
influenced by the partitioning of the energy consumed to ing force’. Therefore, growth is limited by any abiotic or
meet the physiological needs of the organism as defined by biotic factor, or an array of these factors that limit the
many variables in the environment in which it lives. For rate of consumption of food. Temperature is termed a
example, at higher temperatures, BMR and SDA demands rate‐controlling factor as the amount of food consumed
increase but rates of food (energy) consumption will cor- generally increases with a concomitant increase in tem-
respondingly increase and sufficiently offset those needs. perature. Weight, commonly related to size and/or age, is
Hence, there is a temperature window for organisms termed a ‘scaling’ factor because size, in association with
wherein growth increases with temperature until meta- temperature, influences the gross amount of food
bolic needs do not leave any energy available for growth. (energy) consumed. Growth/ration or G/R curves are
Hence, optimal culture conditions for aquatic organisms often used to describe the relationship between growth
are characterised by the most efficient use of energy and ration as influenced by both abiotic and biotic fac-
c oupled with the satisfaction of nutrient requirements to tors. Under an array of described conditions for culture
Figure 8.2 Paths to the production of Protein Carbohydrate Lipid
energy derived from the digestion of Amino acids
protein, lipid and carbohydrate via the Deamination Digestion Digestion
production of pyruvate from different Fatty acids and
processes. Glucose glycerol
Glycolysis Oxidation
ADP ATP
Pyruvate
CO2
Acetyl CoA
Citric acid O2
cycle NADH
Oxidative ADP
phosphorylation
CO2 H2O ATP
160 Aquaculture They will, however, reduce the cost per unit of product
(protein) produced, an example of economic sustainabil-
and size/age of the organism, certain rations will result in ity. Feed conversion efficiency (FCR) is a measure of the
different growth responses. Some rations will result in a amount of feed fed divided by the amount of weight gain
negative growth rate (R0), no growth (Rmaint) when all during a specified time of feeding, and is lowest when the
energy derived from the ration is exclusively used to feed efficiency, the inverse of FCR × 100, is highest. To
meet metabolic needs, optimal growth (Ropt), where the measure the response to the efficacy of the feed and asso-
ratio, i.e., slope, of growth to ration is greatest, and maxi- ciated ration, both researchers in aquatic animal nutrition
mum growth (Rmax) (Figure 8.3). Subtraction of Rmaint and farm managers often use FCR.
from Rmax yields what is termed the ‘scope for growth’
under the conditions provided for growth. The scope for 8.4 Requirements and Metabolic
growth is that amount of ration (energy) that can poten- Functions of Nutrients
tially be manifested in growth. The scope for growth var-
ies according to level of ration and other factors such as Knowledge of nutrient requirements for species of
temperature, age, aquaculture feeds, i.e., the quality of organisms that have become the focus of aquaculture
the feed being fed, salinity, light (photoperiod or inten- enterprise is essential to achieving economic success.
sity), oxygen concentration and uptake rates. The pro- Most of the nutrient requirement knowledge is confined
portional division of the ration over more than one to only a small subset of fish and crustacean species.
feeding per day and biomass density of the farmed organ- However, hundreds of species are farmed or have been
isms may also change the scope for growth. This array of the subject of evaluation as emerging species. For just
physical, biological and behavioural factors influences marine species, 449 species have been the subject of
both the amount of food consumed and the amount of some type of domestication strategy.
energy required to address metabolic needs which deter-
mine the scope of growth that can be realised. Research on nutrient requirements is commonly based
on the use of purified diets, those that contain chemically
Thus, a major consideration for the feeding and nutri- defined ingredients. Response to graded levels of a nutri-
tion of farmed aquatic organisms is not only the ration ent under investigation, included by supplementing a con-
but also the composition (nutrient quality) of the feed. For trol experimental diet, is commonly evaluated by growth,
example, insufficient protein levels, poor digestibility of other physiological responses and possibly clinical mani-
the feed, etc., and a nutrient requirement being met mar- festations as typically manifested in a dose‐response curve
ginally, can alter how much energy is ultimately available (Figure 8.4). This curve essentially represents an increasing
for growth. Most commercial farming is devoted to max- proportion of the population responding to a dose (dietary
imising the ‘scope for growth’, i.e., providing the ration level) as the dose increases. Therefore, each point gener-
that will maximise growth so that product turnaround is ated on the curve is an observation for that proportion of
minimised. Efforts to feed a ration that will achieve opti- the population that is responding to the dose. A number of
mal growth under existing conditions of culture, the level
at which the feed will be used most efficiently, will not
result in higher turnover to meet the desired market size.
Rmax; Gmax
Growth rate
(% weight/day)
Response
Ropt; Gopt
0
R0; G0
Ration (% weight/day) Dietary level
Figure 8.3 Typical growth‐ration curve illustrating no growth Figure 8.4 Typical dose‐response curve generated by measuring
(G0), optimal growth (Gopt) and maximum growth (Gmax) and responses (weight gain, survival, enzyme activity, etc.) to graded
corresponding ration. Any ration that is below the zero‐growth levels of a dietary nutrient.
line will result in a weight loss.
Nutrition and Feeds 161
methods estimating nutrient requirements, such as the Table 8.1 Essential and non‐essential amino acids.
broken‐stick regression and quadratic regression, have
been proposed. The at times wide variability in reported Essential Amino Acids Non‐essential Amino Acids
requirements of nutrients is probably the result of short- (EAAs) (NEAAs)
comings in experimental design or the choice of the model
to estimate the requirement. The varied results arising Arginine (Arg) Alanine (Ala)
from differences in approach were addressed by Shearer Histidine (His) Asparagine (Asn)
(2000) who provided a ‘set of protocols’ to achieve greater Isoleucine (Ile) Aspartic Acid (Asp)
accuracy and therefore comparative value. Leucine (Leu) Cysteine (Cys)
Lysine (Lys) Glutamic Acid (Glu)
In contrast to fish, benthic organisms such as crusta- Methionine (Met) Glutamine (Gln)
ceans, molluscs and echinoderms commonly do not con- Phenylalanine (Phe) Glycine (Gly)
sume experimental diets rapidly. Rather, these species Threonine (Thr) Proline (Pro)
tend to manipulate or break it up, given their character- Tryptophan (Trp) Serine (Ser)
istic feeding apparatuses. Thus, a difficulty is often Valine (Val) Tyrosine (Tyr)
encountered in arriving at a true estimate of the require-
ments of water‐soluble nutrients due to, in some cases, can be compensated through its synthesis from methio-
rapid loss caused by differential leaching (dissolution) nine, an essential amino acid. Under these conditions,
into the water. As a result, requirements of water‐soluble the methionine dietary requirement would be higher. A
nutrients such as some vitamins are often overestimates similar relationship exists between the essential amino
for these species. In contrast to terrestrial animals, nutri- acid phenylalanine and the non‐essential amino acid,
ent requirements of aquaculture species are expressed as tyrosine.
a percent dry weight of the diet, rather than as mg/g/day.
Nutrient requirements can be expressed as g/kg of In the literature, taurine is often termed an amino acid,
diet (macronutrients, such as protein, carbohydrate and but is actually an organic acid, lacking the carboxyl group
lipid) or mg/kg diet (micronutrients such as vitamins that is characteristic of all amino acids. Based on the
and minerals). Knowledge of species‐specific nutrient results of several published research investigations, there
requirements is ultimately applied to the formulation of is some evidence to suggest that taurine may be required
practical diets consisting of feedstuffs that are readily by larval and early juvenile stages of fish and shrimp
available, highly digestible and collectively satisfy the (NRC, 2011). For example, a dietary requirement of
requirements. 1.09% taurine was found for juvenile yellow catfish,
Pelteobagrus fulvidraco, fed a diet that contained only
8.4.1 Protein plant‐derived protein. Additional research is needed to
determine whether differences in dietary taurine needs
Although the literature contains descriptions of protein may be related to rate of synthesis from another amino
requirements of aquaculture organisms as a percentage acid, cysteine and/or to diets that contain protein that is
of the diet, there is no true protein requirement. Rather, derived exclusively from plant sources. These conditions
the protein requirement is actually based on the cumula- may be further complicated by differential requirements
tive satisfaction of requirements for essential amino as influenced by specific physiological needs.
acids. There are 20 different amino acids (Table 8.1).
Protein requirements, expressed as a percent of the
●● Ten amino acids are termed essential or indispensable diet, commonly range from 12–25% for mammals and
because animals cannot either synthesise them or syn- birds in contrast to fish and crustaceans which report-
thesise them at a sufficient rate to meet metabolic edly require higher levels (35–55%). These data suggest
demand, thereby invoking a dependence on dietary that the efficiency of protein use, commonly termed
origins to meet the respective individual requirements Protein Efficiency Ratio (PER), which is expressed as the
of these amino acids. body weight increase per amount of protein consumed x
100, is seemingly much lower in mammals and birds
●● Another 10 dietary amino acids are termed non‐essen- than for aquatic organisms. However, these percent
tial or dispensable because organisms have the ability requirements that are used to formulate practical diets
to synthesise them, often from essential amino acids. represent an absolute requirement. If a calculated rela-
tive requirement, expressed as protein intake (at maxi-
The requirement of an essential amino acid can be influ- mum growth) per gram of body weight consumed per
enced by interactions with other essential amino acids, day, is calculated for an array of terrestrial and aquatic
with non‐essential amino acids and even with non‐
protein nutrients. For example, the possible dietary defi-
ciency of a non‐essential amino acid such as cysteine
162 Aquaculture in protein deposition. In addition, lysine is generally rec-
ognised as the first or at least second limiting essential
animal species, the median of values for each group of amino acid in practical feed formulations. Hence, formu-
species (fish versus terrestrial vertebrate species) is lating a feed based on satisfaction of the lysine require-
very similar. The explanation for this similarity is that ment helps to ensure that required dietary levels of other
fish and other aquatic organisms are not consuming as essential amino acids will be provided. Unlike lysine,
much diet to satisfy the protein (array of essential amino some essential amino acids are not used exclusively for
acids) requirement as terrestrial vertebrates. The reduc- protein deposition and have other metabolic roles. In
tion in overall diet consumption is due to a lower need these cases, the values obtained as a percent of lysine
for energy which primarily originates from the dietary would be underestimates. A good example is the arginine
lipid and carbohydrate. Less dietary energy is needed to requirement of crustaceans because arginine is part of
carry out a variety of physiological and behavioural the argino‐phosphate molecule associated with the con-
activities. Aquatic aquaculture organisms are poikilo- traction of crustacean musculature. When feeds are for-
therms and energy is not needed to maintain body tem- mulated to meet requirements of essential amino acids,
perature. The principal excretory product from the digestibility of feedstuffs that contain essential amino
breakdown of protein is ammonia which requires less acids is less than 100%. Therefore, data about digestibil-
energy to produce urea and uric acid (excretory products ity will guide decisions as to how additional amounts to
of terrestrial animals). The need for energy to perform meet required levels will be provided in the diet. Despite
locomotor activity is much less for aquatic organisms all these considerations, the idea of an ideal protein con-
because of the natural buoyancy afforded by the aquatic cept based on the relationship of lysine to other essential
environment. Hence, due to lower needs of dietary amino acids in the tissue has practical merit toward the
energy, less food needs to be consumed per body weight knowledge and selection of ingredients for the formula-
increase in aquaculture organisms, as illustrated by the tion of practical feeds.
feed conversion ratio (FCR) of fish being comparatively
less than that for beef cattle, swine and poultry. Thus, 8.4.2 Lipids
aquaculture organisms are much more efficient in the Lipids are water insoluble organic molecules that con-
conversion of diet consumed to protein deposition in the tain the highest amount of energy per gram among
muscle where most of the growth is expressed. This effi- macronutrients. In addition to them being a source of
ciency of conversion is a defining aspect of highly sus- energy, dietary lipids are used:
tainable farming, providing argument to focus on fish, ●● as structural components of cellular membranes
crustacean, echinoderm and mollusc farming to meet ●● for transport as part of lipoprotein molecules
future animal protein needs globally. Within a group of ●● to regulate metabolic processes as vitamin, hormone
fish species, the existence of higher absolute protein
requirements (% of total diet) and higher protein intake and prostaglandin molecules
(relative requirement) is generally a characteristic of car- ●● for reproduction.
nivores due to the need for protein to serve as a partial Body lipids can also confer greater buoyancy and help to
energy source. The carnivore’s comparatively low ability exclude pathogens by being part of a protective coating
to digest dietary carbohydrate limits the efficient use of on the outer surface of many aquatic organisms.
this macronutrient as a source of dietary energy.
There are two major classes of lipids, saponifiable
For those amino acid requirement values that have (containing fatty acids) and non‐saponifiable (not con-
been determined for certain species, it is clear that a taining fatty acids). Fatty acids are long chain carbon
good correlation exists between essential amino acid lev- molecules that are composed of single or double bond
els in the diet and those in the tissue where most of the carbon chains that have a carboxyl (COOH) end and a
dietary protein is deposited. This correlation has led to methyl (CH3) end. Due to the nature of the biosynthetic
the Ideal Protein Concept, provision of the ideal dietary process, double bonds which confer the unsaturated
protein that would provide the correct proportional rela- nature to fatty acids are located between every other car-
tionship (balance) of essential amino acids whereby pro- bon atom. A certain nomenclature is used in the identifi-
tein in the diet would be used most efficiently. The high cation of fatty acids. For example, a 16:1n‐9 fatty acid is
correlation of the proportional relationships among die- composed of 16 carbon atoms and one unsaturated bond
tary and tissue essential amino acids can be used to help that is located at the 9th carbon (n‐9) counting from the
estimate the dietary requirements of other amino acids. methyl end. Those fatty acids that have been determined
If the requirement for lysine is determined and it is as essential (EFAs) are composed of at least 18 carbon
assigned a value of 100%, then the requirements for the atoms and two unsaturated bonds. The most common
other essential amino acids can be expressed as a per-
centage of lysine. Lysine is selected because it apparently
has no other metabolic role than that of being included
essential fatty acids (common name in parenthesis) are Nutrition and Feeds 163
either polyunsaturated fatty acids (PUFAs) such as
18:2n‐6 (linoleic acid) and 18:3n‐3 (linolenic acid), or form of triglycerides that serve as a concentrated source
long chain polyunsaturated fatty acids (LC‐PUFAs), also of energy. Qualitative and quantitative fatty acid require-
termed highly unsaturated fatty acids (HUFAs) such as ments derived from consumption of triglycerides or other
20:4n‐6 (arachidonic acid, ARA), 20:5n‐3 (eicosapentae- lipid compounds vary based on trophic level/feeding hab-
noic acid, EPA) and 22:6n‐3 (docosahexaenoic acid, its, environmental temperature, and whether the aquatic
DHA). Sources of the essential PUFAs and LC‐PUFA are organism lives in a freshwater, estuarine, or high salinity
confined to diet. Some aquaculture organisms can syn- environment. An overview of fatty acid classes, nomen-
thesise LC‐PUFAs from PUFAs, a metabolic ability that clature, metabolic use and biosynthetic pathways is
is confined to fatty acids that are common to either the shown in Figure 8.5. Based on published data of several
n‐6 or n‐3 fatty acid family. LC‐PUFAs are very impor- species, some guidelines can emerge. For channel catfish
tant as components of lipid molecules that compose part (Ictalurus punctatus), a freshwater and warm water fish
of the structure of cellular membranes, or are used in that is omnivorous, the requirement is 1% of PUFAs with
lipid transport, spawning, hatching, fertilisation and either 18:3n‐3 (linolenic acid) or 18:2n‐6 being equally
other physiological processes. effective. However, at levels less than 1%, dietary LC‐
PUFAs will produce the same response, indicating that
Triglyceride compounds, termed triacylglycerols (neu- LC‐PUFAs have a greater EFA activity than that of PUFAs.
tral lipids), are composed of a glycerol molecule with fatty Rainbow trout have a 1 % dietary PUFA requirement, but
acids attached to each of its three hydroxyl parts through in contrast to channel catfish, 18:6n‐2 provided at the
an ester bond. Phospholipids (polar lipids) are of a similar same level does not exhibit an equal activity response,
structure but phosphate replaces one fatty acid, being most probably attributable to its being a carnivorous,
similarly bound by an ester bond to one of the three coldwater species. Generally, the dietary EFA require-
hydroxyl parts of the molecule. Another organic molecule ment for marine species of fish and crustaceans is 0.5% to
in turn is attached by an ester bond to the phosphate part 2.0% of the diet contributed by 20:5n‐3 and 22:6n‐3, indi-
which thereby confers a certain identity to the phospho- vidually or in combination, with 20:4n‐6 sometimes man-
lipid molecule. For example, if the attached molecule is ifesting equal activity. Juveniles require the higher 1%
choline, ethanolamine or inositol, the respective phos- dietary level. In contrast, juveniles of the omnivorous
pholipid is called phosphatidylcholine, phosphatidyletha- freshwater prawn, Macrobrachium rosenbergii, require
nolamine and phosphatidylinositol. Phospholipids are only 0.05% of dietary LC‐PUFAs to achieve significantly
important molecules composing the membrane of a cell higher weight gain, and there is no difference in EFA
and as part of lipoprotein molecules that are used in the activity relative to growth response among the 20:4n‐6,
circulatory transport of lipids. 20:5n‐3, and 22:6n‐3 fatty acids. This requirement of LC‐
PUFAs from either the n‐3 or n‐6 family probably remains
Non‐saponifiable lipids are derived from isoprene and because of its omnivorous natural diet and phylogenetic
are termed isoprenoid lipids. These lipids include choles- ancestors that lived exclusively in saltwater.
terol, sex hormones such as testosterone and oestrogen,
some vitamins (D, E, A and K) and pigments. Steroids The ability of marine sea urchins and abalone (gastro-
such as cholesterol and sex hormones are cyclic com- pod) to elongate and desaturate PUFA to LC‐PUFA
pounds and pigments are isoprenoids that are responsible starkly contrasts with the absence of this biosynthetic
for the colour in aquatic organisms. ability in marine fish and crustaceans. Juvenile sea
urchins, Strongylocentrotus droebachiensis, that are char-
There is no true lipid requirement for an aquatic spe- acteristically herbivorous have the ability to elongate and
cies as the dietary requirement can vary, in accordance desaturate n‐3 and n‐6 PUFA to n‐3 and n‐6 LC‐PUFA
(20:5n‐3 and 20:4n‐6), respectively. The abalone Haliotis
with the meeting of a composite of several objectives, i.e., fulgens has the same intrinsic biosynthetic ability, affirmed
provision of sufficient lipid that will minimise the use of by the lack of synthesis by gut microflora. Fatty acid com-
protein as an energy source while still meeting the dietary position differed among different tissues as well as within
the different classes of lipid that composed the tissues.
requirements of essential fatty acids (EFAs), polar lipids Growth of both Haliotis tuberculata and H. discus hannai
and some isoprenoids. Generally, in aquaculture the need is positively influenced by dietary LC‐PUFA, specifically
to provide EFAs and other dietary lipids originates from 20:4n‐6, 20:5n‐3, and 22:6n‐3. Levels (mg of fatty acid/g
an evolutionary loss of the inability to synthesise them at dry weight) of LC‐PUFA (20:5n‐3, 22:6n‐3 and 20:4n‐6)
all or at a rate insufficient to meet requirements. The nat- within the polar lipids in the tissue of H. fulgens were
selectively retained during starvation, suggesting the
ural availability and comparatively high dietary levels of importance of these fatty acids as cellular components and
these compounds, by transfer up through the food chain, essential nutrients. In contrast, LC‐PUFA in the n eutral
either directly or indirectly, is responsible for the loss of
this biosynthetic ability. The greatest amount of dietary
lipid naturally consumed by aquatic organisms is in the
164 Aquaculture Essential Fatty Acids (EFAs)
(Required Dietary Source)
10:0
12:0 14:1n-5 18:2n-6
14:0 16:1n-7 18:3n-3
Short chain fatty acids 18:1n-9 18:3n-6
(SCFAs)
16:0 Monounsaturated Fatty Acids Polyunsaturated Fatty Acids (PUFAs)
18:0 Growth, survival
Saturated Fatty Acids (SFAs) (MUFAs)
Probiotics (SCFAs)
Sources of Energy
BIOSYNTHESIS NO BIOSYNTHESIS
Sea urchins Marine fish
Abalone Crustaceans
(Omnivores/Carnivores)
Herbivorous fish
20:4n-6
20:5n-3
22:5n-3
22:6n-3
Long Chain Polyunsaturated Fatty Acids
(LC-PUFAs)
Sources of energy
Cellular membrane structure
Growth, survival
Reproduction
Figure 8.5 Classes, biosynthetic pathways, sources and physiological roles of fatty acids.
lipids of tissue decreased, probably due to a lack of dietary comparatively inexpensive sources of energy in the diets
PUFAs as precursors for synthesis. formulated for fish, crustaceans, echinoids and gastro-
pods. This energy is derived from the oxidation of the
Takeuchi (1997) has addressed the wide EFA require- monosaccharide glucose, the primary building block of
ment patterns for aquatic species, particularly fish, as starches, that is ultimately derived from a sequence of
associated with life history stage, feeding, salinity and enzymatically effected digestive processes. Glucose
temperature. A detailed compilation of reported species‐ undergoes glycolysis to produce ATP from ADP with
specific fatty acid requirements of fish and shrimp can be the concomitant formation of pyruvate. With the release
found in NRC (2011). Glencross (2009) reviewed the of carbon dioxide, pyruvate is transformed into acetyl
requirements and metabolism of EFAs for fish, crusta- coenzyme A which enters the citric acid cycle to pro-
ceans and other species and addressed prospects for the duce additional ATP.
use of grain, animal and algal derived oils as sources of
EFAs in the formulation of aquafeeds. Unlike many terrestrial homoeothermic species,
fishes, being mostly carnivorous, do not naturally con-
8.4.3 Carbohydrates sume carbohydrates and accordingly a reduced ability to
Carbohydrates are synthesised by plants and consist of enzymatically digest carbohydrate has evolved. This lack
simple and complex molecules. Six simple sugars such of efficient use of dietary carbohydrate reduces its poten-
as glucose and fructose are called monosaccharides, and tial role as a source of energy in formulated diets for fish
when bonded together produce oligosaccharides con- as well as crustaceans that have carnivorous feeding hab-
sisting of three groups, disaccharides, trisaccharides and its. For those aquatic species that are either omnivorous
tetrasaccharides. Much larger ‘complex’ carbohydrate or herbivorous, most carbohydrates are readily digestible
molecules consisting of chains of repeating monosac- and can serve as a comparatively inexpensive source of
charides are termed polysaccharides and are repre- energy in formulated feeds. However, for some species,
sented by such compounds as starch, glycogen, dextrin, dietary cellulose and fibre in aquafeeds do not serve as
mannan, fibre and cellulose. These polysaccharides are effective sources of energy due to the lack of enzymes to
present in plants and plant‐derived feedstuffs, serving as confer efficient digestion and they often cause reduced
growth rates.
Due to these physiological differences imposed by Nutrition and Feeds 165
feeding habits, there can be no true requirement for car-
bohydrate among aquatic species. However, some empir- ratios are surprisingly consistent, generally ranging
ically‐based guidelines for inclusion of carbohydrates in between 20–30 mg/kJ (33 to 50 kJ/g) for crustaceans and
feed formulations have been established. For carnivo- 23.8 to 30.0 mg/kJ (33 to 42 kJ/g) for fish.
rous species, the total amount of dietary carbohydrate
should not exceed approximately 20% of soluble carbo- As previously stated, carnivorous species are generally
hydrate. Feed formulations for herbivorous and omnivo- less capable of digesting carbohydrate so that feed formu-
rous species can typically range from 25 to 45% (dry lation must compensate for this physiological character-
weight), somewhat imposed by the amount of lipid that istic. Carbohydrate containing, plant‐derived ingredients
can be included in the diet. As carbohydrate in the form as sources of energy must be carefully evaluated relative
of fibre is not digestible and therefore unavailable, efforts to amount and digestibility. Lipid levels higher than those
are typically directed at limiting the dietary level to included in diets for herbivores and omnivores are com-
5–7%. The results of some investigations have suggested monly needed. In studies evaluating lipid to carbohydrate
that the presence of fibre may enhance the digestibility of ratios in diets containing three levels of protein fed to the
other nutrients by reducing the rate of gut passage. Some herbivorous pink abalone Haliotis corrugata protein and
carbohydrates are added to formulated feeds to act as carbohydrate appear to be the principal sources of energy.
binders to preserve the stability in water or, if provided in Thus, for this abalone species, the focus would be on pro-
sufficient quantity, to render a feed to float under par- vision of highly digestible carbohydrate to provide energy
ticular conditions (temperature and pressure) of feed to spare use of protein as an energy source. The amount
manufacture termed extrusion (see section 8.6.2). of dietary lipid could therefore be comparatively low, but
at a level sufficient to satisfy the essential fatty acid
8.4.4 Dietary Macronutrient Relationships requirements. The P: E ratios that yielded the highest
(Protein: Energy Ratio) growth were 23.1 and 24 mg/kJ (41.7 to 43.5 kJ/g).
A dietary protein‐energy ratio is expressed as the propor- Age/life history‐specific changes in protein and/or
tional amount of dietary protein relative to the total energy demands would be expected as observed for lar-
amount of macronutrient energy as calculated from the val forms (more protein for growth) and those individu-
energy equivalents of protein, lipid and carbohydrates. als entering reproduction (more lipid for storage) and
Organisms do use some dietary protein as an energy these changes must be accommodated to retain effi-
source, but that amount can be ‘spared’ if sufficient quan- ciency of protein metabolism.
tities of lipid and carbohydrate are provided. Thus, pro-
tein requirements are not only determined by the 8.4.5 Vitamins
composite satisfaction of the essential amino acids, but
also some species‐specific need for protein as an energy Vitamins are organic molecules that are essential for
source, despite other energy sources being provided in growth, most serving as components of coenzymes for
sufficient quantity. Providing highly digestible lipid and the catalysis of metabolic reactions. They cannot be syn-
carbohydrates in sufficient quantities (combination) will thesised and are termed essential micronutrients because
therefore allow for the most efficient use of protein, i.e., they are required in small amounts (mg/kg or µg/kg).
its deposition as tissue (growth). The goal is the formula- Vitamins are divided into two groups based on their sol-
tion of diets whereby energy derived from protein is min- ubility in either water or lipid. Four ‘lipid‐soluble’ vita-
imised and all remaining energy needs are typically mins, E, A, D and K are absorbed with lipid from the
satisfied by dietary lipids and carbohydrates. Thus, for intestine into the circulatory system. A group of 11 vita-
particular feed formulations, there are protein: energy mins, biotin, niacin, folic acid, thiamine, riboflavin, pan-
ratios that will yield the highest growth. If the digestibility tothenic acid, pyridoxine, cyanocobalamin, ascorbic
of the protein and energy sources are known, then these acid, choline and myo‐inositol are water‐soluble vita-
ratios can be expressed as the ratio of digestible protein to mins. These vitamins are transferred from the intestine
digestible energy. There have been numerous studies that into the circulatory system via water. Choline, ascorbic
have evaluated dietary protein to energy ratios for fish acid and myo‐inositol are required at relatively higher
and crustaceans and they are generally expressed without concentrations. They are called megavitamins and are
reference to the digestibility of the protein and energy not components of coenzymes.
sources. Some ratios are inversely expressed as energy
to protein ratios. Upon review, despite the variety of car- Dietary excesses of water‐soluble vitamins are not
bohydrate, protein and lipid sources used, the reported stored, but rapidly excreted from the body. Therefore,
deficiencies will rapidly appear, often causing a variety of
metabolic problems, some of which are manifested as
clinical responses. For example, a dietary deficiency of
ascorbic acid in fish is commonly manifested as ‘broken
back syndrome’ (scoliosis/lordosis) which arises from a
166 Aquaculture selected as the cut‐off year for reporting requirements
because by then studies had assumed a more controlled
deficiency of collagen that serves as the organic matrix of and rigorous methodology that presumably yielded more
bones. Specifically, the absence of sufficient collagen accurate results. When a range was reported, the mid‐
causes a decalcification of the bone which leads to the point was used as an independent value in the calcula-
lack of ‘rigidity’ of the backbone, often manifested as sco- tion of the mean and the level of variation. If there were
liosis. Other water‐soluble vitamin deficiencies that are only two published estimates of a requirement, both val-
clinically exhibited by aquaculture species include poor ues were listed rather than calculating a mean and a
appetite, haemorrhaging, fatty liver, hyperirritability, an standard deviation. The measure of variation (standard
array of aberrant haematological changes and others deviation) is typically high and the range of values are
(NRC, 2011). Although each vitamin supports specific most probably the result of a combination of factors that
structural or metabolic functions, clinical manifestation include species, the experimental diet, feeding behav-
of deficiencies may be the same for different vitamins. In iour, response criteria used to discriminate deficiency
contrast, lipid‐soluble vitamins are stored such that die- and the method used to estimate a requirement.
tary requirements are difficult to determine. However,
when tissue levels reach a threshold caused by excessive Despite the variation, the levels of water‐soluble vita-
dietary levels, a condition termed hypervitaminosis mins reported among an array of freshwater species are
occurs and clinical responses, such as reduced appetite comparable. This observation is not unexpected because
and degeneration of the liver, are manifested. of the commonality of the metabolic roles of these vita-
mins among species. For marine fish, little information is
Vitamins are commonly added as premixes in formula- known about requirements of water‐soluble vitamins
tions to manufacture feeds for aquaculture species. except for ascorbic acid and the reported requirements for
Table 8.2 shows vitamin requirements of species of fish this vitamin are very similar among several species. There
and shrimp reported from 1980 through 2010 and com- is little information about requirements for lipid‐soluble
piled in NRC (2011). Although published reports of vita-
min requirements precede 1980, 1980 was arbitrarily
Table 8.2 Mean ± SD vitamin (water‐soluble and lipid‐soluble) requirements (mg/kg) of species of fish and shrimp published
from 1980 to 2011 (as reported in NRC, 2011). (n) = number of reported values. If estimates were limited to one or two values,
no mean was calculated. If the estimated value was presented as a range, the mid‐point was used in the calculation. B12 and D
values are presented as µg/kg.
Water Soluble
Vitamin Fish Shrimp Shrimp/Fish Ratio
Thiamin 1; 11.2 (2) 68.0 ± 47.0 (3) x
Riboflavin 6.0 ± 2.4 (9) 22.5; 80 x
Pyridoxine 6.6 ± 4.4 (10) 110.1 ± 31.5 16.7
Pantothenic acid 20.8 ± 8.3 (7) 323.3 ± 369.6 (3) 15.5
Niacin 19.1 ± 9.6 (7) 219.1 ± 198.2 (3) 11.5
Biotin 0.77 ± 1.1 (8) 2.2; 0.4 x
B12 (µg) 54.2 ± 39.3 (3) 200; 10 x
Folic acid 1.6 ± 1.1 (7) 1.9; 21 x
Choline 1260.5 ± 1059.6 (8) 6200; 600 x
Myo‐inositol 396.0 ± 153.4 (5) 1351.0 ± 1659.2 (3) 3.4
Ascorbic Acid 101.4 ± 133.1 (75) 159.4 ± 131.8 (20) 1.6
Mean = 9.7
Lipid Soluble 16.3 ± 24.9 (3)
A 87, 99
E 7.8 ± 13.0 (5) 10
D (µg) 72.8 ± 46.3 (18) 35, 185
K 22.7 ± 20.0 (4)
1.9, 0.2, <10
vitamins; nonetheless reported estimates for both fresh- Nutrition and Feeds 167
water and marine fish are similar. The means of reported
water‐soluble vitamin requirements of shrimp species are Cu), that include haemoglobin (fish) and haemocyanin
commonly greater (1.6 to 16.7 X) than those reported for (crustaceans). Minerals are also components of vitamins
fish. This difference most probably does not reside in phy- and hormones, and serve as cofactors that are essential
logeny, but rather loss due to leaching during a protracted for the enzyme catalysis of specific chemical reactions.
period of food consumption that is characteristic of Although minerals are naturally present in feedstuff ingre-
shrimp species. Loss would naturally lead to an overesti- dients that are part of formulations of manufactured feeds
mation of the true requirement with greater departures for aquaculture organisms, mineral mixes need to be
corresponding to those vitamins with higher solubility. included to compensate for anticipated deficiencies or
Vitamin requirements of other potential aquaculture spe- reduced bioavailability of some minerals. Various sources
cies are becoming the subject of investigation. The inclu- of phosphorous such as fishmeal, soybean meal and cal-
sion of a vitamin pre‐mix in a diet for the sea urchin, cium phosphate are differentially available to cultured
Lytechinus variegatus, was found to be a necessary ingre- aquatic organisms. For example, phosphorous derived
dient for weight gain and organ production. from monobasic calcium phosphate, Ca (H2PO4)2, is much
more available to fish than the dibasic form, CaHO4P.
8.4.6 Minerals
There are approximately 20 inorganic elements (minerals) Controlled experiments have achieved good estima-
that are considered to be essential nutrients. They are tions of some mineral requirements. These experiments
divided into two groups, macro‐elements, those required have generally been designed with recognition of
at levels of g/kg of diet, and micro‐elements or trace ele- uniquely external factors that may influence estimates.
ments, those required at levels of mg/kg of diet. Macro‐ Dietary supplementation of macro‐ and micro‐elements
elements and micro‐elements considered essential for is based on a variety of factors. In some cases, rearing
aquatic animals are listed in Table 8.3. These nutrients are condition, i.e., fresh, marine or estuarine water may serve
required for the successful function of a variety of physio- as a significant source to meet a requirement and must
logical processes such a muscle contraction (Mg, P), be considered in the overall estimate. Sources of dietary
production of skeletal structures (Ca, P), osmoregulation protein that have notable mineral components can also
(Na, Cl) and as components of respiratory pigments (Fe, influence requirements. In addition, caution needs to be
exercised so that experimental diets do not include feed-
Table 8.3 Essential macro‐elements and micro‐elements stuffs that contain compounds that render dietary min-
for animal aquaculture species. erals unavailable to the organism. For example, feedstuffs
derived from cereal grains and oil seed meals contain
Macro‐elements Micro‐elements (Trace elements) phytate which binds with (sequesters) calcium, thereby
reducing bioavailability for use in meeting the require-
Cations Cobalt (Co) ment of the organism. Reported requirements of fish and
Calcium (Ca) Copper (Cu) shrimp are generally within the same order of magnitude
Magnesium (Mg) Chromium (Cr) (NRC, 2011).
Potassium (K) Fluorine (Fl)
Sodium (Na) Iodine (I) 8.4.7 Life History and Reproductive Stage
Iron (Fe) Dependent Nutrient Requirements
Anions Manganese (Mn) 8.4.7.1 Larvae
Chlorine (Cl) Molybdenum (Mo) Larval fish and crustaceans are typically fed cultured
Phosphorous (P) Nickel (Ni) live food such as algae, rotifers, copepods, and Artemia
Sulphur (S) Selenium (Se) nauplii (section 9.2). In some cases, enrichment of the
Silicon (Si) nutritional quality of these live organisms, commonly
Tin (Sn) LC‐PUFAs, is frequently accomplished through differ-
Vanadium (V) ent approaches, affording the ability to identify qualita-
Zinc (Zn) tive dietary requirements through observations of
growth and survival responses. Larvae of the northern
rock sole Lepidopsetta polyxystra have been fed rotifers
that have consumed liposomes enriched with taurine.
The use of formulated diets as substitutes for live food
in larval nutrition offers the advantages of control of
nutrient composition, convenience of storage and use,
and a reduction of cost of production associated with the
labour‐intensive effort required to successfully maintain
168 Aquaculture (NRC, 2011). This requirement is not related to the pos-
sible need for additional choline or inositol, two required
mass culture of live organisms. Moreover, the use of for- water‐soluble vitamins that are part of the phospholipid
mulated diets eliminates the potential introduction of molecules. Quantity and quality of dietary fatty acids
pathogenic organisms originating from live foods. affect food intake and absorption efficiency of nutrients
in larvae of gilthead seabream, Sparus auratus and the
Despite noteworthy advances over the past 30 years, Senegalese sole, Solea senegalensis.
challenges remain in achieving an understanding of the
nutrition of larval forms of fish and crustaceans and the Both larval and juvenile crustacean species exhibit a
development of successful feed formulations and feeding unique requirement for cholesterol. Attempts to com-
practices for them (Hamre et al., 2013), As a result, con- pletely or partially substitute dietary cholesterol with
siderable constraints on their production and availability plant‐derived sterols (phytosterols) have not been suc-
for intensive production practices exist. With the advan- cessful. Cholesterol is synthesised from dietary phytos-
tage of nutrient control afforded by formulated diets, a terols, as indicated by the exclusive presence of cholesterol
corresponding opportunity to determine qualitative and in body tissue of those larvae/juveniles fed diets contain-
quantitative nutritional requirements becomes possible. ing phytosterols. Apparently, the rate of synthesis is
Although promising, knowledge of nutrient require- insufficient to satisfy a specific cholesterol requirement
ments of larvae is substantially lacking, mostly qualita- that cannot be ‘spared’. The deficiency response is
tive in nature. Lack of specific quantitative knowledge commonly manifested in reductions in growth rates.
remains plagued by poor consumption, particularly early
larval stages and/or digestion. Only when a water‐stable, 8.4.7.2 Broodstock
formulated diet that is readily consumed, digested and A reliable supply of larvae to produce juveniles (postlar-
assimilated with an equivalency to live feeds will it be vae) for stocking into production systems is integral to
possible to investigate specific nutrient requirements the success of an aquaculture enterprise. Therefore, pro-
with a high level of accuracy and confidence. In particu- viding essential nutrients to broodstock to reach repro-
lar, estimation of requirements of water‐soluble nutri- ductive maturity and for transfer to gametes (egg and
ents is plagued by leaching from diets that lack physical sperm) to produce viable eggs and larvae is very impor-
integrity and have a characteristically high surface area tant. However, broodstock nutrition is the least studied
to volume ratio. area of aquatic animal nutrition and along with larval
nutrition definitely represent an impediment to confer-
A chronological progression toward the successful use ring greater efficiency (sustainability) in aquaculture
of microparticulate diets for the culture of larval forms of enterprises. For example, the nutritional status of females
aquaculture species has occurred over the recent two influences the time to reproductive maturity, the num-
decades. In some cases, the success of complete replace- ber of eggs produced per spawn or per unit of body
ment of live food is limited to certain stages of larval weight (fecundity) and egg size, egg hatchability and sur-
development. Use of commercially available larval feeds vival of larvae through the early developmental stages.
is commonly recommended as supplements rather than
full substitutes for live food (see section 9.5). The evident lack of widespread research focusing on
broodstock nutrition appears to be limited by the length
The successful production of larvae through use of for- of time and corresponding resources, such as holding
mulated diets has rarely translated to their modification units and number of broodstock needed to conduct rig-
and use for the determination of nutrient requirements. orous and meaningful research. This lack of information
However, some progress has revealed that larvae do has imposed a dependency on fresh food as feed, exclu-
exhibit some unique differences in nutrient require- sively or in association with formulated diets as part of
ments. These distinctions are probably due to a greater broodstock husbandry. A detailed review of the role of
demand associated with early growth and development the nutrition of broodstock on reproductive perfor-
combined with a reduced ability to digest and absorb mance is found in Izquierdo et al. (2001) for fish and in
particular macronutrients. It is commonly acknowl- Wouters et al. (2001) for species of penaeid shrimp.
edged that both fish and crustacean (penaeid shrimp) Salient details are presented in the following generalised
larvae have greater requirements for dietary EPA (at summary, but reported species‐specific requirements
least % LC‐PUFA) and also exhibit a requirement are not addressed.
for phospholipids, specifically phosphatidylcholine. As
these species are known to synthesise PC, the existing An increased demand for energy is needed for not only
rate of synthesis probably falls short of satisfying this the production of viable gametes but also expression of
early growth stage requirement. A dietary need of at secondary sexual characteristics and activities associated
least 2 % of dietary phospholipids for good growth and with reproductive behaviour. If more dietary energy is
survival of larval fish and crustaceans has been often needed to meet the demands of the reproductive state,
reported with phosphatidylcholine (PC) and phosphati-
dylinositol (PI) having the greatest nutritional value
then less net energy is available to channel to growth. For Nutrition and Feeds 169
macronutrients, the dietary protein level for broodstock
is recommended to be aligned with those levels fed to percentage of normal larvae) and pyridoxine (synthesis of
grow‐out fish, but higher levels of dietary protein have steroid hormones) have led to recommended inclusion.
been studied and used. Different dietary protein levels The required qualitative and quantitative dietary nutri-
can exert different responses in broodstock. Intermediate ent enhancements needed by broodstock will increase
levels of protein yielded higher relative fecundity (num- feed cost; however, the higher cost should be compen-
ber of eggs per 100 g of female) in a species of tilapia, sated by healthy broodstock yielding higher egg produc-
Oreochromis niloticus. For tilapia, the total eggs and the tion and survival of larvae, particularly for marine fish.
number of eggs per kg female were higher from fish fed Reliable availability of juveniles for stocking into farming
with medium dietary protein (27.6 and 35%) than those systems continues to be a major bottleneck in increasing
fed with higher protein levels (42.6 and 50.1%). The marine fish production and contribution to animal pro-
higher protein diet yields heavier and larger eggs that are tein food security globally.
spawned between longer time intervals. Dietary lipid
levels in formulated diets are commonly increased to During the maturation of the reproductive organs of
meet the increased metabolic demand for energy and a crustaceans, nutrients are primarily transported from
commonly used level is 18%. Certain nutrients have been the hepatopancreas (mid‐gut gland). Therefore, those
repeatedly shown to be required at dietary levels higher nutrients have been identified as important because they
than those for juveniles and subadults and specifically characteristically accumulate in the ovary during repro-
include n‐3 EFAs, alpha‐tocopherol (vitamin E), ascorbic ductive maturation and are notably found in natural food
acid (vitamin C) and vitamin B6 (pyridoxine). organisms that serve as successful broodstock feeds. As
gonadogenesis is induced by the cutting (ablation) of the
The EFA content of gonads corresponds well to the eyestalk (see section 6.2.2), a demand on good nutrition
dietary EFA content and n‐3 LC‐PUFAs are essential for arises immediately. There is a demand for lipid, both as a
normal development of eggs and embryos, and improve source of energy and as EFAs, particularly LC‐PUFAs
the overall quality of eggs. These fatty acids may also such as 20:5n‐3, 22:6n‐3 and 20:4n‐6 which characteris-
serve as a source of energy during embryonic develop- tically accumulate in comparatively high concentrations
ment. Dietary eicosapentaenoic acid (EPA) and arachi- in ovarian tissue. During maturation of the ovary, triglyc-
donic acid (AA) are correlated with higher rates of eride content increases and this lipid is transferred to the
fertilization, possibly due to their observed role in ensur- eggs that are ultimately produced. The ovarian triglycer-
ing good sperm motility. The quantitative LC‐PUFA con- ides principally serve as energy sources, but certain die-
tent of formulated broodstock diets generally falls within tary fatty acids, particularly 20:5n‐3 and 22:6n‐3, are
the range of 1–2% with qualitative needs demonstrating important in maturation of ovaries, fecundity and egg
some species‐specificity. Docosahexaenoic acid (22:6n‐3) quality. In addition, arachidonic acid (20:4n‐6) and other
is an important dietary component especially for transfer n‐6 fatty acids are known to be precursors of prostaglan-
to larvae where the importance of this essential fatty acid dins which are assumed to play an important role in
for larval development has been documented. Higher reproductive processes such as vitellogenesis. Based on
dietary levels of vitamins E and C are important in ensur- presence and concentration in the ovary, phospholipids,
ing high fecundity and high rates of fertilization. Vitamin particularly phosphatidylcholine and phosphatidyletha-
C also manifests a role in vitellogenesis (egg quantity and nolamine, and cholesterol are considered to be important
quality) and steroidogenesis. Both vitamins C and E are in the processes of reproductive maturation. Carotenoids,
antioxidants and presumably serve in a protective role for particularly the xanthophyll astaxanthin, accumulate in
sperm cells during steroidogenesis and in ensuring good the ovaries as part of a lipovitellin protein. These lipid
motility of sperm. Insufficient levels of dietary vitamin E compounds have substantial antioxidant properties and
have been associated with immature gonads and reduc- confer colour to the ovaries which becomes a pigment
tions of both hatching rates and survival of offspring. source for transfer to eggs and larvae. In general, the rec-
Viable eggs, fertilised eggs and larval survival were used ommended levels for inclusion in broodstock diets are
as indicators to evaluate the combined roles of LC‐PUFA 2% phospholipids, 50 mg/kg astaxanthin and a total lipid
and vitamin E in affecting broodstock performance of level of 10%, higher than that found in grow‐out feeds
gilthead seabream, Sparus aurata. Based on the available (~7%). Crustaceans are unable to synthesise LC‐PUFAs
research results, specific nutrient recommendations for and carotenoids de novo, so diet must be the source.
broodstock diets include 250 mg/kg of vitamin E and Cholesterol can be synthesised from phytosterols, but
100‐250 mg/kg of vitamin C. The observed positive the rate of synthesis is insufficient to support the dietary
effects of dietary phospholipids (egg quality), the carote- requirement.
noid astaxanthin (buoyant and hatched eggs, increased
Protein levels in the ovary also increase during matu-
ration, probably arising from the transfer of vitellogenin
from the hepatopancreas where it is synthesised. It
170 Aquaculture process large quantities of nutrient poor food to derive
the required amounts of nutrients. Accordingly, they
appears, therefore, that higher protein levels should be characteristically have long intestines where detrital
included in a formulated diet for broodstock and this material is moved through quickly. The intestines have a
need is also supported by the high levels found in fresh thin mucosal lining to facilitate rapid uptake of nutrients.
food that are fed as ‘maturation diets’. Evidence for the Representative species of aquaculture interest are carp,
essentiality of a specific quantity or quality of carbohy- tilapia and milkfish. Carnivorous species generally have
drate in formulated diets for crustacean broodstock has short (absolute length) intestines with thick mucosal lin-
yet to be observed. ings and the rate of transport through is comparatively
longer. However, along the proximal portion of the intes-
Similar to fish, vitamins, A, C and E have been found to tine of some of these fish there are pyloric caeca which are
be required dietary ingredients to achieve desired repro- characterised by having a large surface area to assist in
ductive performance in broodstock. Vitamin E defi- the efficient uptake of available nutrients. Food passes
ciency has resulted in abnormalities in sperm and a through the caeca at the same rate as that of the intestine.
reduction in the hatching rate of eggs. High hatching Representative species include trout, tuna and hybrid
rates have also been observed in response to increases in striped bass. Many herbivores have a thick‐walled modi-
dietary ascorbic acid (vitamin C) a compound with anti- fied stomach call a gizzard that is primarily used to physi-
oxidant properties. cally break down food in conjunction with chemical
digestion effected by enzymes. The intestinal length of
8.5 Digestion and Assimilation of Food herbivores commonly falls between those of detritivores
and carnivores.
An understanding of the digestion and assimilation of
food is important as choice of highly digestible feedstuffs Most of the digestion of foods consumed by fish occurs
as sources of nutrients for aquaculture species is essen- in the intestine followed by absorption through colum-
tial in achieving low food conversion ratios or high feed nar epithelial cells that have a ‘brush border’ composed
efficiencies. Maximizing nutrient availability and assimi- of microvilli. Based on their morphology they contribute
lation for growth and other essential metabolic processes about 90% of the total absorptive surface area of a fish’s
achieves high growth efficiency, a critical component of gastrointestinal tract.
sustainability. Hence, a summary of the knowledge of the
processing of food consumed by fish, crustaceans and Prior to entrance into the stomach (if one exists), some
echinoderms/molluscs will serve as an important foun- physical breakdown of food may be accomplished by
dation of the understanding of nutrient availability and teeth, if present, in the mouth or the pharynx. The food,
metabolism. The following descriptions of digestion and reduced to smaller particles, is combined with mucous
assimilation of nutrients are derived from more exten- originating from goblet cells which are specialised epi-
sive descriptions found in Ceccaldi (1997) for crusta- thelial cells of the oesophagus. For those species that
ceans and Horn (1998) for fish. The processes of digestion have stomachs there is both physical digestion, effected
and assimilation of nutrients by fish and crustaceans are by the muscular tissue and enzymatic digestion. The ini-
collectively shared with monogastric (one stomach) ter- tial stages of chemical digestion focus on protein that is
restrial animals. broken into smaller chains of amino acids by the action
of the enzyme pepsin in association with HCl, both orig-
8.5.1 Fish inating from the oxyntopeptic cells of the mucosa of the
Fish initially evolved as carnivores followed by the evolu- stomach. Pepsin is an endopeptidase that hydrolyses
tion of herbivory, omnivory and detrivory in some spe- peptide bonds (bonds between amino acids) in the inte-
cies. However, 85% of all species of fish remain carnivores. rior of protein molecules. With the secretion of HCl, the
Carnivores do not have the ability to digest carbohy- pH of the stomach contents decreases to 1–2 and returns
drates effectively, so dietary protein and lipid collectively to between 4 and 7 when this partially digested food
serve as the source of energy. In contrast, carbohydrates mixture moves out of the stomach into the intestine
can serve as an excellent source of energy for herbivores, where the remaining digestion of protein and other
omnivores and detritivores and therefore formulated macronutrients (lipid, carbohydrate) occurs. Bicarbonate
diets can contain less proportional protein and lipid, from the pancreas neutralises the acidic mix from the
commonly resulting in feed formulations that are less stomach whereby the pH of the intestine falls within a
expensive. range of 7 to 9. Most of the digestive enzymes present
within the intestine are secreted from the exocrine cells
The morphology of the guts of species differs according of the pancreas and the brush border cells of the intes-
to feeding strategy and length is generally a defining char- tine itself.
acteristic. For example, detritivores need to efficiently
The pancreas secretes two major endopeptidases, trypsin
and chymotrypsin, which operate at a pH o ptimum of 7–7.5
and continue the digestion of proteins by specifically Nutrition and Feeds 171
hydrolysing peptide bonds that remain in the interior of
protein molecules. These endopeptidases are highly spe- 8.5.2 Crustaceans
cific, executing the hydrolysis of peptide bonds where the As monogastric (single stomach) animals, the digestive
carboxyl group of arginine and lysine is combined with the morphology and enzyme controlled digestive processes
amino group of another amino acid. Trypsin activates chy- of crustaceans are similar to those of fish. Nonetheless,
motrypsin through proteolysis (breakdown) of its inactive some noteworthy differences exist. Prior to transfer to
form (chymotrypsinogen) and chymotrypsin A specifically the mouth, crustaceans physically manipulate and reduce
hydrolyses peptide bonds at the carboxyl end of amino the size of food obtained through external mouthparts,
acids with an aromatic side chain such as tyrosine and specifically mandibles and maxillipeds. Joining the
phenyalanine. Carboxypeptidases A and B continue the mouth to the stomach is the oesophagus which is short,
hydrolysis of peptide bonds of the polypeptide molecules straight and vertically oriented. The inner surface of the
created by the endopeptidases, focusing on the peptide stomach is coated by a chitin‐protein complex. The ante-
bonds that have free carboxyl and amino groups located at rior region of the stomach has a thin epithelium whereas
the end of the molecules. The peptide bonds of the result- the posterior region that includes the cardiac and pyloric
ing tripeptides and dipeptides are further hydrolysed by the regions is also reinforced with calcified ‘teeth’ that are
action of aminopeptidases that originate from the brush used to break down the entering food into smaller parti-
border cells of the mucosal layer of the intestine, reducing cles. The particles are then separated by size, either
them to their constituent amino acids. transported out of the stomach or physically retained by
filters in the stomach for additional breakdown. The very
Other enzymes produced by and released from the fine food particles released from the stomach are ulti-
pancreas are amylases which hydrolyse the glycolytic mately passed to an important organ, the hepatopan-
bonds in polysaccharides to produce oligosaccharides creas, which is also termed the mid‐gut gland. This tissue
and then disaccharides. Disaccharides are further hydro- is composed of two sections and each section, consisting
lysed to monosaccharides by disaccharidases which are of 2–3 lobes, opens into a digestive tract that contains
released from the brush border of the mucosal cells. In secondary and tertiary tubules. These tubules are the
preparation for digestion, lipid in the intestine is physi- glandular part of the mid‐gut and produce an array of
cally reduced in size to droplets that are emulsified by a enzymes to facilitate chemical digestion. Trypsin is the
mixture of phospholipids and bile salts produced by the principal proteolytic enzyme and chymotrypsin is
liver and delivered via the gall bladder. The ester bonds either not present or present in very small amounts.
of the emulsified lipid are then hydrolysed by lipases Carboxypeptidases, aminopeptidases and dipeptidases
delivered from the pancreas and triglycerides are reduced complete the protein digestion. Enzymes to affect the
to diglycerides and then monoglycerides, fatty acids and chemical digestion of carbohydrates and lipids are also
glycerol. Phospholipases are also present to hydrolyse present. Amino acids and molecules that are the prod-
the phosphorous and glycerol ester bond found in phos- ucts of lipid and carbohydrate digestion are absorbed at
pholipid molecules. the brush border cells of the intestine. The mid‐gut gland
manifests liver‐like functions, both storing and metabo-
The absorption of nutrients in the form of the building lizing organic compounds and storing minerals.
blocks of macronutrients, i.e., amino acids, fatty acids
and monosaccharides, occurs in the intestine, either 8.6 Formulation, Manufacture
actively (via carrier molecules) or passively (diffusion). and Digestibility of Feeds
The cellular sites of the absorption of the nutrients are
called enterocytes. Fatty acids, monoglycerides, fat solu- A variety of ingredients, feedstuffs, premixes and supple-
ments generally compose an aquafeed either research or
ble vitamins, cholesterol and other lipophilic nutrients practical use (Table 8.4). The qualitative and quantitative
combine with bile salts and phospholipid to form a ingredient composition of a feed is termed a formula-
micelle whereby these nutrients are transported to the tion. Ingredients used in formulations to produce feed
for aquatic organisms are often the by‐products of the
brush border of the enterocyte into which they are processing of food for humans. The choice of feedstuff
released via passive diffusion. Glucose and other mono- ingredients must ultimately be governed by production
saccharides, amino acids, small peptides and even some practices that are sustainable and not used for human
intact proteins are engulfed at the brush border of the consumption. A primary goal in the development of a
enterocytes through a process called pinocytosis. Amino formulation is to minimise the number of ingredients
while ensuring that they are highly digestible to achieve
acids, glucose, di‐ and tripeptides and water‐soluble vita- efficient satisfaction of nutrient requirements. Glencross
mins, aligned with specific proteinaceous carrier or
transporter molecules, are actively absorbed across the
membranes of the brush border cells into the circulatory
system. These nutrients are then transported to the liver
to be either stored or oxidised to produce energy.
172 Aquaculture
Table 8.4 Examples of ingredients typically found in arrays of laboratory/research diets, farm‐made feeds and commercial feeds
for aquatic animals. Proportional amounts vary dependent on choice of other ingredients, production systems used, type of
manufacture and species.
Laboratory Research Farm‐made/Supplemental Commercial Extruded/Pelleted
Vitamin‐free casein Fishmeal Fishmeal
Isolated soy protein Rice bran Soybean meal
Gelatine Coconut meal/Coconut oil cake Poultry by-product meal
Refined soy lecithin Lupine seed Poultry feather meal
Dextrin Cassava leaves flour Wheat grain/
Wheat middlings
Phosphatidylcholine Peanut meal Corn gluten meal
Wheat starch Soybean meal Blood meal
Alpha‐cellulose Trash fish Ground wheat
Egg albumin Coconut/ground nut oil Wheat flour
Fish protein concentrate Chicken feed Squid meal
Fish/vegetable oil Fish/vegetable oil Fish/vegetable oil
Vitamin/mineral premixes Vitamin/mineral premixes Vitamin/mineral premixes
Canthaxanthin Soybean hulls Astaxanthin
et al. (2007) compiled a group of strategies designed to amounts as well as inconsistent nutrient composition
evaluate ingredients for use in aquafeeds and this work is caused by differences in collection time or vagaries in
an excellent foundation to guide the development of preparation practices are foundation for reservations
international standards for choice and use of feedstuffs about use.
in aquafeeds. Ideally, manufacturers will recognise the
benefits of these standards/practices and accordingly The use of farm‐made feeds for small farms continues
accept and implement them. and production methods that will improve consistency
and efficiency are the ongoing subject of investigation.
8.6.1 Feed Manufacture (Farm‐Made Feeds) For example, the desire to use floating feeds that do not
Farm‐made feeds are still produced for use in small‐scale require the costly use of the feed manufacturing technol-
culture operations in many countries due to either the ogy of extrusion (see following section) has led to the
scarce availability of commercially produced pelleted feed testing of different arrays of ingredients to produce a pel-
or the prohibitive cost of these feeds for use in such rudi- leted dough that has positive buoyancy characteristics.
mentary, sometimes subsistence farming, endeavours. The use of farm‐made feeds will continue. However, with
Examples of ingredients in farm‐made feeds include anticipated increases in scales of production throughout
shrimp waste, fresh leaves, corn silage and beef liver. the world to meet future per capita protein demand,
Choice of ingredients is governed by availability and cost. commercially‐manufactured aquafeeds will replace
Generally, the formulations of farm‐made feeds are spe- farm‐made feeds in those instances where the economic
cies‐specific, but recommended ingredient compositions advantages of such a feeding practice is revealed. The
are lacking. The use of unprocessed animal ingredients, goal of environmentally and economically sustainable
the extensive variety of conventional and raw materials production practices will ultimately be realised through
that may be used, and the lack of consistent quality and adoption of such practices that will dramatically reduce
nutrient composition of locally available ingredients cause waste and disease transmission and limit vagaries in
concern about the ongoing value of farm‐made feeds. annual production.
Quality assurance is not a component of production prac-
tices. Use of some locally available feedstuffs as ingredi- 8.6.2 Feed Manufacture (Dry Pelleting)
ents in formulations of commercially‐m anufactured diets The manufacture of pelleted feed consists of a sequence
are appealing. However, the common lack of sufficient of steps. Held in storage facilities, the feedstuffs intended
for use as ingredients of a formulation may be subject to
a preliminary grinding process. The goal of this process Nutrition and Feeds 173
is to enhance digestibility as well as establish a homoge-
neous ingredient mixture. Subsequent to the grinding digestibility of nutrients) characteristics have been
process, the ingredients are batched together and micro- defined as qualitative measures of high energy extruded
nutrients such a mineral and vitamin premixes are added feed. Differences in the water stability of feeds can confer
separately. Mixing and additional grinding of the entire different digestibility and feed intake characteristics rela-
mixture follows. Pelleted feeds are produced with the aid tive to the species under study. For example, harder pel-
of steam (15–18 %) to moisten the mixture and a simul- lets have been found to both reduce and increase feed
taneous heating between 65 and 85 °C before passage intake and are also characterised by higher retention
through a pellet die to provide the desired size of a com- times in the gastrointestinal tract.
pressed sinking pellet. Pelleted feeds are also produced
by subjecting the final mixture to a slightly different pro- The principal feedstuff components of a feed are added
cedure whereby a greater amount of moisture (~25%) is to serve as protein and energy sources. Examples of com-
added to the mixture followed by exposure to a higher mon sources of protein used in feed formulations include
range of temperature (90–150 °C) in an extruder barrel. fishmeal, soybean meal and poultry by‐product meal. A
This mixture is then forced through a pellet die (extru- variety of fishmeal sources such as menhaden, anchovy
sion pelleting). As the pellet passes through the die, some and herring are used. The best plant protein source is
of the moisture is vaporised, causing the pellet to expand. soybean meal, but other plant oil seed sources that are
Pellets are dried to contain between 8 to 10% moisture used include cottonseed meal and canola meal. The con-
and can be either sinking or floating, as determined by tent of protein in animal‐derived feedstuffs ranges from
the ingredient composition of the mixture. 50 to 85 %, whereas protein content of plant‐derived
sources ranges from 20 to 50 %. Mixtures of different
The type of manufacturing process itself can be a posi- protein sources are commonly used in an effort to pro-
tive factor through enhancement of the nutritive value vide sufficient and balanced levels of available amino
of the feed. Through the addition of comparatively acids. Sources of lipid to provide both energy and a
higher amounts of water combined with the use of a source of EFAs are generally marine‐derived oils or com-
higher manufacture temperature prior to extrusion, the binations of marine‐derived and plant‐derived oils to
starch contained within the feedstuff ingredients is provide a mixture of n‐3 LC‐PUFA and PUFA and n‐6
gelatinised. Gelatinised starch has been shown to be LC‐PUFA and PUFA, respectively. A greater total pro-
more digestible than other forms of starch for trout and portion of plant‐derived feedstuffs can be used in feed
carp, thereby conferring a greater amount of available formulation for herbivorous and omnivorous species
dietary energy. The pellet expansion that occurs during because of their ability to digest carbohydrates more effi-
extrusion also produces a surface on which a lipid coat- ciently. These carbohydrates serve as complementary
ing can be applied. The ability to introduce lipids in this sources of energy.
manner offers the opportunity to increase the energy
content of feed, enhance palatability, and substantially During the past 30 years, considerable effort has been
reduce the quantity of fines (dust) that can accumulate directed toward the reduction or elimination of marine‐
after manufacture and during storage/transport, thereby derived meals and oils from diets because both these
minimizing the amount of feed that is unavailable for feedstuffs are not sustainable. In addition, unpredictable
use by the farmer. availability of these feedstuffs accompanied by fluctuat-
ing costs is not conducive to sustaining a successful
Certain feed ingredients may interfere with or be par- aquaculture enterprise that requires feed. Therefore,
tially subject to loss in conjunction with the feed manu- feedstuffs selected to compose formulations must be
continuously and readily available while contributing to
facturing process. For example, rice bran is not a preferred the lowest possible cost of production expressed as per
feed ingredient because it can be abrasive on equipment unit of protein. To attend to this goal, feed manufacture
used in the manufacturing process. Extrusion manufac- may involve the use of computer software to achieve
‘least cost’ formulations which are based on the selection
ture of feed causes a loss (elimination of activity) of of a combination of feedstuffs guided by an array of
approximately 50% of ascorbic acid. To provide for the restrictions in meeting the fulfilment of nutrient require-
desired dietary level, some type of compensation such as ments. These restrictions include maximum and mini-
the addition of higher amounts or the presence of a stabi- mum limits that are imposed to avoid problems arising
liser needs to be addressed. in manufacture or in the introduction of properties/
characteristics that reduce the availability of the nutri-
Physical quality of the feed will vary according to ingre- ents to the aquatic species. Thus, the availability of phos-
dient composition and processing. The differences can phorous, often present in plant‐derived ingredients, may
affect growth by negatively affecting consumption and be reduced by up to 33 % if sufficient phytate, which
binds with phosphorous, is present in the combination of
the digestibility of the feed. Physical (hardness, integrity,
density, oil absorption) and nutritional (availability and
174 Aquaculture A 0.2% addition of saponins from soybean to diets con-
taining plant meals and fed to Atlantic salmon caused
plant‐derived feedstuffs used in a specific formulation. inflammation in the distal intestine and reduced digesti-
Provisions to avoid this extant lack of variability can be bility of lipid, fatty acids and minerals. In some cases,
part of the least cost formulation. minimum levels of these digestion inhibitors have been
recommended for inclusion in diets. Thus, the level of
8.6.3 Digestibility of Feedstuffs plant‐derived feedstuffs that can be used as alternatives
and Nutrients to fishmeal may be influenced by their respective dietary
levels of saponins as well as provision of the proper
Digestibility of a feed, different feedstuffs and specific amounts and balance of essential amino acids.
nutrients by most species of farmed fish and shrimp is
principally determined by an indirect method that Inhibitors of lipase digestion are commonly found in
involves the addition of an indigestible marker to an feedstuffs derived from seeds that have characteristically
experimental diet. The most common marker used is high levels of lipid (soybeans, sunflower and peanut/
chromic oxide, but others include ash, silica, crude fibre, groundnut). Also included in this group of feedstuffs
polythene, stable isotopes and titanium oxide. To yield that contain lipase inhibitors are cereals such as wheat,
accurate determinations of digestibility, the marker must barley and sorghum. These inhibitors interfere with the
be non‐toxic, inert (not metabolised) and not alter the digestion process by binding to emulsified lipid droplets
process of digestion and rate of flow through the diges- or they can actually form a complex with the enzyme
tive tract. The relative increase of the marker in the fae- itself. Generally, lipid digestion in fish is very high, but
ces versus the amount in the diet is used to calculate a exceeding particular levels of cereals or seed meals in
percentage which is called the apparent digestibility co‐ formulated diets will probably exert a deleterious effect
efficient. The term ‘apparent’ is used because faeces con- unless some neutralizing treatment can be introduced or
tain organic material such as enzymes and epithelial cells applied.
of the intestine that are the respective products of the
digestion process and the movement through the intesti- Phytic acids or salts of phytic acid (phytate) are natu-
nal tract. These minor additions contribute to a slight rally found in plant feedstuffs often used in feed formula-
reduction in calculated digestibility coefficients., Among tions. These compounds characteristically reduce the
nine different feedstuffs of marine origin (fish, crab, scal- availability of minerals for absorption. Phytic acid mainly
lop, shrimp head, and squid meals) apparent digestibility binds with K+ and Mg2+ and can precipitate out as a salt
coefficients for dry matter ranged from 46 to 102% and with other cations such as Mn2+ and Ca2+. Soybean meal
protein digestibility coefficients ranged from 64 to 89% and rapeseed meal contain 10–15 g/kg and 50–75 g/kg of
for the marine shrimp, Litopenaeus vannamei. phytate, respectively. A diet containing 0.5% (5 g/kg) of
phytic acid and fed to rainbow trout reduced growth by
8.6.4 Anti‐nutrients and Contaminants 10%. Feeding a diet containing 0.21% (2.1 g/kg) of phytic
acid adversely affected the activity of trypsin in the intes-
Choices of feedstuffs for inclusion in formulated feeds tinal tract of Atlantic salmon. Elimination of the adverse
must also consider the presence of naturally occurring effects of phytic acid and phytate can be effectively
compounds that prevent/inhibit the efficiency of diges- addressed through the inclusion of the enzyme phytase
tion or the uptake of particular nutrients. Compounds in formulated pelleted diets for fish and crustaceans (see
that can adversely affect the activity of digestive enzymes the following section).
are generally proteins, both simple and complex, and this
undesirable characteristic can be neutralised through Gossypol is a yellow lipid‐soluble compound that is
treatments such as heat, fermentation or alcohol extrac- found in seeds of cotton plants of the genus Gossypium,
tion. A good example of a current effort to circumvent except for a ‘glandless’ variety, and has been connected to
the introduction of potential anti‐nutrients into feeds is a variety of toxic effects in several species of fish. The
manifested by the evaluation of fermented soybean meal amount of gossypol present in cottonseed meal varies,
as a partial replacement for fishmeal. depending on species and respective growth under dif-
ferent climatic conditions. Toxicity is defined through a
One group of ‘anti‐nutritional’ factors inhibit the variety of responses that include reductions in growth,
activity of proteinases during the digestive process. Some feed consumption, reproduction and the percentage of
of these factors are broadly active, affecting an array of red blood cells in the plasma. Histological indicators of
proteinase enzymes, whereas others specifically focus on gossypol toxicity are lesions on the liver, kidney spleen
trypsin and carboxypeptidases. The activity of amylases, and reproductive organs. Toxic levels of gossypol appear
needed for carbohydrate digestion, is restricted by the to vary with species. Age and size of the organisms as
presence of saponins that commonly have their source in well as other ingredients of manufactured feeds can
feedstuffs that originate from beans, grains and tubers. influence the magnitude of response to the same level of
dietary gossypol. Potential toxicity of gossypol can be Nutrition and Feeds 175
avoided through use of either proportionately low levels
of cottonseed meal or glandless varieties containing no However, fishmeal and fish oil are ingredients that inflict
gossypol. dramatic pressure on the sustainability of forage (pelagic)
fisheries, such as menhaden and anchovy to meet the
The development of accurate methods to identify con- demand. Progress toward the use of both plant and ani-
taminants in feeds is an important area of future research mal‐derived alternative feedstuffs, in concert with
that can positively impact efficiency of production. The appropriate economic and regulatory incentives, will
ultimate goal is the definition of limits of their content substantially improve the perception of aquaculture as
whereby such information is effectively delivered to feed being a highly favourable complement to capture fisher-
manufacturers. This information would hopefully serve ies to meet global protein demand through the use of
as the foundation of universal (global) acceptance of reg- feeds containing sustainable ingredients.
ulations for quality control of manufacture of aquafeeds.
A number of potential sources of alternatives to the
8.6.5 Feedstuff Alternatives and Additives use of fishmeal have been identified. Plant-based protein
sources include canola, soybeans, peas, lupines and
The feed industry and animal food producers continu- wheat. Soy protein concentrate and wheat are potentially
ally evaluate alternative feedstuffs and additives that good options, but their use is not cost‐effective. During
appear to have potential to increase efficiency of produc- the past 20 years, an abundance of published investiga-
tion through reduction of cost per unit of animal protein. tions using different fed aquatic species has focused on
Glencross et al. (2007) provide a review of strategies to the use of different soybean meals to serve as a complete
evaluate ingredient suitability for aquaculture feeds or partial replacement of fishmeal. These studies have
based on the foremost considerations of digestibility, revealed that generally soybean meal protein can effec-
palatability and nutrient utilization and interference. tively substitute for up to 50% of the fishmeal protein.
The utility of an alternative feedstuff also lies in charac- Plant‐derived protein sources are not as digestible as
teristics of consistent availability and easy storage, han- equivalent amounts of animal‐derived sources because
dling and shipping. Finally, use of any feedstuff alternative they contain fibre and insoluble starches that are not eas-
and/or additive must be judged by whether the environ- ily digested by carnivores and some omnivores, and
mental footprint associated with its manufacture or pro- therefore cannot be used as a source of energy. Exclusive
curement is comparatively low. These considerations use of protein from one plant source results in a defi-
contribute to goals of economic and environmental sus- ciency in one or more essential amino acids to meet the
tainability. Comprehensive assessments of possible dietary requirements. Therefore, successful use of plant‐
changes in the protein source on the health of the animal, derived sources of dietary protein will most probably be
the consumer and the environment relative to inclusion realised through the inclusion of other‐sources of plant
of the alternative feedstuff must be conducted. Another protein in combination with dietary supplements of indi-
guiding criterion that governs the use of alternatives vidual amino acids to ultimately meet species‐specific
would be lack of competition for use as sources of human requirements.
food. An array of aquafeed additives, such as enzymes,
carotenoids, prebiotics and probiotics, has become the Use of plant‐derived protein sources in aquafeeds is
subject of increased interest by aquafeed manufacturers also associated with other detrimental consequences
and commercial growers to increase efficiency of pro- such as the absence or reduced levels of LC‐PUFAs, the
duction. Some are already being added into commer- reduction of palatability and corresponding reduction
cially produced feeds, whereas others lack a conclusive in feed consumption, and nutrient interaction whereby,
demonstration of value or common application. for example, dietary phosphorous is nutritionally una-
vailable to the farmed organisms and consequently
8.6.5.1 Alternative Feedstuffs – The Search released into the environment. Inclusion of the widely
for Fishmeal and Fish Oil Replacements used soybean meal as a substitute could also be limited
Despite successful efforts to reduce levels of fishmeal by lack of availability due to demands for its use in the
and fish oil in aquafeeds, demand may, at least temporar- manufacture of feeds for terrestrial animal production
ily, increase due to the need to adopt intensive produc- species and foods for human consumption.
tion systems to address resource conservation, combined
with the dramatic increase in the farming of fed carnivo- Rendered meals derived from waste products of land
rous marine species. animal processing as well as defatted meal prepared from
insects fed fish processing wastes establish them as
Both feedstuffs are excellent sources of required nutri- strong replacements for fishmeal. They are attractive
ents, specifically essential amino acids and fatty acids. options because they share important characteristics of
high protein content, the desired balance of essential
amino acids, and high digestibility. Fishmeal derived
from the wastes of the processing of both cultured and
176 Aquaculture 8.6.5.2 Feed Additives
captured fish also represents at least a partial substitute The aquafeed industry uses certain feed additives with
of fishmeal derived from the processing of pelagic fish the principal intent to increase production efficiency
like menhaden or sardines. Recycling of wastes to pro- through improvement of growth and production, and
duce these feedstuffs contribute to both environmental enhancement of resistance to pathogens. Additives are
and economical sustainability. also used to achieve a colouration that will be appealing
to the consumer. This section will focus on carotenoids,
The use of plant‐derived oils such as soybean oil as a enzymes, prebiotics and probiotics.
1:1 substitute for fish oil is not possible due to the
absence of essential LC‐PUFAs that are characteristic of Carotenoids are compounds that are responsible for
marine fish oils, i.e., marine food chains. A combination the pigmentation found in farmed species. Lacking the
of plant and fish oils, thereby reducing the use of fish biochemical ability to synthesise these compounds de
oils, has been the feed formulation strategy in the pro- novo, animals are dependent on an exogenous source
duction of effective salmonid feeds. The fish oil portion (food) of them to achieve colouration. Astaxanthin, a
of the total oil content of the feed proportionately xanthophyll, is the primary carotenoid found in the tissue
increases during the few months prior to the intended of fish and crustaceans. Therefore, to achieve the appro-
harvest. This feed management practice is designed to priate level of pigmentation in meeting consumer accept-
enhance the content of LC‐PUFAs in the muscle tissue ance of farmed aquatic species, the feed must contain a
and thereby enhance nutritional value of the fillets sufficient concentration of carotenoids. The combined
sought by the consumer. Finishing diets containing a fish dietary level of carotenoids derived from feedstuffs is
oil have been successfully used to ‘restore’ the fatty acid commonly insufficient to produce the desired colour and
composition of fish tissue after feeding a diet containing intensity of pigmentation. Therefore, a carotenoid is gen-
plant‐based oil for most of the grow‐out period. Fish oil erally added to the feed. Different astaxanthin‐c ontaining
derived from the waste left from the processing of fish animal‐derived products are used in aquafeeds (NRC
from the culture and capture fisheries also appears to be 2011) and copepod, shrimp, krill or crab oil have the
an attractive, cost effective source of required LC‐PUFAs highest concentrations, ranging from 50–150 mg/kg.
to include in diets.
The reddish orange colouration of the flesh of salmon
Sources of protein and oil derived from single cell cul- is due to the presence of two oxygenated carotenoids,
ture, provided as the entire organism or as an extract astaxanthin and canthaxanthin (xanthophylls) which are
(lipid), are receiving considerable attention. Promising commonly added as synthetic forms in the feed fed to
results have been realised under small‐scale laboratory salmon. Recommended dietary levels of these specific
production; extrapolation to large scale production for xanthophylls range from 50 to 150 mg/kg to maintain
cost-effective use in aquafeeds will likely become a reality. tissue levels that yield consumer acceptance. Natural
Future provision of dietary LC‐PUFAs could ultimately sources of astaxanthin which may ultimately become
reside in plants that have been genetically modified to cost‐effective additives include the red yeast, Phaffia
produce LC‐PUFAs. rhodozyma and Hematococcus species of algae. The dif-
ferent carotenoids found in plants such as lutein and
The use of a meal derived from the culture of insects zeaxanthin are metabolic precursors in the synthesis of
has become the subject of interest as an alternative ingre- astaxanthin and canthaxanthin but use of these xantho-
dient in feeds for aquatic organisms (see section 27.3.3.1; phylls as dietary additions does not yield pigmentation
Figure 27.9). Deficiencies in essential amino acid or LC‐ that is commensurate with animal‐derived carotenoids.
PUFA content can be rectified by the type of food fed to Therefore, the most efficient method to attain the desired
larval forms of insects. When fish offal was provided as pigmentation in a farmed species is to provide the carot-
food for soldier fly larvae in different proportions of the enoid that is principally found in the tissue. When singu-
total diet for 21 days, tissue levels of LC‐PUFA expressed lar sources of different carotenoids were included in
as weight percent of the total lipid, increased by 2.3 to diets fed to juvenile lobsters, Homarus sp. which contain
2.7 %. This notable level of change was also achieved astaxanthin as the tissue carotenoid, the intensity of pig-
when larvae were fed for only 24 hours after being mentation in the shell and tissue was found to be directly
removed from a control diet consisting of no fish offal. related to the proximity of the specific dietary carote-
This specific insect meal effectively replaced 15 % of the noid to astaxanthin in the biosynthetic pathway. This
fishmeal protein and 38 % of the fish oil ingredient in a c arotenoid‐specific graded response would most proba-
diet fed to rainbow trout Oncorhynchus mykiss. Total bly be manifested in other aquaculture species in other
lipid in insect meal characteristically exceeds that of fish- phyla such as molluscs and echinoderms. The ability of
meal (~30% vs. ~ 10 %) and may need to be reduced by an organisms to retain the desired pigmentation is influ-
extraction procedure if an economically and environ- enced by an array of factors which include rates of
mentally acceptable product for use in aquafeeds is to
be developed.
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