Aquaculture, 33 (1983) 215-227

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

215
INBREEDING IN FISH POPULATIONS USED FOR AQUACULTURE
HAROLD L. KINCAID
Fish Genetics Station, U.S. Fish and Wildlife Service, Box 700, Kearneysville, WV 25430
(U.S.A.)
(Accepted 1 December 1982)
ABSTRACT
Kincaid, H.L., 1983. Inbreeding in fish populations used for aquaculture. Aquaculture,
33: 215-227.
The theoretical causes of inbreeding depression are reviewed. Studies to measure de-
pression at a series of inbreeding levels in rainbow trout populations using the method of
inbred-outbred half-sib families are discussed. While the actual depression estimates
varied widely between populations and inbreeding levels, significant levels of depression
were found in many hatchery, field, and brood stock performance traits after only one
generation of brother--sister mating. Especially susceptible to inbreeding depression, were
measures of growth based upon attained fish weight at a given age in each test situation
examined: 147 day weight (0.0 to 19.1%). 364 day weight (6.2 to 62.8%), weight after
6 months in fishing pond (-4.6 to 25.4%), weight after 12 months in fishing pond (13.0
to 29.1%), a-year male weight (11.3 to 55.3%), and a-year female weight (10.2 to 57.0%).
Other hatchery performance traits that showed inbreeding depression were: egg hatch-
ability (-0.2 to 53.1%), fry survival (0.4 to 8.2%), feed conversion efficiency (5.0 to
9.0%), fiih length at a-years of age (4.7 to 36.9%). and egg mass produced at %-years
(12.1 to 57.0%). Field performance traits that yielded inbreeding depression were per-
cent recovery of fish stocked (-2.5 to 41.1%) and biomass index (16.2 to 47.7%).
Literature on the effects of inbreeding in fish populations is reviewed and the breed-
ing approaches for controlling the rate of inbreeding accumulation in brood stock popu-
lations are outlined and discussed.
INTRODUCTION
The increasing role of aquaculture as a source of protein and other pro-
ducts during the past decade has forced fishery managers to begin to address
many problems associated with the intensive management of small popula-
tions. Any fishery operation that results in a limited number of fish being
available to produce progeny for use as brood stock in the next generation
can lead to a constriction in the gene pool of that population, either in a
hatchery or a natural fishery. These constrictions can occur as a result of
breeding practices such as: the selection of a small number of superior
individuals as brood stock, the use of fish taken from a small segment of the
spawning season, the use of limited numbers of individuals that survive a
0044-8486/83/$03.00 0 1983 Elsevier Science Publishers B.V.
216
major collapse of the fishery, or the use of a small number of brood fish be-
cause they provide adequate numbers of eggs or progeny to meet manage-
ment needs. The potential for acceleration of the natural rate of inbreeding
is presented by each of these breeding practices.
Current understanding of the causes and effects of inbreeding in fish pop-
ulations are reviewed in this paper and general breeding methods available to
the brood stock manager to minimize rates of inbreeding are outlined. Dur-
ing recent years the techniques of gynogenesis and sex reversal have been ap-
plied as an approach for the production and study of inbreeding effects in
aquatic populations. However, further examination of sex control techniques
and their application to inbreeding questions is beyond the scope of this
paper. The reader is referred to the review by Yamazaki (1983, this volume)
on sex control and manipulation.
THEORETICAL APPROACH TO INBREEDING
Inbreeding in an infinitely large population is defined as the mating of in-
dividuals that are more closely related to each other than individuals mating
at random within a population. However, the populations actua.lly used in
most aquacultural programs are finite populations because they possess a
limited number of members. All finite populations experience some degree
of inbreeding that is based on the number of individuals that contribute pro-
geny to each succeeding generation. Inbreeding is measured by a value called
the inbreeding coefficient which is normally represented by the symbol F.
The inbreeding coefficient (F) is the probability that two alleles at any locus
are identical and descended from a common ancestor. Since the inbreeding
coefficient is a probability, it can assume only the values within the range
from zero to 1.0.
Inbreeding coefficients express the amount of inbreeding that has accumu-
lated starting from a specific point in the ancestry of the population. Be-
cause the number of independent ancestors is limited in any finite popula-
tion, all alleles of a single form would be identical and descended from a
common ancestor if they were traced far enough into the past. Therefore,
the inbreeding coefficient is meaningful only if a specific time in the past is
chosen beyond which ancestries will not be considered, and at which time
all alleles are considered to be independent. This point is called the base
population, and by convention it is considered to have an inbreeding coeffi-
cient of zero. The inbreeding coefficient of any given generation therefore
expresses the fraction or percent of heterozygosity that has been lost since
the base population generation. When an F-value is given for a brood stock
population, the specific base population is not always identified but the ex-
istence of a base population is always implied.
Inbreeding depression is the effect of inbreeding normally measured as a
reduction in the expected performance of the affected trait. Inbreeding de-
pression is measured as the average performance difference between an in-
217
bred population and the base population. Traits that frequently exhibit in-
breeding depression are multi-locus or quantitative traits associated with re-
productive capacity (e.g. fecundity, egg size, hatchability) and physiological
efficiency (e.g. fry deformities, growth rate, feed conversion efficiency, sur-
vival). Other traits may or may not show depression with inbreeding.
The characteristics of inbreeding can be studied by examination of what
happens to gene frequencies in a brood stock population that is randomly
divided into a series of mating pairs which are then isolated for several gene-
rations to establish inbred lines. This procedure yields a series of inbred lines
that collectively represent the gene pool of the original generation. Gene fre-
quencies in the separate inbred lines would vary widely as random genetic
drift caused fixation of some alleles and the loss of some alleles. This process
leads to reduced heterozygosity in the individual lines, however, with a large
number of inbred lines to represent the origmal gene pool and random fixa-
tion of alleles within each line, the gene frequencies collectively across all in-
bred lines should be the same as in the original population. This would be
the case within sampling errors and provided that no lines were lost during
the inbred line development process. When lines are lost, the effect is to pro-
duce a modified population that is changed by the selective loss of the un-
fit lines. The characteristics of inbreeding shown in this ideal population
demonstrate that inbreeding depression is the result of differences in the
genetic value of homozygotes relative to heterozygotes. The primary cause
for this difference is the within locus interaction of alleles, a phenomenon
known as dominance. The effects of inbreeding depression in quantitative
traits can be accounted for largely by the phenotypic expression of increas-
ing numbers of unmasked recessive alleles and the reduced frequency of
heterozygous loci expressing codominance and overdominance.
Inbreeding depression tends to increase in proportion to the inbreeding
coefficient during the initial inbreeding generations as reproductive capacity
and viability are reduced and some inbred lines are lost. However, as lines
are lost, the surviving lines become a selected population and the theoretical
expectations for rate of inbreeding are no longer directly applicable. As a
result the prediction of increasing rate of inbreeding depression from the in-
creasing inbreeding level is limited to the initial generations before the in-
breeding coefficient reaches a high level. Traits measured during egg or fry
stages, such as egg size, hatchability, fry mortality, or fry growth rate are
often affected by maternal influences. Such traits are doubly sensitive to
inbreeding depression because of the influence not only of the inbred geno-
type of the individual, but also the genotype of the inbred female parent
as it effects the maternal environment provided during the egg development
stage. As a result of changes in the total gene pool caused by the collective
effects of natural selection and maternal influences, the relationship between
inbreeding depression measured in a trait and the coefficient of inbreeding is
usually non-linear, after the initial generations.
A frequent problem when working with fish brood stocks is that available
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219
information on the breeding history simply is inadequate to determine the
level of inbreeding. Information on the genealogy of individual fish is almost
never known, thereby eliminating most classical methods for the calculation
of inbreeding coefficients. When information on the actual number of fish
of each sex in the breeding population is known for several generations, an
estimate of the inbreeding accumulated during that period can be calculated
using approximation methods (Falconer, 1981). One formula frequently
used when working with random mating populations is
AF=
L+L
slv, 8Nf
Where, AF = the expected increase in the inbreeding coefficient per genera-
tion, N, = number of male parents actually used to produce the next
brood stock generation, and ZVf = number of female parents actually used
to produce the next brood stock generation. The calculated AF value for
each generation is added to the inbreeding coefficient of the preceeding
generation to yield the new inbreeding coefficient. Inbreeding coefficients
estimated by this approximation method yield overestimates of the actual
inbreeding rate after the first generation with the magnitude of the over
estimation decreasing as the effective population size increases. The esti-
mated rate of inbreeding accumulation for a range of male and female parent
number combinations is given in Table I. When historic records on breeding
practices and brood stock parent numbers are unavailable, the inbreeding
coefficient can not be calculated directly.
INBREEDING DEPRESSION IN AQUATIC ORGANISMS
The deleterious effects of inbreeding depression in domestic and labora-
tory animal species have long been recognized by research workers (Fisher,
1949; Robinson and Bray, 1965; Hill and Robertson, 1968; Falconer, 1981).
In recent years a few reports on the effect of inbreeding in fish species have
begun to appear in the published literature. Moav and Wohlfarth (1963) re-
ported a 15% reduction in relative growth rate in inbred carp (Cyprinus
carpi o) produced from full-sib parents as well as an increased frequency of
fish with dorsal fin anomalies. Aulstad and Kittelsen (1971) described an in-
crease in the occurrence of fry deformities in rainbow trout (S&no gaird-
neri) that was associated with an inbreeding coefficient of F = 0.25. Ryman
(1970) found lower recapture frequencies from inbred families of Atlantic
salmon (3. s&r) suggesting that lower .survival rates were associated with
inbreeding. Inbreeding depression estimates, per 10% inbreeding, of 5.12%
in fish weight and 0.44% in formalin tolerance at 150 days of age was re-
ported by Bridges (1973) in rainbow trout. Gjerde et al. (1983) working
with three generations of inbreeding in rainbow trout reported inbreeding
depression per 10% inbreeding in egg mortality to the eye stage (2.5%), ale-
220
vin mortality (1.9%), fry mortality (3.2%), fingerling growth rate (3.0%),
and growth rate to 18 months in seawater (5.1%). Estimates of inbreeding
depression in body weight of brook trout (Salvelinus fontinalis) after one
generation of brothel--sister mating were found to be 27.7% at 7 months
and 34.4% at 19 months (Cooper, 1961).
Kincaid (1976a, b) reported that one generation of brothersister mating
in rainbow trout produced an increase in fry deformities (37.6%) and de-
creased feed conversion efficiency (5.6%), fry survival (19%), and fish weight
at 147 days of age (11.0%) and 364 days of age (23.2%). After two genera-
tions of brother-sister mating even greater changes were measured: fry de-
formities increased (191%) while decreases were found in feed conversion
efficiency (14.9%), fry survival (29.7%), and weight attained by fish at 147
days (13.4%) and at 364 days (33.5%). This trend of increasing depression
in body weight with fish age appears to be associated with the cubic nature
of the growth curve magnifying the reduced growth rate of the inbred fish.
Fujino (1978) reported inbreeding depression in body weight in some in-
dividuals of a wild population of Pacific abalone (Haliotis discus). Individuals
expressing reduced body weight were found to have a much higher fre-
quency of homozygosity at two e&erase loci than normal animals. Longwell
and Stiles (1973) and Stiles (1981) working with the American oyster (Crass-
ostrea uirginica) reported that progeny from full-sib matings produced signi-
ficantly lower survival of larvae to metamorphosis and higher frequencies of
larval abnormalities than the outbred control lines. Lannan (1979, 1980)
working with the Pacific oyster (Crassostrea gigas) found no depression in
larval survival through two generations of inbreeding. Mrakovcic and Haley
(1979) examined half-sib and full-sib families of the zebra fish (Brachydanio
rerio) and reported no effect of inbreeding on hatchability in either group.
Inbreeding depression was observed in egg fertility, frequency of abnormal
fry, fry survival to 30 days, and fish length at 30 days in both inbreeding
levels.
From the work reviewed here as well as work reported on numerous do-
mestic and laboratory animals, it would appear that in most diploid bisexual
animal species, increasing levels of inbreeding yield reduced performance in
a variety of traits. The type of traits most frequently reported to show in-
breeding depression in fish species have been: increased fry abnormalities, re-
duced survival, reduced growth rate, and lowered reproductive success. The
magnitude of the depression observed in a particular strain within a species
varies widely depending on genetic background, historic inbreeding, cultural
history, and rearing environment.
INBREEDING DEPRESSION IN A NATURAL FISHERY
Studies conducted by the U.S. Fish and Wildlife Services Fish Genetics
Laboratory between 1975 and 1980 to evaluate the performance of inbred
and outbred half-sib families stocked in a l-ha spring-fed fishing pond as
221
fingerlings provide further evidence of inbreeding depression in a natural en-
vironment. Fish were stocked in the pond at approximately 7 months of age
with each family identified by the use of numbered stainless steel tags in-
serted in the body cavity. Recovery periods were conducted at 6-month
intervals after stocking. Each recovery period consisted of an angling period
during which 1000 angler hours of fishing pressure was applied and a gill
netting period when four experimental gillnets were placed in the pond for
a total of 100 net hours. All fish captured were removed from the fishery,
identified by family, and individually weighed. In addition, samples from
each family were also reared to 1 year of age in a standardized rearing envi-
ronment. Inbreeding depression was measured as the average difference in
performance of inbred and outbred half-sib families.
One experiment using two groups of fish inbred for one and three genera-
tions from the winter 1977 year class showed depression in both hatchery
and field performance traits (Table II). After one generation of inbreeding,
hatchery performance traits showed inbreeding depression in hatchability,
fry survival, feed conversion efficiency, and 364 day weight with actual de-
pressions being similar to that previously reported (Kin&d, 1976a). Depres-
sion in the group inbred for three generations of brother-sister mating was
not markedly different than that found after one generation except for an
increased effect on weight at 147 days (16.0%) and 364 days (41.7%). How-
ever, since the two groups were derived from different strains it is not pos-
sible to compare the relationship of depression to increasing level of inbreed-
ing.
Inbreeding depression in field performance traits was evident in both
groups after the fish had been in the pond for 6 months (Table II). Average
fish weight in inbred groups was lower at both the 6 and 12 months recovery
periods compared to outbred controls. Inbreds of the F = 0.25 group had a
recapture rate that was lower than outbreds in the first recovery period and
equal in the second period. This recapture pattern shows the proportion of
the total fish recovered during each recovery period but does not give the
actual survival rate of the inbred and outbred groups for each period. Be-
cause a higher proportion of fish from the outbred group was removed du-
ring the first period, fewer fish of that group were left in the fishery to
compete for survival in the pond environment and to be available for har-
vest during the second recovery period. Therefore, the percent recovered
in the second period relative to the number of fish potentially available for
recapture, was lower in the inbred group. Measures of inbreeding depres-
sion in traits such as total percent recovery of fish planted and biomass in-
dex are used to indicate the net effect of inbreeding on production in this
fishery. Since each of these traits reflects the contribution of many other
traits (i.e., survival, predator avoidance, feeding behavior, social behavior,
adaptability to environment, and growth rate) they must be considered to
be composite performance traits. Depression in total percent recovery (6.9
to 21.2%) reflects the reduction in survival during the test period while de-
222
TABLE II
Effects of inbreeding by brothemister mating for one and three generations in winter
spawning rainbow trout. Growth and survival traits were evaluated in the hatchery, and
during a la-month field test in a l-h fishing pond after stocking as fingerlings. Fish were
from the winter 1977 year class
Trait Inbreeding coefficient and number of inbred-outbred half-sib
family pairs
F = 0.25 (16 pairs) F =0.50 (13 pairs)
Inbred Outbred % de- * Inbred Outbred % de- *
mean mean pression mean mean depression
Hatchery performance
Hatchability (%) 69.1 83.5 17.2 84.2 84.0 -0.2
Fry survival to 84 days
days (%) 89.2 94.7 5.8 89.3 89.7 0.4
Weight (g), 147 days 3.4 3.4 0.0 3.0 3.6 16.0
Feed conversion
147 days 2.3 2.2 -6.7 2.1 2.0 -5.0
Weight (g), 364 days 68.0 91.0 25.0 85.1 145.9 41.7
Field performance
Weight (g), at planting 28.6 31.4 8.8 36.4 35.1 -3.6
B-month recovery
percent 24.5 33.0 25.8 36.5 38.4 5.0
mean weight (g) 72.4 79.9 9.4 65.2 74.5 12.5
1 a-month recovery
percent 7.2 7.2 0.0 5.9 7.1 16.9
mean weight (g) 150.7 173.2 13.0 132.6 187.0 29.1
Total percent recovery 31.7 40.2 21.2 42.4 45.5 6.9
Biomass indexb 2858.8 3883.7 26.7 3162.1 4188.5 24.5
aPercent depression is calculated as outbred mean minus inbred mean divided by outbred
mean.
bBiomass index is the total biomass recovered per 100 fish planted.
pression in biomass index (24.5 to 26.7%) reflects both the reduction in
survival and growth rate. Both traits demonstrate a significant reduction in
the productivity of the inbred groups.
The effect of inbreeding on seven growth and maturity traits at first ma-
turity was measured in inbred lines produced by one, three, four and five
generations of brother--sister mating (Table III). The measures of growth
evaluated in this study - weight at 1 year and length and weight at 2
years - uniformly showed large and highly significant inbreeding depres-
sion at all four inbreeding levels. In addition, the magnitude of depres-
sion increased with each generation of inbreeding. The depression mea-
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224
sured in total weight of eggs produced (egg mass) was large and statistically
significant in all inbreeding groups except the first generation of inbreeding
where the half-sib deviations were extremely variable. Depression in egg mass
weight was closely related to depression in Z-year length of the female parent
suggesting that the smaller size of the inbred female provided proportionate-
ly less body cavity volume for the developing ovaries. Since the traits egg size
and number in each egg mass were not evaluated, the effect of the reduced
egg mass on egg number and egg size is unknown. If the smaller egg mass re-
sulted from reduced egg size, then the expected effect would be reduced
hatchability and smaller fry with reduced fry survival If the smaller egg mass
results from reduced fecundity, then the expected effect would be reduced
numbers of eggs and lower numbers of live fry per breeding female. It is
highly probable that both reduced egg size and reduced egg numbers contri-
bute to the smaller egg mass found in the inbred groups.
Studies completed in the rainbow trout demonstrate that inbreeding de-
pression is expressed in a variety of performance traits throughout the life
cycle from the egg stage to first sexual maturity. Investigations into the ef-
fects of inbreeding have been initiated in several aquacultural species, how-
ever, only limited information has been published to date. Knowledge of
inbreeding effects in rainbow trout, while not directly applicable to other
species, can provide an indication of the kinds of the traits that may be sus-
ceptible to inbreeding depression and the approximate magnitude of depres-
sion to be expected in other fish species. The fishery manager needs to know
as much as possible about the effects of inbreeding because he is the one
who will make decisions about: What source of brood stock will be used?
How many individuals are needed in the brood stock to maintain the genetic
diversity of the gene pool? What are the effects of a selection program?
What are the risks of stocking wild fisheries with fish taken from a small frac-
tion of the total spawning run? The answers to these questions and many
others directly affect the long term vitality and productivity of most brood
stocks.
BREEDING APPROACHES TO CONTROL INBREEDING
Fisheries personnel responsible for maintaining brood stocks must become
more aware of the serious problems that can result from inbreeding so they
can adopt breeding methods that will minimize future inbreeding problems.
While inbreeding can be a powerful technique for developing new and im-
proved strains, the negative aspect of inbreeding is what is most frequently
seen by the individual fish breeder.
Current breeding approaches to avoid inbreeding fall into three general
categories: (1) the use of large random mating populations; (2) the use of
systematic line crossing schemes to eliminate the mating of close relatives;
and (3) strain crossing to produce hybrid populations. The use of large ran-
dom mating populations is the simplest approach and requires only that the
225
breeder take steps to insure that a large number of fish contribute progeny
to the next brood stock generation. One source of confusion frequently ex-
perienced by the breeder using large random mating populations is that the
breeding population is defined as the number of fish used to produce the
next brood stock generation, not the total number of fish in the population.
The rate of inbreeding accumulation in a random mating population may
also be seriously effected by the number of fish of each sex actually used to
produce the progeny generation (Table I).
An important factor in determining just how large a random mating brood
stock population needs to be is the expected rate of inbreeding. The meth-
ods normally used to study the effects of inbreeding involve the production
of many inbred lines by mating close relatives to produce a rapid rate of in-
breeding. In actual brood stock management situations, the mating system
would be expected to provide a much slower rate of inbreeding accumula-
tion. The rate of inbreeding increase is extremely important because it indi-
cates the time in generations required for inbreeding to increase to critical
levels. It also determines how effectively selection pressures can be used to
mitigate the effects of inbreeding. When the inbreeding rate is low the homo-
zygosity level increases slowly, therefore, allowing time for selection to
lower the frequency of undesirable genotypes through natural mortality and
the discard of individuals that fail to meet performance criteria. On the other
hand, when inbreeding rates are high and homozygosity levels increase rapid-
ly, selection would not be less effective because of the expected higher
egg and fry mortality rates and general reduction in performance
throughout the population. One study that applied this principal to rainbow
trout inbred for three generations and simultaneously selected for rapid
growth rate found that the effects of inbreeding depression and selection
essentially neutralized each other (Anderson and Woods, 1979).
The number of fish used each generation to maintain a brood stock by
the random mating method should be the largest number of breeding adults
possible with multiple spawns taken throughout the spawning season. This
ideal is often limited, however, by the practical considerations of available
brood stock, spawning facilities, manpower, etc. The minimum number of
breeding adults for maintaining a random mating brood stock should be at
least 50 pairs or 50 of the least numerous sex with spawning adults taken
throughout the spawning period. A breeding population of 50 adult pairs
yields an expected rate of inbreeding increase of 0.5% (Table I) per genera-
tion provided that mating adults are randomly paired and that each mating
pair contributes equal numbers of progeny to the succeeding generation of
brood stock. To the extent these assumptions are not fully achieved because
of the range in time of individual maturity throughout the spawning season,
differential egg hatchability between families, and differential survival of
families to the adult stage, the actual rate of inbreeding will be somewhat
higher than predicted. The recommended minimum number of 50 pairs to
maintain a random mating population considers the inability of most aqua-
226
culture brood stocks to be truly random mating and the need to keep the
rate of inbreeding accumulation at a low level. Brood stock managers who
want to apply selection practices on the population can easily do so provid-
ing steps are taken to insure that selected fish represent many different
families.
The second approach - systematic line crossing schemes - serves to elimi-
nate the mating of full sibs and therefore, effectively to reduce the rate of
inbreeding accumulation below that found in random mating populations of
equal population size at least during the early generations (Kimura and
Crow, 1963; Robinson and Bray, 1965). The rotational line crossing system
proposed by Kincaid (1977) is one mating system that uses this approach.
Selection programs can be incorporated into most systematic line crossing
schemes.
These two approaches, large random mating populations and systematic
line crossing reduce the rate of inbreeding accumulation but do not prevent
further inbreeding. All breeding systems applied to a closed population will
experience some inbreeding over a period of time. If the population is large,
however, inbreeding will occur slowly and the normal effects of selection
may prevent a serious build-up. The practice of introducing an unrelated
brood stock to cross with an existing inbred brood stock to produce a new
hybrid brood stock serves to break up the gene combinations (homozy-
gosity) that cause inbreeding depression. However, if that hybrid brood
stock is maintained as a closed random mating population after the initial
hybridiation, then, inbreeding will again begin to accumulate. For this rea-
son, when a different strain is introduced and hybridized with the existing
brood stock, the breeder should consider incorporating a breeding scheme
to minimize future inbreeding. A second problem area with strain introduc-
tions is that the new strain should be carefully chosen to complement the
present strain and to avoid the introduction of undesirable characteristics.
The breeding approach and specific breeding program adopted by the breed-
er will depend on many different factors including the rate of inbreeding and
the planned use of the brood stock for present and future production.
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