Genetic parameters of fillet fatty acids

and fat deposition in gilthead seabream

( Sparus aurata ) using the novel 30 k
Medfish SNP array S.S. Horn
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 Aquaculture 556 (2022) 738292

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Genetic parameters of fillet fatty acids and fat deposition in gilthead

seabream (Sparus aurata) using the novel 30 k Medfish SNP array
S.S. Horn a, *, M.L. Aslam a, G.F. Difford a, K. Tsakoniti b, S. Karapanagiotis b, B. Gulzari c,
J.W.M. Bastiaansen c, C. Peñaloza d, R. Houston d, B. Ruyter a, A.K. Sonesson a
a
Nofima, Norwegian Institute for Food, Fisheries and Aquaculture Research, NO-1433 Ås, Norway
b
Galaxidi Marine Farm S.A., Galaxidi 330 52, Greece
c
Wageningen University & Research, Animal Breeding and Genomics, PO Box 338, 6700 AH Wageningen, the Netherlands
d
The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian EH25 9RG, United Kingdom

A R T I C L E I N F O A B S T R A C T

Keywords: Lipid-related traits are important candidates for a breeding goal for gilthead seabream, because they affect both
Omega-3 fish and human health, as well as production efficiency. However, to date there have been very few estimates of
Gilthead seabream genetic parameters for these traits, and the genetic relationship between fatty acids and other important traits
Lipid metabolism
have never been reported for gilthead seabream. Therefore, the aim of this study was to estimate genomic
Lipid deposition
Genetic parameters
heritability and genetic relationships of fat deposition traits and individual muscle fatty acids in a commercial
population of gilthead seabream using the novel ~30 k MedFish SNP array.
In total 967 gilthead seabream fed with a commercial feed were genotyped with the MedFish SNP chip which
included ~30 K informative markers for this species. On average, the fish weighed 372 g. The mean content of
eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) was 822 mg per 100 g fillet. The heritability of
muscle fat, viscera weight and percentage viscera were in the range of 0.34–0.46. The genetic correlation of body
weight with muscle fat was 0.12, indicating that genetic variation in muscle fat is largely independent of the
weight of the fish. The heritability of the product of endogenous fatty acid synthesis (n = 240), palmitoleic acid
(16:1n-7), was high (0.43). The estimated heritability of EPA (%) and DHA (%) was 0.39 and 0.33, respectively.
Both EPA and DHA had low, non-significant genetic correlations with body weight, and DHA had a negative
genetic correlation with muscle fat (− 0.53).
It is possible to increase EPA and DHA content in gilthead seabream fillets by selective breeding. The high
heritability of 16:1n-7, a marker of de novo lipogenesis, suggests that there is a strong genetic component to this
metabolic pathway in gilthead seabream. Muscle fat deposition and body weight seem to be independent traits,
and selective breeding for faster growth is not likely to influence the proportional content of EPA and DHA.

1. Introduction Lipids are important in production of all farmed animals because

Gilthead seabream is one of the major aquaculture species in Europe,
ranked as number four, with a total production of almost 200,000 tons in
2019 (FEAP, 2020), but is at an early stage regarding selective breeding.
The first commercial breeding programs of seabream were initiated in
the early 2000's and have primarily focussed on improving growth rate
(Brown, 2004; Janssen et al., 2017; Thorland et al., 2007).
they are linked to production efficiency, health, and product quality.
Excess dietary lipids that are not deposited in the edible muscle or used
as energy source for growth are considered a loss and an indicator of low
efficiency. Constant energy excess may lead to excessive fat deposition
in and around internal organs and thereby increase the risk of metabolic
disorders, oxidative stress and inflammation (Fontana et al., 2007;
Todorčević et al., 2010).

* Corresponding author.
E-mail addresses: siri.storteig.horn@nofima.no (S.S. Horn), Luqman.Aslam@Nofima.no (M.L. Aslam), gareth.difford@nofima.no (G.F. Difford), k.tsakoniti@
galaxidimarine.farm (K. Tsakoniti), s.karapanagiotis@galaxidimarine.farm (S. Karapanagiotis), benan.gulzari@wur.nl (B. Gulzari), john.bastiaansen@wur.nl
(J.W.M. Bastiaansen), Carolina.Penaloza@roslin.ed.ac.uk (C. Peñaloza), ross.houston@roslin.ed.ac.uk (R. Houston), Bente.Ruyter@Nofima.no (B. Ruyter), anna.
sonesson@nofima.no (A.K. Sonesson).

https://doi.org/10.1016/j.aquaculture.2022.738292
Received 22 December 2021; Received in revised form 22 April 2022; Accepted 23 April 2022
Available online 27 April 2022
0044-8486/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
S.S. Horn et al.
The lipid content of muscle tissue affects organoleptic and processing
qualities of fish fillets. In seabream, it has been shown that the taste,
colour, and juiciness of fillets are affected by the fat content (Grigorakis
et al., 2003; Grigorakis, 2007). Muscle fat also affects the nutritional
quality of fillets. Fish is an important source of the essential omega-3
long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic
acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in human
diets. EPA and DHA have several beneficial health effects, such as pre­
venting and attenuating a range of inflammatory disorders, including
cardiovascular disease, immune dysfunction, and obesity (Calder, 2015;
Thota et al., 2018; Todorcevic and Hodson, 2015). Hence, improving the
omega-3 content of fish fillet is a point of focus in aquaculture. As a
significant part of the Mediterranean diet, seabream is an important
source of these essential and health-promoting fatty acids.
In general, omega-3 fatty acid content of fish muscle is largely
determined by the dietary fatty acid composition, which is well docu­
mented also for gilthead seabream (Ballester-Lozano et al., 2011; Gri­
gorakis et al., 2002; Izquierdo et al., 2003; Mnari et al., 2007). In
addition to feed, other factors including environmental factors (e.g.
water temperature) have also been shown to influence the omega-3 fatty
acid content of seabream muscle (Ibarz et al., 2005). Moreover, meta­
bolic factors are likely to play a role, as higher DHA level in muscle
compared to the feed have been observed in gilthead seabream
(Izquierdo et al., 2003). Omega-3 content in fish fillets can also be
regulated genetically: a recent study in gilthead seabream showed that
there is a heritable component to variation in muscle content of EPA and
DHA in gilthead seabream (Vallecillos et al., 2021). Dong et al. (2016)
estimated a heritability of 0.44 for the omega-3 levels of fillets of the
marine fish species large yellow croaker. Studies in Atlantic salmon have
revealed medium to high heritability for the omega-3 levels of fillets and
showed that genetic variation is possibly linked to omega-3 bioconver­
sion capacity (Horn et al., 2018; Leaver et al., 2011). Although marine
species have very limited capacities for omega-3 bioconversion (con­
version of the shorter chain alpha-linolenic acid (ALA) to the longer
chain EPA and DHA), some species like gilthead seabream and European
seabass do express several of the required genes. Fatty acid elongase
genes, as well as Delta-6 desaturase have been described, and are up-
regulated by dietary factors in the same manner as in salmonids (Sei­
liez et al., 2003; Tocher and Ghioni, 1999). Still, in gilthead seabream,
the omega-3 fatty acid bioconversion capacity is shown to be limited due
to a deficiency in Delta5 desaturase activity (Tocher and Ghioni, 1999).
There are, however, other metabolic factors that can influence the
omega-3 fatty acid composition in fish, such as fatty acid uptake,
deposition, and beta-oxidation (Henderson and Sargent, 1985; Horn
et al., 2019; Vegusdal et al., 2004). Although knowledge is limited in
gilthead seabream, studies have shown a preferential retention of
arachidonic acid (20:4 n-6) and DHA over EPA (Izquierdo, 1996; Kalo­
geropoulos et al., 1992; Koven et al., 2001).
The first publicly available combined-species SNP array for European
seabass and gilthead seabream, the new Axiom® MedFish SNP array,
was recently developed (Peñaloza et al., 2021). This array enables the
accurate high-throughput genotyping of ~ 30 K SNPs distributed
throughout the seabream genome. The availability of genotype data on
individuals allows the computation of genomic relationship matrix
(GRM) where the coefficient of relationships between relatives are
described more precisely with possibility of covering the deviations in
relatedness caused by Mendelian sampling (Houston et al., 2020; Niel­
sen et al., 2009). Although heritability can be underestimated or over­
estimated in influential regions with high or low linkage disequilibrium
(LD), GRM reflects more precise relatedness among individuals than the
pedigree-based relationships (Da et al., 2014). The genetic variance
components estimated using GRM are therefore possibly more precise
than pedigree-based estimates, especially when the available pedigree is
not very deep.
A key factor in the rate of genetic improvement possible for any given
trait in a breeding programme is its heritability, and its genetic
2
Aquaculture 556 (2022) 738292
correlations with other key traits in the breeding goal. Only a few studies
have reported genetic parameters of fat deposition traits in gilthead
seabream (Elalfy et al., 2021; García-Celdrán et al., 2015b; Navarro
et al., 2009b), but never using high-throughput genotyping. One study
has reported heritability of muscle fatty acids (Vallecillos et al., 2021),
but the genetic correlations between fatty acids and other important
traits have never been reported for gilthead seabream.
The aim of the current study was to estimate genomic heritability
and genetic relationships of fat deposition traits and individual muscle
fatty acids in gilthead seabream using the novel ~30 k MedFish SNP
array.
2. Material and methods
Gilthead seabream originating from the selective breeding program
of Galaxidi Marine Farm SA (Galaxidi, Greece) were used in this
experiment. All fish used in this study were the offspring of 33 males and
20 females mass spawned in a single broodstock tank over one day. The
fish were stocked in a sea cage at an average weight of 2.6 g. During the
sea phase, the fish were fed commercial feed and kept in commercial sea
cages in Galaxidi, Greece. They were slaughtered in September 2019,
after 15 months at sea and fasted prior to slaughter. From the average
body weight of ≈165 g to 270 g, the fish were fed a diet with a protein/
lipid content of 45/20, that only contained fish oil. From six to eight
months, the fish were fed a second experimental diet of a 45/16 protein/
lipid content, that included some sunflower oil. During the last two-
week phase of the trial before slaughter, the fish returned to the first
diet. Due to the logistics of sampling, slaughtering took place over
several days, therefore the number of days fasted prior to slaughter
varied from 4 to 12, which is considered in the statistical analysis of the
data.
At the time of slaughter, body weight, viscera weight, and muscle fat
measured by using Distell meter were recorded on 959 fish. Total viscera
weight was measured directly after slaughter by removing and weighing
the entire viscera, including liver and heart. The liver and heart were
also weighed separately, so for the analyses in the current study, the
viscera weight was calculated by subtracting heart weight and liver
weight from total viscera weight. Visceral fat was not measured directly,
but viscera percentage of body weight (viscera weight/body
weight*100) was used as an indicator of amount of visceral fat in the
current study (Viscera %).
2.1. Lipid measurements
Two methods of measuring muscle fat percentage were applied on
the fish studied, the non-invasive Distell fat meter (Distell (Model-FM
692, www.distell.com), and the gold standard laboratory Folch method
(MFATF). Directly after slaughter, a non-invasive fat measurement was
made on each of the 959 fish using the Distell fat meter, a handheld
microwave dielectric spectrometer. Two measurements were recorded
above the lateral line and two below, repeated on both sides of the fish.
The average of these eight measurements was used as a measure of the
total muscle fat percentage (MFATD). A calibration equation for muscle
fat in sea bream using the internal “custom calibration” setting was
generated as per the manufacturer's specifications (Distell, 2011). The
resulting calibration equation on 20 fish had a coefficient of determi­
nation of R2 = 0.79. The average of the eight measurements per fish after
use of the custom calibration was used as measure of the total muscle fat
percentage (MFATD).
The whole skinless fillet of 240 of the 959 fish were individually
homogenized, and total lipids were extracted using the Folch extraction
method (Folch et al., 1957) to record total muscle fat percentage
(MFATF). The fatty acid composition of the total lipids was determined
using the Mason & Waller method by means of methyl ester gas chro­
matography separation and flame ionization detection (Mason and
Waller, 1964). This gave fatty acid composition estimates for both
S.S. Horn et al.
proportional (% of total fatty acids) and quantitative (mg per g fillet,
based on internal standard) content of each fatty acid. For the data
analysis, outliers and error datapoints were removed through the
Interquartile Range (IQR) method. The results of the current study will
focus on the five most prevalent fatty acids of the muscle (18:1n-9, 16:0,
18:2n-6, 22:6n-3 (DHA) and 16:1n-7), as well as the omega-3 fatty acids
20:5n-3 (EPA) and 18:3n-3 (ALA).
2.2. DNA extraction and genotyping
DNA was extracted from fin clips by IdentiGEN (Dublin, Ireland),
using a crude DNA isolation method consisting of PK/Chelex extraction
followed by a dilution step. The fish were genotyped using the MedFish
array, which contains approximately 29,800 validated SNPs for gilthead
seabream (Peñaloza et al., 2021).
Genotype data were filtered using Plink software (Purcell et al.,
2007), excluding SNPs with minor allele frequency (MAF) lower than
2%, missing call rates exceeding 10%, and variants which had Hardy-
Weinberg equilibrium exact test p-value below 1e-15. In total 25,919
SNPs passed filters and quality control.
2.3. Statistical analysis
The genomic relationship matrix was generated with the GCTA
software, following the method by Yang et al. (2011), using the
following equation to estimate the genetic relationship between in­
dividuals j and k:
( )
1 ∑N xij − 2pi (xik − 2pi )
Gjk = ,
N i=1 2pi (1 − pi )
where xij is the number of copies of the reference allele for the ith SNP of
the jth individual, xik is the number of copies of the reference allele for
the ith SNP of the kth individual, and pi is the frequency of the reference
allele, estimated from the observed allele frequencies.
Variance and covariance components were estimated by residual
maximum likelihood procedures using the CGTA program. Bivariate
analyses were performed to estimate genetic correlations between traits,
using the following bivariate animal model (Henderson, 1984):
Y = XB + U + E
where Y is a matrix of phenotypic records for individuals i = 1, 2, …, n
and traits j = 1, 2, X is a matrix of the fixed effects of animal i on trait j, B
is a matrix of fxed efect solutions. U is a matrix containing the random
effects of animal i on trait j, with variance with variance G0⊗ G, where G
is the genomic relationship matrix between individuals and G0 is a ge­
netic variance–covariance matrix among traits. E is a matrix of residual
effects, that is assumed to have a variance of
[ ]
σ2e1 σ e1,e2
R = , where 1 and 2 indicate traits.
σ e1,e2 σ 2e2
Univariate analyses were performed to estimate heritability for all
traits. For the univariate analyses, matrices Y, B, U and E were reduced
to vectors and matrices G and R were reduced to scalars. Heritability
(narrow sense) was estimated as the ratio of additive genetic variance to
total phenotypic variance. Slaughter day (representing number of days
fasted) was included as a fixed effect for all traits.
Genetic coefficient of variation was determined based on the method
suggested by Burton and Devane (1953) as follows:
√̅̅̅̅̅
σ2 g
GCV = *100,
μ
Where μ is the mean of the trait, and σ 2g is the genetic variance.
As parent fish were not genotyped, it was not possible to perform
parentage assignment and develop pedigree, therefore, principal
component analysis (PCA) was performed to evaluate population
3
Aquaculture 556 (2022) 738292
structure and get an idea on number of clusters possibly representing full
and/or half-sib families. The PCA based cluster analysis showed that the
population is quite homogenous (little to no cluster differentiation),
with relatively low variation explained by first two PCAs, 4.9% and
3.8% variation explained by PCA1 and PCA2, respectively (Fig. 1). The
cluster analysis showed ~42 clusters with number of individuals
ranging from 2 to 53 individuals per cluster with a mean cluster size of
23 individuals.
3. Results and discussion
The body size and muscle fat level were within the normal range for
commercially produced harvest sized gilthead seabream, with a mean
body weight of 372 g, ranging from 124 to 546 g (Table 1). The coef­
ficient of variation for all production traits surpassed 0.15, showing that
there was substantial individual variation in these traits.
3.1. Genetic parameters of lipid related production traits
The heritability of muscle fat, viscera weight and viscera % were all
above 0.3, showing that there is a substantial genetic component to
these traits (see Table 2). Viscera % had an especially high heritability
estimate of 0.46, which was almost equal to the estimated heritability of
viscera weight with body weight included as covariate in the statistical
model (0.45 ± 0.06, result not shown). This result is in line with the
previously reported heritability of 0.5 for visceral fat in gilthead seab­
ream by Navarro et al. (2009b), although García-Celdrán et al. (2015a)
reported a heritability of 0.2 for visceral fat with body weight as a co­
variate in the model. In the current study, the observed variation in
viscera weight was assumed to mainly be due to the size of the fish and
the amount of lipids stored here; hence the viscera weight expressed as
% of body weight was used as an indicator of amount of visceral fat. The
genetic correlation between body weight and viscera weight was 0.72,
supporting that there is some genetic variation in viscera weight that is
independent of the weight of the fish, which could be variation in dis­
tribution of lipid deposition.
Previously reported heritability of muscle fat ranges from 0.05 to
0.31 in gilthead seabream (Elalfy et al., 2021; García-Celdrán et al.,
2015a; Navarro et al., 2009b; Vallecillos et al., 2021). In the current
study, both methods of recording muscle fat showed heritability slightly
higher than previous estimates: MFATD had a heritability of 0.46, and
MFATF of 0.34. The considerably smaller dataset is reflected in the
larger standard error for MFATF (Table 2). The differences in heritability
estimates between studies could be partly related to the accuracy of the
recording technique. Moreover, there are differences in the statistical
models used. García-Celdrán et al. (2015a) and Vallecillos et al. (2021)
included body weight as a covariate. In the current study, body weight
was not included as a covariate in the statistical model, but when tested,
body weight as covariate did not have a significant effect on heritability
estimates of the muscle fat traits (results not shown). This is due to that
the genetic correlation between body weight and muscle fat was very
low (rg = 0.12 for MFATD and rg = 0.13 for MFATF), which was in
accordance with the low phenotypic correlations observed (Fig. 2 &
Table 3). A low genetic correlation between body weight and muscle fat
is in agreement with the estimate reported by Navarro et al. (2009a) (rg
= 0.12). However, estimates reported by García-Celdrán et al. (2015b)
and Vallecillos et al. (2021) are higher (0.29 and 0.59, respectively).
This could explain the differences in statistical models and heritability
estimates. Furthermore, these differences in estimates of heritability and
genetic correlations may be due to genetic differences among the studied
populations, as well as genotype-by-environment interactions (Gulzari
et al., 2022; Kause et al., 2002).
The low genetic correlation between body weight and muscle fat
observed in the current study of gilthead seabream is contrary to find­
ings of salmonid species such as Atlantic salmon where muscle fat per­
centage is highly genetically correlated with body weight, i.e. rg =
S.S. Horn et al. Aquaculture 556 (2022) 738292

Fig. 1. PCA plot of the population structure.

Table 1
Descriptive statistics for production traits.
N Mean (SD) Min Max CV (%)

Body weight (g) 967 372 (64) 124 546 17

Viscera weight (g) 911 22.9 (6.0) 5.4 39.6 26
Viscera % 911 6.1 (1.1) 3.3 9.8 18
MFATD (%) 960 7.6 (1.54) 2.8 11.9 18
MFATF (%)* 238 9.4 (0.1) 3.9 14.8 20

SD = standard deviation. CV = coefficient of variation. Viscera % = (viscera

weight/body weight), MFATD = muscle fat % by Distell meter, MFATF = muscle
fat % by Folch chemical analysis.

Table 2
Heritability (h2) of production traits.
Trait N Va Ve h2 GCV %

Body weight 967 1435 (271) 2842 (187) 0.34 (0.05) 10

Viscera weight 911 14.87 (2.73) 23.31 (1.7) 0.39 (0.06) 17
Viscera % 911 0.58 (0.09) 0.67 (0.05) 0.46 (0.05) 12
MFATD 960 1.03 (0.16) 1.21 (0.09) 0.46 (0.05) 13
MFATF 238 1.13 (0.46) 2.2 (0.38) 0.34 (0.12) 11

Standard errors in brackets. Va: Additive genetic variance. Ve: Residual vari­
ance. GCV = Genetic coefficient of variation. Viscera % = (viscera weight/body
weight), MFATD = muscle fat % by Distell meter, MFATF = muscle fat % by Folch
chemical analysis. Fig. 2. Scatter plot of body weight against muscle fat, including the regression
line and correlation coefficient, n = 238.
0.45–0.84 (Powell et al., 2008; Tsai et al., 2015; Vieira et al., 2007).
There are similar findings in the salmonids Rainbow trout (rg =
0.30–0.52) (Blay et al., 2021) and European white fish (rg = 0.62–0.64) Table 3
Genetic (upper triangle) and phenotypic (bottom triangle) correlations.
(Janhunen et al., 2017). This difference is likely explained by the
inherent differences in the mechanisms of lipid deposition between the Body Viscera Viscera % MFATD MFATF
fish species. For example, in Atlantic salmon, the muscle is a main lipid weight weight

storage site, while in gilthead seabream very little fat is stored in muscle, Body 0.72 (0.06) 0.25 0.12 0.13
and the viscera is the main lipid storage site (McClelland et al., 1995). weight (0.12) (0.12) (0.20)*
Viscera 0.74 0.86 0.20 0.19
These differences in lipid storage are likely due to differences in
weight (0.03) (0.12) (0.20)*
behavioural adaptation to their natural habitats; seabream is a bottom- Viscera % 0.22 0.81 0.23 0.11
dwelling sedentary fish species, while Atlantic salmon is an active sur­ (0.11) (0.20)*
face feeder species, and needs energy available in their locomotory MFATD 0.28 0.24 0.18 0.82
muscles (McClelland et al., 1995). (0.09)*
MFATF 0.15** 0.21 0.18 0.60
There was a low genetic correlation between the two fat deposition
traits MFATD and Viscera % (rg = 0.23). This agrees with Navarro et al. Standard errors in brackets.
*
(2009a), who found an even lower correlation of 0.05, and used a direct Only converged with no covariates in the statistical model.
**
measure of visceral fat - manual removing and weighing of visceral fat p > 0.01.

4
S.S. Horn et al. Aquaculture 556 (2022) 738292

deposits. This shows that deposition of fat in these two lipid deposits with body weight (Table 6). However, DHA (but not EPA) was signifi­
(muscle and viscera) are genetically different traits, similar to what has cantly correlated with muscle fat, which is in agreement with Vallecillos
been described in Rainbow trout (Kause et al., 2007). et al. (2021). Thus, including muscle fat as a covariate in the model
The genetic correlation between the two methods of measuring would likely reduce the heritability estimate for DHA in this data
muscle fat was high; 0.82 (Table 3), but lower than reported by a recent material.
study on European seabass, where the genetic correlation between the One limitation of this study is the lack of pedigree, which may have
same two methods on the same trait was close to unity at 0.96 ± 0.03 biased the estimation of genetic parameters, as population stratification
(Difford et al., 2021). Pearson's correlation coefficient for the two can inflate estimates of genetic relatedness (Dandine-Roulland et al.,
methods was 0.6, showing that the Distell meter was not highly accurate 2016). However, the evaluation of population structure by principal
for phenotypic recording of muscle fat percentage in this dataset. component analysis showed that the population is quite homogenous
with little to no cluster differentiation (Fig. 1). Also, previous studies
3.2. Muscle fatty acid composition have shown that genomic information can more accurately reflect the
relationships between individuals than pedigree information (Da et al.,
The fatty acid composition of muscle was recorded as both propor­ 2014; Wang and Da, 2014). The increased accuracy of genomic pre­
tional content (% of total fatty acids) and quantitative content (mg/g diction compared with pedigree prediction is evident in a range of
tissue) on 240 fish (Table 4). The phenotypic variation in proportional aquaculture species (reviewed in Houston et al. (2020)).
content was low, while the variation in quantitative content was higher, The high heritability for both EPA and DHA estimated in the current
reflecting the variation in total muscle fat content. The major fatty acids study demonstrate that selective breeding is a promising tool for
in the muscle, constituting more than 60% of muscle fatty acids, were increasing muscle content of these essential nutrients in the fillet of this
oleic acid (18:1n-9), palmitic acid (16:0), and linoleic acid (18:2n-6). species. This agrees with studies in Atlantic salmon that have shown
Oleic and linoleic acid are the major fatty acids from plant oils in fish significant heritability of EPA and DHA content of muscle (Leaver et al.,
feed, while palmitic acid is the most common saturated fatty acid found 2011; Horn et al., 2018). Wang et al. (2019) also showed that there is a
in all animals. The mean proportional content of EPA and DHA in the genetic component to omega-3 content of Asian seabass fillets. Herita­
gilthead seabream muscle was 2.43% and 6.95% of total lipids, bility estimates of DHA in tilapia and common carp have been close to
respectively. The mean quantitative content of EPA and DHA was 2.14 zero, which could be due to the very low quantities of DHA detected
and 6.08 mg/g, respectively, i.e. 822 mg per 100 g fillet in total. (Nguyen et al., 2010; Prchal et al., 2018). The estimates for both fish
Considering the recommendations on daily intake of omega-3 FAs for species indicated that EPA had a considerably higher heritability than
the general human public of 250–500 mg EPA + DHA (Cunnane, 2004; DHA, while in Atlantic salmon, the heritability of EPA was lower than
GOED, 2014), farmed seabream is a good source of the essential nutri­ DHA (Horn et al., 2018).
ents EPA and DHA. Alpha-linolenic acid (18:3n-3; ALA) had the lowest heritability of all
fatty acids (0.03). The ratio of DHA/ALA was included in the analysis as
an indicator-trait for the conversion of ALA to DHA (omega-3 biocon­
3.3. Heritability and genetic correlations of fatty acids in gilthead
version), but is in this case likely reflecting variation in DHA due to the
seabream fillets
very low variation in ALA. This low variation, together with the low
general content of ALA in the muscle of gilthead seabream, indicates
The estimated heritability of the proportional content of both EPA
that omega-3 bioconversion of ALA to DHA is not substantial, and not
and DHA was relatively high, at 0.39 and 0.33, respectively (Table 5).
very important for the overall DHA content in the muscle of this species.
These estimates are substantially higher than those recently reported by
This implies that the phenotypic and genetic variation observed in EPA
Vallecillos et al. (2021), at 0.05 for EPA and 0.11 for DHA. This differ­
and DHA levels are linked to other metabolic factors, such as deposition
ence is likely due to the different statistical models used, as Vallecillos
or beta-oxidation, rather than omega-3 bioconversion. This is in accor­
et al. (2021) used a Bayesian model, which is quite conservative, and
dance with a study reporting that the omega-3 bioconversion capacity is
included body weight as a covariate in the model, which could remove
limited in gilthead seabream due to a deficiency in Delta5 desaturase
some of the genetic variation in the fatty acid traits. In the current study,
activity, catalyzing conversion of 20:4n-3 to EPA (Tocher and Ghioni,
we chose not to include body weight as a covariate for fatty acids, as
1999).
both % EPA and % DHA had low and non-significant genetic correlations
The estimates of genetic correlations showed that the proportional
content of each fatty acid was differently correlated with muscle fat
Table 4 (Table 6). The standard errors on the estimates were large due to the
Descriptive statistics of proportional (% of total fatty acids) and quantitative
small dataset on fatty acids, but the genetic correlations agreed with the
(mg/g tissue) content of the top five most prevalent fatty acids in muscle, and the
phenotypic correlations. The fatty acids with the strongest positive
omega-3 fatty acids EPA (20:5n-3) and ALA (18:3n-3).
correlations with muscle fat were 18:1n-9 and 16:1n-7 (Table 6), both of
Fatty Proportional content (%) Quantitative content (mg/g) which are products of de novo lipogenesis, synthesis of fatty acids,
acid
Mean Min Max CV Mean Min Max CV although 18:1n-9 is also found in the feed. This indicates that a genetic
(SD) (%) (SD) (%) predisposition for higher muscle fat is due to a higher inherent de novo
18:1 n-9 29.0 27.4 30.6 2 25.6 12.5 39.3 20 lipogenesis activity. The high heritability of % 16:1n-7, which is almost
(0.05) (0.33) exclusively a product of de novo lipogenesis, also indicates that the rate
16:0 15.7 14.4 16.9 3 13.8 7.1 21.2 19 of de novo lipogenesis in gilthead seabream muscle is a heritable trait
(0.03) (0.17)
18:2 n-6 15.6 13.9 17.3 4 13.8 7.1 21 19
(Table 5).
(0.04) (0.17) The genetic correlations indicated that the observed genetic varia­
22:6 n-3 7.0 6.1 7.9 6 6.1 3.5 8.5 16 tion in % EPA and % DHA is independent of body weight, as both % EPA
(DHA) (0.03) (0.06) and % DHA had low and non-significant genetic correlations with body
16:1 n-7 5.1 4.4 5.9 6 4.5 2.1 7 22
weight (Table 6). The proportional content of EPA had a weak positive
(0.02) (0.06)
20:5 n-3 2.4 2 2.9 7 2.1 1.1 3.2 20 (non-significant) correlation with muscle fat, while DHA had a negative
(EPA) (0.01) (0.03) genetic correlation. This difference between EPA and DHA likely reflects
18:3 n-3 2.4 2.2 2.6 3 2.1 1.1 3.2 20 the metabolic differences between the two fatty acids, and their roles in
(ALA) (0.01) (0.03) muscle tissue (Calder, 2006; Stillwell and Wassall, 2003). The observed
N = 240. CV = coefficient of variation. SD = standard deviation. relationship between DHA and muscle fat is seen in several species and

5
S.S. Horn et al. Aquaculture 556 (2022) 738292

Table 5
Heritability (h2) of fatty acid traits.
Trait % mg/g
2
Va Ve h GCV (%) Va Ve h2 GCV (%)

16:0 0.06 (0.03) 0.16 (0.02) 0.28 (0.11) 1.57 2.17 (0.84) 4.21 (0.70) 0.33 (0.11) 10.67
16:1n-7 0.04 (0.01) 0.05 (0.01) 0.43 (0.12) 3.75 0.34 (0.12) 0.56 (0.10) 0.38 (0.12) 12.88
18:1n-9 0.16 (0.07) 0.32 (0.06) 0.34 (0.12) 1.40 9.25 (3.33) 15.65 (2.68) 0.36 (0.11) 11.88
18:2n-6 0.13 (0.06) 0.32 (0.05) 0.28 (0.12) 2.27 2.07 (0.78) 4.27 (0.66) 0.33 (0.11) 10.44
ALA 0.00 (0.00) 0.01 (0.00) 0.03 (0.08) 0. 59 0.06 (0.02) 0.10 (0.02) 0.35 (0.11) 11.40
EPA 0.01 (0.00) 0.02 (0.00) 0.39 (0.11) 4.28 0.07 (0.02) 0.12 (0.02) 0.37 (0.12) 12.56
DHA 0.05 (0.02) 0.10 (0.02) 0.33 (0.12) 3.14 0.32 (0.13) 0.61 (0.10) 0.35 (0.12) 9.21
DHA/ALA 0.01 (0.00) 0.03 (0.00) 0.25 (0.11) 3.40 – – – –

% = proportional content of fatty acids, mg/g = quantitative content of fatty acids, Va: Additive genetic variance. Ve: Residual variance. Standard errors in brackets.
GCV = genetic coefficient of variation. N = 240.

correlations indicated that increasing muscle fat would actually reduce

Table 6
the proportional content of DHA in the muscle, leading to a less desir­
Phenotypic (rP) and genetic (rG) correlations between production traits and fatty
able fatty acid profile of the seabream fillet (Table 6). On the other hand,
acids.
the genetic correlations between proportional contents of muscle fatty
Trait rP rG (SE)
acids showed a potential to select for an overall healthier fatty acid
BW * % EPA 0.24 0.17 (0.18) profile in muscle (Table 6). DHA was positively correlated with EPA (rg
BW * % DHA 0.08** − 0.03 (0.20) = 0.55), and negatively correlated with the main saturated fatty acid
MFATF * % EPA − 0.05** 0.11 (0.25)
(16:0) and the main omega-6 fatty acid (18:2n-6) in muscle (rg = − 0.48
MFATF * % DHA − 0.56 − 0.53 (0.20)
MFATF * % 18:1n-9 0.5 0.81 (0.17) and − 0.37, respectively). Thus, according to these results, selection for
MFATF * % 16:1n-7 0.4 0.51 (0.20) increased proportional content of DHA will result in a fish fillet with a
MFATF * 18:2n-6 − 0.07** − 0.20 (0.29) higher proportion of marine omega-3 fatty acids, and lower proportion
MFATF * 16:0 0** − 0.15 (0.30)
of saturated fatty acids and pro-inflammatory omega-6 fatty acids.
DHA * EPA 0.42 0.55 (0.19)
DHA * 18:2n-6 − 0.22 − 0.37 (0.29)
However, a lower selection response may be expected using this strategy
DHA * 16:0 − 0.23 − 0.48 (0.25) as the genetic coefficients of variation was lower for proportional
DHA * 16:1n-7 − 0.04** 0.05 (0.26) compared to quantitative content of EPA and DHA (Table 5).
DHA * 18:3n-3 − 0.22 nc Even though both quantitative and proportional content of EPA and
DHA * 18:1n-9 − 0.63 nc
DHA are highly heritable, getting genetic response in fatty acid traits is
MFATF = Muscle fat % by Folch chemical analysis, BW = body weight. SE = not straight forward. With low phenotypic and genetic coefficients of
standard error. EPA = 20:5n-3. DHA = 22:6n-3. Nc = no convergence. ** = p > variation it is difficult to get a reasonable selection response, even when
0.01. heritability is high. We recommend future research into economic
weights and selection indices which are outside the scope of this paper.
tissues and can be explained by DHA being a PUFA with important roles Another challenge with implementing selection for fatty acid traits is
in cell membranes that is therefore found in phospholipids (PL) to a that phenotyping requires costly and time-consuming chemical ana­
larger degree than in triglycerides (TG). When muscle fat increases, the lyses, which make large-scale data recording challenging. New rapid
proportion of PL relative to TG decreases, and therefore the % DHA methods for predicting fatty acid composition in fish fillet are being
decreases (Tocher, 2003). developed, such as Raman and near infrared spectroscopy (e.g. EWOS
SalmoNIR technology from Cargill) (Afseth et al., 2006; Bekhit et al.,
3.4. Selection strategies for increased omega-3 fatty acid content of fillets 2014; Blay et al., 2021), which can make practical implementation of
selection for these traits more feasible.
The heritability of EPA and DHA indicated that the content of
healthy omega-3 fatty acids in seabream fillets can be increased through 4. Conclusions
selective breeding, and there are two possible strategies to achieve this:
by increasing the quantitative content (mg per g muscle), or by In the gilthead seabream, muscle fat deposition and body weight
increasing the proportional content (% of total muscle fatty acids). seem to be independent traits, and selective breeding for faster growth is
As muscle fat percentage increases, so does the quantitative content not likely to influence the proportional content of EPA and DHA. The
of all fatty acids. Therefore, the heritability of the quantitative content of fatty acid 16:1n-7, a marker of de novo lipogenesis, had a high herita­
all fatty acids mirrored the heritability of muscle fat and were all close to bility (0.43), indicating that there is a strong genetic component to this
0.34 (Table 5). This was confirmed in the estimates of genetic correla­ metabolic pathway in seabream.
tions, where the quantitative content of all fatty acids had a genetic It is possible to increase EPA and DHA content in gilthead seabream
correlation to muscle fat close to unity (results not shown). This implies fillets by selective breeding, as the estimated heritability of EPA (%) and
that an increase in the quantitative content of EPA and DHA can be DHA (%) was 0.39 and 0.33, respectively, and there was a positive ge­
achieved by selective breeding for increased muscle fat content, which netic correlation between the two fatty acids (0.55). There is a potential
could be achieved by e.g. the Distell fat meter for non-invasive pheno­ to select for a healthier overall fatty acid profile in muscle of seabream
typic recording. It should then be considered that increased muscle fat by selection for increased proportional content of DHA, as DHA was
would influence the organoleptic qualities of the fish (Grigorakis, 2007). negatively correlated with the major omega-6 fatty acid (18:2n-6) and
It should also be considered that selective breeding for increased muscle the major saturated fatty acid (16:0).
fat content would result in an increase in all fatty acids, including the
less desirable omega-6 fatty acids. This is critical from a nutritional CRediT authorship contribution statement
health perspective, as it is not only the amount of EPA and DHA that is
important, but also the ratio of omega-3 to omega-6 fatty acids (Saini S.S. Horn: Methodology, Writing – original draft, Visualization,
and Keum, 2018; Simopoulos, 2002). In addition, the genetic Writing – review & editing, Formal analysis. M.L. Aslam: Methodology,

6
S.S. Horn et al.
Writing – review & editing, Formal analysis. G.F. Difford: Methodology,
Writing – review & editing, Formal analysis. K. Tsakoniti: Resources,
Conceptualization, Data curation. S. Karapanagiotis: Resources, Data
curation, Writing – review & editing. B. Gulzari: Writing – review &
editing. J.W.M. Bastiaansen: Writing – review & editing. C. Peñaloza:
Resources, Writing – review & editing. R. Houston: Resources, Writing
– review & editing. B. Ruyter: Data curation, Conceptualization,
Writing – review & editing. A.K. Sonesson: Funding acquisition, Project
administration, Supervision, Conceptualization, Methodology, Writing –
review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This study was made possible by the EU project MedAID (H2020
grant agreement No 727315). Authors would like to thank Galaxidi
Marine Farm S.A. for their participation in the study and its staff for
providing necessary help and facilities for raising the population and the
experimental data collection. Authors are grateful to Målfrid Tofteberg
Bjerke and Anne Marie Langseter for skilful technical assistance.
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Another random document with
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The Project Gutenberg eBook of Ozymandias
This ebook is for the use of anyone anywhere in the United States
and most other parts of the world at no cost and with almost no
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under the terms of the Project Gutenberg License included with this
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you are located before using this eBook.

Title: Ozymandias

Author: Ivar Jorgensen

Illustrator: Dan Adkins

Release date: November 9, 2023 [eBook #72080]

Language: English

Original publication: New York, NY: Royal Publications, Inc, 1958

Credits: Greg Weeks, Mary Meehan and the Online Distributed

Proofreading Team at http://www.pgdp.net

*** START OF THE PROJECT GUTENBERG EBOOK

OZYMANDIAS ***
 OZYMANDIAS

By IVAR JORGENSON

Illustrated by DAN ADKINS

There was open strife between the military

and scientific staffs. But which was mightier?

[Transcriber's Note: This etext was produced from

Infinity November 1958.
Extensive research did not uncover any evidence that
the U.S. copyright on this publication was renewed.]
The planet had been dead about a million years. That was our first
impression, as our ship orbited down to its sere brown surface, and
as it happened our first impression turned out to be right. There had
been a civilization here once—but Earth had swung around Sol ten-
to-the-sixth times since the last living being of this world had drawn
breath.
"A dead planet," Colonel Mattern exclaimed bitterly. "Nothing here
that's of any use. We might as well pack up and move on."
It was hardly surprising that Mattern would feel that way. In urging a
quick departure and an immediate removal to some world of greater
utilitarian value, Mattern was, after all, only serving the best interests
of his employers. His employers were the General Staff of the Armed
Forces of the United States of America. They expected Mattern and
his half of the crew to produce results, and by way of results they
meant new weapons and sources of strategic materials. They hadn't
tossed in 70% of the budget for this trip just to sponsor a lot of
archaeological putterings.
But luckily for our half of the outfit—the archaeological putterers' half
—Mattern did not have an absolute voice in the affairs of the outfit.
Perhaps the General Staff had kicked in for 70% of our budget, but
the cautious men of the military's Public Liaison branch had seen to
it that we had at least some rights.
Dr. Leopold, head of the non-military segment of the expedition, said
brusquely, "Sorry, Mattern, but I'll have to apply the limiting clause
here."
Mattern started to sputter. "But—"
"But nothing, Mattern. We're here. We've spent a good chunk of
American cash in getting here. I insist that we spend the minimum
time allotted for scientific research, as long as we are here."
Mattern scowled, looking down at the table, supporting his chin in his
thumbs and digging the rest of his fingers in hard back of his
jawbone. He was annoyed, but he was smart enough to know he
didn't have much of a case to make against Leopold.
The rest of us—four archaeologists and seven military men; they
outnumbered us a trifle—watched eagerly as our superiors battled.
My eyes strayed through the porthole and I looked at the dry
windblown plain, marked here and there with the stumps of what
might have been massive monuments millennia ago.
Mattern said bleakly, "The world is of utterly no strategic
consequence. Why, it's so old that even the vestiges of civilization
have turned to dust!"
"Nevertheless, I reserve the right granted to me to explore any world
we land on, for a period of at least one hundred sixty-eight hours,"
Leopold returned implacably.
Exasperated, Mattern burst out, "Dammit, why? Just to spite me?
Just to prove the innate intellectual superiority of the scientist to the
man of war?"
"Mattern, I'm not injecting personalities into this."
"I'd like to know what you are doing, then? Here we are on a world
that's obviously useless to me and probably just as useless to you.
Yet you stick me on a technicality and force me to waste a week
here. Why, if not out of spite?"
"We've made only the most superficial reconnaissance so far,"
Leopold said. "For all we know this place may be the answer to
many questions of galactic history. It may even be a treasure-trove of
superbombs, for all—"
"Pretty damned likely!" Mattern exploded. He glared around the
conference room, fixing each of the scientific members of the
committee with a baleful stare. He was making it quite clear that he
was trapped into a wasteful expense of time by our foggy-eyed
desire for Knowledge.
Useless knowledge. Not good hard practical knowledge of the kind
he valued.
"All right," he said finally. "I've protested and I've lost, Leopold. You're
within your rights in insisting on remaining here one week. But you'd
damned well better be ready to blast off when your time's up!"

It had been foregone all along, of course. The charter of our

expedition was explicit on the matter. We had been sent out to comb
a stretch of worlds near the Galactic Rim that had already been
brushed over hastily by a survey mission.
The surveyors had been looking simply for signs of life, and, finding
none (of course), they had moved on. We were entrusted with the
task of investigating in detail. Some of the planets in the group had
been inhabited once, the surveyors had reported. None bore present
life. None of the planets we had ever visited had been found to hold
intelligent life, though many had in the past.
Our job was to comb through the assigned worlds with diligence.
Leopold, leading our group, had the task of doing pure
archaeological research on the dead civilizations; Mattern and his
men had the more immediately practical job of looking for fissionable
material, leftover alien weapons, possible sources of lithium or tritium
for fusion, and other such militarily useful things. You might argue
that in a strictly pragmatic sense our segment of the group was just
dead weight, carted along for the ride at great expense, and you
would be right.
But the public temper over the last few hundred years in America
has frowned on purely military expeditions. And so, as a sop to the
nation's conscience, five archaeologists of little empirical
consequence so far as national security mattered were tacked onto
the expedition.
Us.
Mattern made it quite clear at the outset that his boys were the
Really Important members of the expedition, and that we were
simply ballast. In a way, we had to agree. Tension was mounting
once again on our sadly disunited planet; there was no telling when
the Other Hemisphere would rouse from its quiescence of a hundred
years and decide to plunge once more into space. If anything of
military value lay out here, we knew we had to find it before They
did.
The good old armaments race. Hi-ho! The old space stories used to
talk about expeditions from Earth. Well, we were from Earth,
abstractly speaking—but in actuality we were from America, period.
Global unity was as much of a pipedream as it had been three
hundred years earlier, in the remote and primitive chemical-rocket
era of space travel. Amen. End of sermon. We got to work.

The planet had no name, and we didn't give it one; a special

commission of what was laughably termed the United Nations
Organization was working on the problem of assigning names to the
hundreds of worlds of the galaxy, using the old idea of borrowing
from ancient Terran mythologies in analogy to the Mercury-Venus-
Mars nomenclature of our own system.
Probably they would end up saddling this world with something like
Thoth or Bel-Marduk or perhaps Avalokitesvara. We knew it simply
as Planet Four of the system belonging to a yellow-white F5 IV
Procyonoid sun, Revised HD Catalog #170861.
It was roughly Earthtype, with a diameter of 6100 miles, a gravity
index of .93, a mean temperature of 45 degrees F. with a daily
fluctuation range of about ten degrees, and a thin, nasty atmosphere
composed mostly of carbon dioxide with wisps of helium and
nitrogen and the barest smidgeon of oxygen. Quite possibly the air
had been breathable by humanoid life a million years ago—but that
was a million years ago. We took good care to practice our
breathing-mask drills before we ventured out of the ship.
The sun, as noted, was an F5 IV and fairly hot, but Planet Four was
a hundred eighty-five million miles away from it at perihelion and a
good deal farther when it was at the other swing of its rather
eccentric orbit; the good old Keplerian ellipse took quite a bit of
punishment in this system. Planet Four reminded me in many ways
of Mars—except that Mars, of course, had never known intelligent
life of any kind, at least none that had troubled to leave a hint of its
existence, while this planet had obviously had a flourishing
civilization at a time when Pithecanthropus was Earth's noblest
being.

In any event, once we had thrashed out the matter of whether or not
we were going to stay here or pull up and head for the next planet on
our schedule, the five of us set to work. We knew we had only a
week—Mattern would never grant us an extension unless we came
up with something good enough to change his mind, which was
improbable—and we wanted to get as much done in that week as
possible. With the sky as full of worlds as it is, this planet might
never be visited by Earth scientists again.
Mattern and his men served notice right away that they were going
to help us, but reluctantly and minimally. We unlimbered the three
small halftracks carried aboard ship and got them into functioning
order. We stowed our gear—cameras, pick-&-shovels, camel's-hair
brushes—and donned our breathing-masks, and Mattern's men
helped us get the halftracks out of the ship and pointed in the right
direction.
Then they stood back and waited for us to shove off.
"Don't any of you plan to accompany us?" Leopold asked. The
halftracks each held up to four men.
Mattern shook his head. "You fellows go out by yourselves today and
let us know what you find. We can make better use of the time filing
and catching up on back log entries."
I saw Leopold start to scowl. Mattern was being openly
contemptuous; the least he could do was have his men make a
token search for fissionable or fusionable matter! But Leopold
swallowed down his anger.
"Okay," he said. "You do that. If we come across any raw veins of
plutonium I'll radio back."
"Sure," Mattern said. "Thanks for the favor. Let me know if you find a
brass mine, too." He laughed harshly. "Raw plutonium! I half believe
you're serious!"

We had worked out a rough sketch of the area, and we split up into
three units. Leopold, alone, headed straight due west, toward the dry
riverbed we had spotted from the air. He intended to check alluvial
deposits, I guess.
Marshall and Webster, sharing one halftrack, struck out to the hilly
country southeast of our landing point. A substantial city appeared to
be buried under the sand there. Gerhardt and I, in the other vehicle,
made off to the north, where we hoped to find remnants of yet
another city. It was a bleak, windy day; the endless sand that
covered this world mounted into little dunes before us, and the wind
picked up handfuls and tossed it against the plastite dome that
covered our truck. Underneath the steel cleats of our tractor-belt,
there was a steady crunch-crunch of metal coming down on sand
that hadn't been disturbed in millennia.
Neither of us spoke for a while. Then Gerhardt said, "I hope the
ship's still there when we get back to the base."
Frowning, I turned to look at him as I drove. Gerhardt had always
been an enigma: a small scrunchy guy with untidy brown hair
flapping in his eyes, eyes that were set a little too close together. He
had a degree from the University of Kansas and had put in some
time on their field staff with distinction, or so his references said.
I said, "What the hell do you mean?"
"I don't trust Mattern. He hates us."
"He doesn't. Mattern's no villain—just a fellow who wants to do his
job and go home. But what do you mean, the ship not being there?"
"He'll blast off without us. You see the way he sent us all out into the
desert, and kept his own men back. I tell you, he'll strand us here!"
I snorted. "Don't be a paranoid. Mattern won't do anything of the
sort."
"He thinks we're dead weight on the expedition," Gerhardt insisted.
"What better way to get rid of us?"
The halftrack breasted a hump in the desert. I kept wishing a vulture
would squeal somewhere, but there was not even that. Life had left
this world ages ago. I said, "Mattern doesn't have much use for us,
sure. But would he blast off and leave three perfectly good halftracks
behind? Would he?"
It was a good point. Gerhardt grunted agreement after a while.
Mattern would never toss equipment away, though he might not have
such scruples about five surplus archaeologists.
We rode along silently for a while longer. By now we had covered
twenty miles through this utterly barren land. As far as I could see,
we might just as well have stayed at the ship. At least there we had a
surface lie of building foundations.
But another ten miles and we came across our city. It seemed to be
of linear form, no more than half a mile wide and stretching out as far
as we could see—maybe six or seven hundred miles; if we had time,
we would check the dimensions from the air.
Of course it wasn't much of a city. The sand had pretty well covered
everything, but we could see foundations jutting up here and there,
weathered lumps of structural concrete and reinforced metal. We got
out and unpacked the power-shovel.
An hour later, we were sticky with sweat under our thin spacesuits
and we had succeeded in transferring a few thousand cubic yards of
soil from the ground to an area a dozen yards away. We had dug
one devil of a big hole in the ground.
And we had nothing.
Nothing. Not an artifact, not a skull, not a yellowed tooth. No spoons,
no knives, no baby-rattles.
Nothing.
The foundations of some of the buildings had endured, though
whittled down to stumps by a million years of sand and wind and
rain. But nothing else of this civilization had survived. Mattern, in his
scorn, had been right, I admitted ruefully: this planet was as useless
to us as it was to them. Weathered foundations could tell us little
except that there had once been a civilization here. An imaginative
paleontologist can reconstruct a dinosaur from a fragment of a thigh-
bone, can sketch out a presentable saurian with only a fossilized
ischium to guide him. But could we extrapolate a culture, a code of
laws, a technology, a philosophy, from bare weathered building
foundations?
Not very likely.
We moved on and dug somewhere else half a mile away, hoping at
least to unearth one tangible remnant of the civilization that had
been. But time had done its work; we were lucky to have the building
foundations. All else was gone.
"Boundless and bare, the lone and level sands stretch far away," I
muttered.
Gerhardt looked up from his digging. "Eh? What's that?" he
demanded.
"Shelley," I told him.
"Oh. Him."
He went back to digging.

Late in the afternoon we finally decided to call it quits and head back
to the base. We had been in the field for seven hours, and had
nothing to show for it except a few hundred feet of tridim films of
building foundations.
The sun was beginning to set; Planet Four had a thirty-five hour day,
and it was coming to its end. The sky, always somber, was darkening
now. There was no moon to be still as bright. Planet Four had no
satellites. It seemed a bit unfair; Three and Five of the system each
had four moons, while around the massive gas giant that was Eight a
cluster of thirteen moonlets whirled.
We wheeled round and headed back, taking an alternate route three
miles east of the one we had used on the way out, in case we might
spot something. It was a forlorn hope, though.
Six miles along our journey, the truck radio came to life. The dry,
testy voice of Dr. Leopold reached us:
"Calling Trucks Two and Three. Two and Three, do you read me?
Come in, Two and Three."
Gerhardt was driving. I reached across his knee to key in the
response channel and said, "Anderson and Gerhardt in Number
Three, sir. We read you."
A moment later, somewhat more faintly, came the sound of Number
Two keying into the threeway channel, and I heard Marshall saying,
"Marshall and Webster in Two, Dr. Leopold. Is something wrong?"
"I've found something," Leopold said.
From the way Marshall exclaimed "Really!" I knew that Truck
Number Two had had no better luck than we. I said, "That makes
one of us, then."
"You've had no luck, Anderson?"
"Not a scrap. Not a potsherd."
"How about you, Marshall?"
"Check. Scattered signs of a city, but nothing of archaeological
value, sir."
I heard Leopold chuckle before he said, "Well, I've found something.
It's a little too heavy for me to manage by myself. I want both outfits
to come out here and take a look at it."
"What is it, sir?" Marshall and I asked simultaneously, in just about
the same words.
But Leopold was fond of playing the Man of Mystery. He said, "You'll
see when you get here. Take down my coordinates and get a move
on. I want to be back at the base by nightfall."

Shrugging, we changed course to head for Leopold's location. He

was about seventeen miles southwest of us, it seemed. Marshall and
Webster had an equally long trip to make; they were sharply
southeast of Leopold's position.
The sky was fairly dark when we arrived at what Leopold had
computed as his coordinates. The headlamps of the halftrack lit up
the desert for nearly a mile, and at first there was no sign of anyone
or anything. Then I spotted Leopold's halftrack parked off to the east,
and from the south Gerhardt saw the lights of the third truck rolling
toward us.
We reached Leopold at about the same time. He was not alone.
There was an—object—with him.
"Greetings, gentlemen." He had a smug grin on his whiskery face. "I
seem to have made a find."
He stepped back and, as if drawing an imaginary curtain, let us take
a peek at his find. I frowned in surprise and puzzlement. Standing in
the sand behind Leopold's halftrack was something that looked very
much like a robot.
It was tall, seven feet or more, and vaguely humanoid: that is, it had
arms extending from its shoulders, a head on those shoulders, and
legs. The head was furnished with receptor plates where eyes, ears,
and mouth would be on humans. There were no other openings. The
robot's body was massive and squarish, with sloping shoulders, and
its dark metal skin was pitted and corroded as by the workings of the
elements over uncountable centuries.
It was buried up to its knees in sand. Leopold, still grinning smugly
(and understandably proud of his find) said, "Say something to us,
robot."
From the mouth-receptors came a clanking sound, the gnashing of—
what? gears?—and a voice came forth, oddly high-pitched but
audible. The words were alien and were spoken in a slippery
singsong kind of inflection. I felt a chill go quivering down my back.
The Age of Space Exploration was three centuries old—and for the
first time human ears were hearing the sounds of a language that
had not been spawned on Earth.
"It understands what you say?" Gerhardt questioned.
"I don't think so," Leopold said. "Not yet, anyway. But when I address
it directly, it starts spouting. I think it's a kind of—well, guide to the
ruins, so to speak. Built by the ancients to provide information to
passersby; only it seems to have survived the ancients and their
monuments as well."
I studied the thing. It did look incredibly old—and sturdy; it was so
massively solid that it might indeed have outlasted every other
vestige of civilization on this planet. It had stopped talking, now, and
was simply staring ahead. Suddenly it wheeled ponderously on its
base, swung an arm up to take in the landscape nearby, and started
speaking again.
I could almost put the words in its mouth: "—and over here we have
the ruins of the Parthenon, chief temple of Athena on the Acropolis.
Completed in the year 438 B.C., it was partially destroyed by an
explosion in 1687 while in use as a powder magazine by the Turks
—"
"It does seem to be a sort of a guide," Webster remarked. "I get the
definite feeling that we're being given an historical narration now, all
about the wondrous monuments that must have been on this site
once."
"If only we could understand what it's saying!" Marshall exclaimed.
"We can try to decipher the language somehow," Leopold said.
"Anyway, it's a magnificent find, isn't it? And—"
I began to laugh suddenly. Leopold, offended, glared at me and said,
"May I ask what's so funny, Dr. Anderson?"
"Ozymandias!" I said, when I had subsided a bit. "It's a natural!
Ozymandias!"
"I'm afraid I don't—"
"Listen to him," I said. "It's as if he was built and put here for those
who follow after, to explain to us the glories of the race that built the
cities. Only the cities are gone, and the robot is still here! Doesn't he
seem to be saying, 'Look on my works, ye Mighty, and despair!'"
"Nothing besides remains," Webster quoted. "It's apt. Builders and
cities all gone, but the poor robot doesn't know it, and delivers his
spiel nonetheless. Yes. We ought to call him Ozymandias!"
Gerhardt said, "What shall we do with it?"
"You say you couldn't budge it?" Webster asked Leopold.
"It weighs five or six hundred pounds. It can move of its own volition,
but I couldn't move it myself."
"Maybe the five of us—" Webster suggested.
"No," Leopold said. An odd smile crossed his face. "We will leave it
here."
"What?"
"Only temporarily," he added. "We'll save it—as a sort of surprise for
Mattern. We'll spring it on him the final day, letting him think all along
that this planet was worthless. He can rib us all he wants—but when
it's time to go, we'll produce our prize!"
"You think it's safe to leave it out here?" Gerhardt asked.
"Nobody's going to steal it," Marshall said.
"And it won't melt in the rain," Webster added.
"But—suppose it walks away?" Gerhardt demanded. "It can do that,
can it not?"
Leopold said, "Of course. But where would it go? It will remain where
it is, I think. If it moves, we can always trace it with the radar. Back to
the base, now; it grows late."
We climbed back into our halftracks. The robot, silent once again,
planted knee-deep in the sand, outlined against the darkening sky,
swivelled to face us and lifted one thick arm in a kind of salute.
"Remember," Leopold warned us as we left. "Not one word about
this to Mattern!"

At the base that night, Colonel Mattern and his seven aides were
remarkably curious about our day's activities. They tried to make it
seem as if they were taking a sincere interest in our work, but it was
perfectly obvious to us that they were simply goading us into telling
them what they had anticipated—that we had found absolutely
nothing. This was the response they got, since Leopold forbade
mentioning Ozymandias. Aside from the robot, the truth was that we
had found nothing, and when they learned of this they smiled
knowingly, as if saying that had we listened to them in the first place
we would all be back on Earth seven days earlier, with no loss.
The following morning after breakfast Mattern announced that he
was sending out a squad to look for fusionable materials, unless we
objected.
"We'll only need one of the halftracks," he said. "That leaves two for
you. You don't mind, do you?"
"We can get along with two," Leopold replied a little sourly. "Just so
you keep out of our territory."
"Which is?"
Instead of telling him, Leopold merely said, "We've adequately
examined the area to the southeast of here, and found nothing of
note. It won't matter to us if your geological equipment chews the
place up."
Mattern nodded, eyeing Leopold curiously as if the obvious
concealment of our place of operations had aroused suspicions. I
wondered whether it was wise to conceal information from Mattern.
Well, Leopold wanted to play his little game, I thought; and one way
to keep Mattern from seeing Ozymandias was not to tell him where
we would be working.
"I thought you said this planet was useless from your viewpoint,
Colonel," I remarked.
Mattern stared at me. "I'm sure of it. But it would be idiotic of me not
to have a look, wouldn't it—as long as we're spending the time here
anyway?"
I had to admit that he was right. "Do you expect to find anything,
though?"
He shrugged. "No fissionables, certainly. It's a safe bet that
everything radioactive on this planet has long since decomposed.
But there's always the possibility of lithium, you know."
"Or pure tritium," Leopold said acidly. Mattern merely laughed, and
made no reply.
Half an hour later we were bound westward again to the point where
we had left Ozymandias. Gerhardt, Webster and I rode together in
one halftrack, and Leopold and Marshall occupied the other. The
third, with two of Mattern's men and the prospecting equipment,
ventured off to the southeast toward the area Marshall and Webster
had fruitlessly combed the day before.
Ozymandias was where we had left him, with the sun coming up
behind him and glowing round his sides. I wondered how many
sunrises he had seen. Billions, perhaps.
We parked the halftracks not far from the robot and approached,
Webster filming him in the bright light of morning. A wind was
whistling down from the north, kicking up eddies in the sand.
"Ozymandias have remain here," the robot said as we drew near.
In English.
For a moment we didn't realize what had happened, but what
followed afterward was a five-man quadruple-take. While we gabbled