1. Introduction
Aquaculture is the fastest growing food production sector with great potential to supply high-quality food for the expanding world population [
1,
2]. One of the major challenges for the aquaculture industry is to secure feed ingredients of marine origin, fishmeal (FM), and fish oil (FO) that supply high quality protein and lipid [
3,
4,
5,
6]. In fact, capture fisheries—including that for production of FM and FO—remain steady over the last decade [
1]. Both FM and FO are ingredients with a high nutrient bioavailability and adequate nutritional composition, which fulfill essential amino acids and fatty acids requirements of fish species [
7,
8]. Alternatively, oils and meals obtained from terrestrial crops, such as plant based protein sourses (VM) and vegetable oils (VO), are currently used to replace FM and FO in fish feeds [
7,
8]. Complete substitution of FO by VO adversely affects fish immune system and stress and disease resistance [
3,
4,
5,
6]. In addition, it also reduces the muscle content of long-chain
n-3 polyunsaturated fatty acids (
n-3 LC-PUFA), such as EPA (20:5
n-3) and DHA (22:6
n-3) that negatively affect the nutritional value of farmed fish for humans [
4,
9,
10,
11,
12].
Most fish cannot synthesize
n-3 and
n-6 PUFA
de novo and they must be supplied in the diet. Generally, essential fatty acid (EFA) requirements of freshwater fish can be met by the supply of 18:3
n-3 and 18:2
n-6 fatty acids in their diets, whereas the EFA requirement of marine fish can only be met by supplying the LC-PUFA, EPA, and DHA [
13]. Unlike freshwater fish, marine fish either lack or show a low activity of Δ6-desaturase, and thus require the long chain PUFA’s, EPA, and DHA to meet their EFA requirement. The bioconversion of 18:3
n-3 to EPA and DHA involves desaturations at Δ-6 and Δ-5 positions in the carbon backbone and an intermediate 2-carbon chain elongation step. Synthesis of DHA from EPA requires elongation of EPA to 22:5
n-3 and 24:5
n-3 which is then converted to 24:5
n-3 and 24:6
n-3 by Δ6-desaturase and finally shortened to DHA by β-oxidation. Among enzymes involved in
n-3 LC-PUFA synthesis, Δ-6-desaturase enzyme (Fads2), encoded by
fads2 gene, is considered to be the rate-limiting step in the biosynthetic pathway of PUFA in gilthead sea bream [
14,
15]. LC-PUFA synthesis also involves chain elongation catalyzed by elongases (Elovl) with different substrate preferences [
16]. Among them Elovl6 is a key lipogenic enzyme that elongates long-chain saturated and monounsaturated fatty acids of 12, 14, and 16C, and has received much attention due to its link with certain metabolic disorders [
17]. LC-PUFA may have a direct effect on the expression of other genes related to lipid or carbohydrate metabolism [
18]. For instance, lipoprotein lipase (Lpl) facilitates the tissue uptake of circulating fatty acids [
19] from lipoproteins and
lpl gene expression in the liver can be regulated by
n-3 PUFAs [
20]. Also, the energy supplied by β-oxidation of free fatty acids is transported into the mitochondria in the form of fatty acyl-carnitine esters by carnitine acyltransferases such as carnitine palmitoyltransferases (Cpt) [
21]. Replacement of FO by VO changes the fatty acid composition of liver and muscle, affects the β-oxidation capacity as well as regulates the expression of
cptI and
cptII genes [
22,
23,
24,
25]. β-oxidation also takes place in the peroxisome, which is modulated by peroxisome proliferator activator receptors (Ppars). Three different ppar isoforms (𝛼,β,𝜸) have been characterised in gilthead sea bream,
ppar𝛼 being the major form expressed in the liver [
26]. Ppars are nuclear receptors that regulate differentiation, growth, and metabolism, and epigenetic mechanisms have been described to regulate these processes in mammals [
27]. For example, feeding pregnant rats a protein-restricted diet reduces methylation of the Ppar𝛼 promoter in the offspring, and hypomethylation persists into adulthood [
28]. Another gene considered to be potentially regulated by LC-PUFA is cyclooxygenase-2 (
cox2), a key enzyme in prostanoid biosynthesis [
29].
Recent studies conducted on broodstock diets of fish suggest that inclusion of VO as a major source of lipid may alter metabolic pathways in offspring of gilthead sea bream and improve the utilization of VM and VO by their offspring [
30,
31]. Therefore, higher levels up to 80% substitution of FO by VO in broodstock diets may upregulate
fads2 expression in offspring larvae and improve the utilization of low FM/FO diets at the juvenile stages of their life cycle [
30]. This higher utilization of low FM/FO diets was persistent even in the 16-month-old offspring and affected the expression of some key gene encoding enzymes related to lipid utilization and LC-PUFA biosynthesis, including
lpl,
cpt1,
elovl6 [
31]. More recently, it has also been demonstrated that broodstock fish showing high
fads2 expression levels in blood after one month feeding a low FO/high linseed oil (LO) diet had improved spawning performance, as well as growth of the offspring when nutritionally challenged with low FM/FO diets at the age of 6-months (Turkmen et al., submitted). However, increased dietary ALA, without a significant reduction in LC-PUFA, may lead to adverse effects on nutritional programming in offspring, resulting in lower growth as compared to offspring obtained from broodstock fed 100% FO. Such effects of ALA-rich broodstock diet or the selection of fish with a high
fads2 expression on offspring liver biochemical and fatty acid composition, or the potential molecular mechanisms involved, have not yet been studied.
Nutritional programming through parental diets has been well studied in humans, where early nutritional interventions during plastic developmental stages may alter the risk of cardiovascular diseases related to metabolic defects such as type 2 diabetes mellitus, hypertension, obesity, and osteoporosis [
32]. Moreover, LC-PUFA supplementation during early nutrition can lead to long-term effects on metabolism by affecting the epigenome through different epigenetic mechanisms, including DNA methylation [
33]. In murine models, maternal fat intake alters ARA (22:4
n-6) and DHA contents in the liver, which was related to the epigenetic regulation of Fads2 gene promoter and the expression of the gene [
34]. In fish, epigenetic studies are considered a relatively new area of research [
35]. An earlier study showed that there was a negative correlation between the methylation status of the putative promoter region of the
fads2 and
fads2 gene expression in Japanese sea bass (
Lateolabrax japonicus) [
36]. However, in the European sea bass (
Dicentrarchus labrax), the methylation level of several positions examined within the
fads2 promoter did not change after nutritional conditioning of larvae with high- or low-PUFA diets [
37]. When these changes do occur, and whether the transcription of the corresponding gene and metabolic processes may be altered, remains to be established [
33]. To our knowledge, there are no data regarding the epigenetic mechanisms involved in the nutritional programming effect of broodstock diets in gilthead sea bream. The recent publication of the whole genome for this species [
38] and another on-going genome project, such as those of IATS-CSIC-Nutrigroup (
http://nutrigroup-iats.org/seabreamdb/index.php), are opening new opportunities to understand potential epigenetic mechanisms.
The present study was designed to investigate the effects of parental fads2 expression levels, and broodstock feeding a diet rich in VO, on their juvenile offspring response to a low FM and low FO diet. In particular, the changes in liver fatty acid composition, expression of genes involved in lipid metabolism and LC-PUFA biosynthesis, and the methylation status in a region of the fads2 gene promoter were investigated.
2. Results
After one month of feeding the high VO and VM diet (
Table 1), there were significant differences in the
fads2 expression in broodstock fish, which was up to 23 times higher in females (
p = 0.01) and 13 times higher in males (
p > 0.04) than the lowest
fads2 expression of each sex (
Table 2). After dividing the broodstock fish by their
fads2 expression, the resultant high
fads2 expression group showed significantly higher expression than the low
fads2 group (
p < 0.05) (
Table 2). There was no link between fish weight and
fads2 expression of brood fish (
R = 0.0061,
p > 0.05).
The nutritional challenge of the juveniles with the very low amounts of FM and FO diet (5% FM and 3%FO) (
Table 3) resulted in higher SGR values for the offspring of broodstock origin with a higher
fads2 expression (F
HD and V
HD), than for those coming from parents with a lower expression (F
LD and V
LD) (
p < 0.001, two-way ANOVA,
Figure 1), particularly, when parents were fed diet F (
p < 0.05,
Figure 1). Besides, offspring from broodstock fed the V diet showed lower growth than offspring from parents fed the F diet (
p < 0.001, two-way ANOVA,
Figure 1). In offspring obtained from broodstock fed V diet (V
HD and V
LD), those from broodstock with lower
fads2 expression (V
LD) showed significantly higher HSI (
p < 0.05,
Table 4). In addition, offspring from broodstock with lower
fads2 expression (F
LD and V
LD), and fish fed diet V (V
LD) had higher HSI (
p < 0.05,
Table 4). Thus, HSI was increased in offspring of parents with lower
fads2 expression (
p < 0.05, two-way ANOVA), particularly when broodstock were fed diet V, showing an interaction between broodstock
fads2 expression and broodstock diets (
p < 0.05,
Table 4).
Crude protein, crude lipid and ash contents of liver were not significantly different among the experimental groups (
p > 0.05,
Table 4), despite a 6–13% increase in hepatic lipid content of juveniles from broodstock fish with lower
fads2 expression. However, offspring from broodstock with lower
fads2 expression (F
LD and V
LD) showed significantly higher monounsaturated fatty acids 16:1
n-5 and 20:1
n-5, as well as some medium chain PUFAs such as 16:3
n-4, 18:2
n-4 and, particularly, 18:2
n-6 (substrate for Fads2), or the LC-PUFAs 20:2
n-9, 20:3
n-6, 20:5
n-3, 22:5
n-3, or 22:6
n-3 (
p < 0.05,
Table 5).
The comparison of juveniles from broodstock fed the diet V (VHD and VLD) showed that in VLD juveniles, products of Fads2 activity such as 20:2n-9 (p < 0.05) and 20:3n-6 (p < 0.1) were reduced, or did not change, such as 18:2n-9, 18:3n-6, 18:4n-3, or 20:4n-3 (p > 0.05), and Fads2 substrates such as 20:1n-9, 20:2n-6, 18:1n-9, 18:2n-6, 18:3n-3, or 20:3n-3 were also not significantly affected.
A reduction in a substrate for Elovl6, 16:0 (
p = 0.06) was observed in F
LD and V
LD fish, but 18:1 product was similar among the experimental groups (
p > 0.05) (
Table 5). Broodstock diet had no significant (
p > 0.05) effect on fatty acid profiles of the liver tissue of the offspring juveniles (
Table 5), except for a trend towards lower values of saturated fatty acids F
LD and V
LD (
Table 5,
p > 0.05). No interaction between broodstock diet and
fads2 expression was observed on fatty acid profiles, except for the content in a minor fatty acid, 16:0ISO (
p < 0.05,
Table 5).
In terms of the expression of selected genes, juveniles from parents with low
fads2 expression (F
LD and V
LD) showed a significantly (two-way ANOVA,
p < 0.001,
Figure 2) higher expression of
elovl6, particularly when broodstock were fed with diet F. Thus,
elovl6 expression was approximately 2 times higher in F
LD group in comparison with F
HD (
p < 0.05) (
Figure 2), whereas between juveniles from parents fed V diet, the expression of this gene was only 1.3 times higher in V
LD fish than in V
HD group (
p < 0.05) (
Figure 2).
Feeding broodstock with the diet V significantly (two-way ANOVA,
p < 0.01,
Figure 2) reduced
elovl6 expression in the offspring juveniles, particularly in those from broodstock with lower
fads2 expression. Thus, juveniles from lower
fads2 expression broodstock showed a significantly lower expression in
elovl6 when their parents were fed diet V (V
LD juveniles) as compared to the parents were fed diet F (F
LD juveniles,
p < 0.05) (
Figure 2). A significant interaction between broodstock
fads2 expression and broodstock diet on the expression of
elovl6 in their juveniles was observed (two-way ANOVA,
p < 0.01,
Figure 2). The expression of
cpt1 followed a similar trend, although the downregulation effect of broodstock diet V had lower significance (two-way ANOVA,
p < 0.05,
Figure 2). Thus, juveniles from parents with low
fads2 expression (F
LD and V
LD) showed a significantly (two-way ANOVA,
p < 0.001,
Figure 2) higher expression of
cpt1 than those from parents with higher expression (F
HD and V
HD). Despite selection of broodstock for low
fads2 expression and feeding diet V that resulted in downregulation of the expression of
fads2 and
cox2 in juveniles, there were no significant differences among juveniles in the expression of these two genes (
p > 0.05,
Figure 2). Neither there were differences in
lpl or in
ppara gene expressions (
p > 0.05,
Figure 2).
In general, a low level of cytosine methylation was found for the studied fragment of
fads2 promoter. Methylation level was always <10% for individual CpG positions and <4% for the average of all CpG positions examined (
Figure 3). However, few differences were observed in individuals with CpGs; methylation at positions CpG2 and CpG3 in offspring juveniles from broodstock fed V diet was significantly higher when broodstock had a low
fads2 expression (V
LD)(
p < 0.05) (
Figure 3a). Although not statistically significant, the same trend was observed for all other positions analysed and consequently, the average value for V
LD juveniles was higher than for V
HD juveniles (
p < 0.05) (
Figure 3b). Two-way ANOVA of methylation data for CpG2 and CpG3 revealed that in any case the parents’ diet had a significant effect (CpG2:
f = 0.0006,
p = 0.981; CpG3:
f = 3.30,
p = 0.099). The level of
fads2 gene expression of parents (i.e., selection) had no effect on the methylation level at CpG2 (
f = 4.34,
p = 0.058) while it had a significant effect at CpG3 (
f = 14.13,
p = 0.004). For both CpG positions, a significant interaction between parents’ selection and nutrition was found for methylation level (CpG2:
f = 10.11,
p = 0.007; CpG3:
f = 5.75,
p = 0.037).
3. Discussion
Prior to one month of spawning of broodstock fish fed a high VM and VO diet showed a wide variation in
fads2 expression in peripheral blood cells. Some females showed 23 times higher
fads2 expression than the lowest values found in other females. Whereas males had the highest
fads2 expression values, up to 13 times higher than the lowest values for each sex. This variation, and the higher expression in female individuals could be related to a potential higher requirement for DHA in females than in males, since this fatty acid plays an essential role in embryonic development [
40]. Similar to this finding, female mammals maintain higher levels of DHA in liver and plasma phospholipids because of their ability to synthesise higher levels of DHA than male counterparts [
41]. Moreover, studies in mouse have showed a very high correlation between reproductive hormones, such as progesterone and estradiol, and FADS2 expression as well as DHA concentration [
42]. To date, the knowledge of the relationship between reproductive hormones and
fads2 gene expression in fish is scarce. Further studies are needed to clarify this relationship in gilthead sea bream.
After one month of feeding the low FM and low FO diet (5% FM and 3% FO), juvenile offspring obtained from broodstock selected with high
fads2 expression showed better growth and lower liver 18:2
n-6 concentration, a substrate for Fads2, than juveniles from broodstock with low
fads2 expression. Interestingly, juveniles obtained from broodstock with high
fads2 expression showed the largest variation in
fads2 expression in liver, as well as the highest values. However, there were no significant differences among fish groups in
fads2 gene expression, probably due to large variations among individual fish. It appears that the combined genetic and nutritional effects of parents on offspring growth resulted in higher growth of fish obtained from high
fads2 expression and parents fed diet F. Growth was higher in high
fads2 than low
fads2 groups regardless of parental diet intake. Studies in humans showed that LC-PUFA metabolism in babies could be affected by maternal FADS2 genetic and epigenetic status [
43].
However, in the absence of a study on the heritability of
fads2 gene, it is difficult to interpret the reason for these individual differences. Ongoing studies with the known kinship of broodstock fish will reveal more information on the genetic variability and the parental effects of selection based on
fads2 gene expression. The putative epigenetic results are in agreement with previous studies on gilthead sea bream that showed the long-term effects of broodstock feeding diets with FO substitution by LO and a similar diet at the early stages of juvenile offspring. This may also explain part of the variation observed in
fads2 expression in the progeny originated from high
fads2 expression parents [
31]. Nevertheless, it should be taken into account that the substitution levels of FO by LO were not significantly different between earlier and current studies (60% vs. 70%). However, Fads2 products (ARA, EPA, and DHA) were higher in fish fed V diets in this study, which may have led to lower variation in the progeny obtained from V diet fed groups.
Broodstock showing low
fads2 expression associated
elovl6 expression in the offspring is in agreement with lower 16:0 in the liver of juveniles with respect to those from broodstock with high
fads2 expression. Elovl6 is a key rate-limiting enzyme in long-chain fatty acid elongation and is involved in elongation of 16:0 and 16:1 to 18:0 and 18:1 fatty acids. Moreover, it has been shown that it is the sole enzyme with the ability to elongate 16:0, as shown in ELOVL−/− mice [
44]. The content of 16:0 in liver decreased in fish obtained from low
fads2 groups (
p = 0.06) and 16:1
n-5 increased (
p < 0.04)(
Table 5), in line with higher
elovl6 expression. However, 18:1 fatty acid showed no clear trend in relation with the
elovl6 expression, and this could be related to the relatively low levels of 18:0 (3.53%) and the high levels of 18:1
n-9 (15.25%) fatty acids in the juvenile diets.
In juveniles from low
fads2 groups,
cpt1 was also upregulated as compared to those from broodstock with high
fads2 expression. It is weidely recognized that Cpt1 is a key enzyme for energy production through the β-oxidation of fatty acids, which are transported into the mitochondria in the form of fatty acyl-carnitine esters by carnitine acyltransferases in fish [
21]. Therefore, the upregulation of
cpt1 gene would imply an increase in liver β-oxidation, in agreement with a 6–13% reduction in hepatic lipid content of juveniles obtained from low
fads2 groups. A downregulation of both
elovl6 and
cpt1 in these groups were probably linked, since, Cpt1 gene expression in the liver of Elovl6 knock-out rats was also downregulated, leading to specific changes in the fatty acid ratios in liver [
17] similar to those observed in this study. In addition, in the liver of Elovl6 knock-out rats, changes in chain length of fatty acids (decrease in LC-PUFA higher than 18C) and the ratio of fatty acids (C18:0/C16:0, C16:1/C16:0) reduced sterol regulatory element-binding protein 1 (Srebp-1) and Ppara [
17]. PPARa also has an important role in energy metabolism, and its deficiency causes obesity in rats [
45]. Thus, modulation of
elovl6 expression may be partly responsible for the improved growth in offspring of broodstock selected for high
fads2. Moreover, when juveniles were obtained from broodstock fed the V diet, broodstock showing low
fads2 expression had the lowest growth and also increased HSI in comparison to juvenile from broodstock with high
fads2 expression. Besides, juveniles from broodstock with low
fads2 expression showed significantly increased methylation at CpG2 and CpG3 positions in the promotor region of
fads2. These results suggest a lower ability of juveniles to transcribe
fads2 in comparison to those from broodstock with high
fads2 expression and fed the V diet, and requires further investigation. Liver fatty acid profiles of juveniles obtained from broodstock with low
fads2 expression also showed that products from Fads2 activity such as 20:2
n-9 and 20:3
n-6 were significantly reduced and showed no change in 18:2
n-9, 18:3
n-6, 18:4
n-3, and 20:4
n-3 fatty acids as compared to juveniles from broodstock with high
fads2 expression and fed the V diet. Moreover, the substrates for Fads2 such as 20:1
n-9, 20:2
n-6, 18:1
n-9, 18:3
n-3, and 20:3
n-3 did not change, similar to 18:2
n-6. Both of these fatty acid profiles and
fads2 expression, suggest again a lower Fads2 activity in juveniles of the low
fads2 expression parent origin, in comparison to juveniles from broodstock with high
fads2 expression and fed the V diet. Nevertheless, as discussed above, the large variation in the
fads2 expression values limited finding specific changes among juveniles of different origins.
In recent years, fish epigenetics has been an emerging area of research interest from different scientific perspectives. However, studies on epigenetic mechanisms in most farmed fish are few, limited partly due to the limited molecular tools available for research and development. To the best of our knowledge, this is the first study describing the promoter methylation pattern of certain genes related to lipid metabolism (i.e.,
fads2) in gilthead sea bream. A previous study conducted on Japanese seabass showed that fish fed a
n-3 LC-PUFA rich diet showed higher methylation in the promoter of
fads2, while those fed VO showed around 4% lower methylation levels [
36]. The increased methylation of the promoter region of
fads2 led to lower gene expression of
fads2 in negative correlation to methylation levels. These findings agree with the higher methylation at certain positions of the
fads2 promoter in the liver, and the lower liver content of fatty acid products of Fads2 activity found in the progeny of broodstock fish selected for low
fads2 expression. However, in our study, the difference between the lowest (V
HD) and the highest (V
LD) of average methylation of 10 CpG positions only reached 1.5%, and this could contribute to the high variation in the
fads2 expression of the progeny obtained from high
fads2 expression broodstock that showed the lack of significant response of
fads2 expression. Studies in rats have showed differential expression of the Fads2 gene in negative correlation with its promoter methylation. However, the change in methylation pattern was higher (up to 15% change in the methylation on -623 region of the promoter) [
34] in comparison to this study. Therefore, broodstock selection for low
fads2 expression, together with VO broodstock fed diets, only caused epigenetic changes at the
fads2 gene promoter, in the form of relatively small differences in cytosine methylation at certain positions. Indeed, increased promoter methylation of the
fads2 gene in offspring originated from low
fads2 groups, induced a small but statistically significant difference in the methylation of certain CpG positions within the
fads2 promoter that did not affect
fads2 expression levels. This result may suggest that the positions CpG2 and CpG3 are more likely to change in methylation due to their sequence context or other related factors. Since the parents diet did not affect the offspring methylation at these positions whereas the
fads2 expression of parents had a significant effect as well as its interaction with parents diet, it is posible that the selection of parents in this study resulted in the segregation of epialleles (i.e., a specific DNA methylation pattern of a locus) with different susceptibility to change methylation at these positions. Whether the above-mentioned and other CpG positions are involved in the transcriptional regulation of
fads2 under certain nutritional regimes or in particular genotypes remains to be elucidated. Expression of
fads2 among groups was similar, and the higher concentrations of EPA and DHA in offspring from broodstock with lower
fads2 expression and fed VO could be related to the selective retention of these fatty acids in the liver, rather than to
n-6 or
n-3 LC-PUFA synthesis. Other data from the same individuals showed that ARA, EPA, and DHA contents in other tissues—such as muscle content—were not affected (Turkmen et al., submitted). Further studies are needed to fully understand the relationship between
fads2 gene promoter methylation and corresponding gene expression.
Feeding broodstock with diet V produced juveniles with reduced
elovl6 expression in the liver, particularly broodstock with a low
fads2 expression. These results were in agreement with the 15–21% reduction in hepatic lipid contents and a trend towards lower saturated fatty acid content. In addition, juveniles from broodstock fed the V diet also showed a reduction in
cpt1 expression. These results are in agreement with the previous study that showed downregulation of
cpt1 in the liver of progenies from broodstock fed increased contents of dietary VO [
31]. However a negative correlation was found between the level of VO inclusion in the broodstock diets and
cpt1 expression in the offspring juveniles when fed low FM and low FO diets in this report.
Likewise, feeding broodstock with the ALA rich diet (16.3% ALA of the total fatty acid profile, V diet) produced juveniles with a reduced growth when challenged with the low FM and FO diet. In summary, these results indicate a nutritional programming effect of FO replacement by ALA, when the products of Fads2 (ARA, EPA, and DHA) were similar in the eggs [LC-PUFA (% of total fatty acids), F diet: 20.29±2.70 and 18.44±4.02]. In previous studies, feeding gilthead sea bream broodstock a diet with 60% FO replacement by LO increased the ALA content in the eggs (13.08% of the total fatty acids) but reduced products of Δ-6-desaturase enzyme (ARA, EPA, and DHA). Modification of these fatty acids in the eggs generated a nutritional programming effect by producing juveniles that grew better when fed low FM and low FO diets. However, higher (80% and 100%) levels of FO replacement in broodstock diets negatively affected juveniles growth [
30,
31]. In these studies, 60% replacement of FO by VO also resulted in an increase in the amounts of LC-PUFA precursors such as LA and ALA up to 0.5 and 4.8 times in eggs, but it was accompanied by a decrease in products of
n-3 LC-PUFA biosynthesis such as EPA and DHA (0.3 and 0.2 times, respectively) [
30]. However, in the present study, due to the contribution of the
n-3 LC-PUFA fatty acids from the FM source used, the dietary levels of EPA and DHA changed to a lesser extent (0.01 and 0.06 times, respectively) between both diets used in the nutritional programming, while differences between LA and ALA were 0.03 and 2.6 times, respectively. In comparison to our previous studies, [
30,
31,
46], it appears that an increase in precursors, but not a reduction in products of
n-3 LC-PUFA synthesis, resulted in a programming effect as evidenced by a reduction in the juvenile growth when challenged with low FM/FO diets. There are few studies on the effects of the increase in the levels of LA and/or total PUFA intake on the inhibition of the PUFA metabolic pathway. It has been shown that high intake of 18C fatty acids and very low
n-3 LC-PUFA levels might inhibit
fads2 expression in gilthead sea bream [
15,
30]. Moreover, studies on Atlantic salmon have shown that
n-3 LC-PUFA and DHA, but not EPA, were responsible for the downregulation of
fads2 and elovl2-like elongase in liver [
47]. In addition, the ratio of ALA to LA is important because it determines the DHA accumulation in plasma phospholipids [
48]. If the ratio of LA to ALA is low (0.5–0.8), as in diet V of this study, increasing levels of ALA to 2% of dietary energy content increases plasma DHA phospholipid, however, DHA levels declined sharply when ALA was above 6% of total energy content [
48].