Parental LC-PUFA biosynthesis capacity and nutritional intervention with alpha-linolenic acid affect performance of Sparus aurata progeny

Serhat, Turkmen,; J., Zamorano, Maria; Hanlin, Xu,; Hipólito, Fernández-Palacios,; Lidia, Robaina,; Sadasivam, Kaushik,; Marisol, Izquierdo,

doi:10.1242/jeb.214999

Parental LC-PUFA biosynthesis capacity and nutritional intervention with alpha-linolenic acid affect performance of Sparus aurata progeny

Serhat, Turkmen,;J., Zamorano, Maria;Hanlin, Xu,;Hipólito, Fernández-Palacios,;Lidia, Robaina,;Sadasivam, Kaushik,;Marisol, Izquierdo, 2020-12-01 00:00:00 © 2020. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 RESEARCH ARTICLE Parental LC-PUFA biosynthesis capacity and nutritional intervention with alpha-linolenic acid affect performance of Sparus aurata progeny 1,2, 1 1 1 1 Serhat Turkmen *, Maria J. Zamorano , Hanlin Xu , Hipólito Fernández-Palacios , Lidia Robaina , 1 1 Sadasivam Kaushik and Marisol Izquierdo ABSTRACT 2019). Polyunsaturated fatty acids (PUFA) play important roles in programming the metabolism of different organisms (Lillycrop and Environmental factors such as nutritional interventions during early Burdge, 2018). Gilthead seabream (Sparus aurata), a marine fish, is developmental stages affect and establish long-term metabolic an interesting animal model to study the effects of alterations of changes in all animals. Diet during the spawning period has a PUFA during the early stages as the yolk sac composition of the nutritional programming effect in offspring of gilthead seabream and offspring depends on the continuous uptake of nutrients and can be affects long-term metabolism. Studies showed modulation of genes modified to some extent through the diets supplied during the such as fads2, which is considered to be a rate-limiting step in the spawning period (Fernández-Palacios et al., 1995). In recent years, synthesis of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). there has been increasing interest in the replacement (either partial or However, it is still unknown whether this adaptation is related to the complete) of fishmeal (FM) and fish oil (FO) with more readily presence of precursors or to limitations in the pre-formed products, n-3 available plant protein sources and vegetable oils (VO) in diets used LC-PUFA, contained in the diets used during nutritional programming. for aquaculture (Turchini et al., 2009; Vestergren et al., 2012). This study investigated the combined effects of nutritional However, high substitution levels may negatively affect a range of programming on Sparus aurata through broodstock diets during the different parameters such as growth or health (Izquierdo et al., 2005; spawning period and in broodfish showing higher or lower fads2 Montero and Izquierdo, 2010; Rosenlund et al., 2010; Torrecillas expression levels in the blood after 1 month of feeding with a diet et al., 2017a; Torrecillas et al., 2017b). While marine oils are rich containing high levels of plant protein sources and vegetable oils (VM/ sources of n-3 long-chain polyunsaturated fatty acids (LC-PUFA), VO). Broodfish showing high fads2 expression had a noticeable oils extracted from conventional terrestrial oil seeds – namely VO – improvement in spawning quality parameters as well as in the growth of rarely have ≥20 carbon fatty acids and totally lack essential 6 month old offspring when challenged with a high VM/VO diet. fatty acids, such as eicosapentaenoic acid (20:5n-3, EPA) and Further, nutritional conditioning with 18:3n-3-rich diets had an adverse docosahexaenoic acid (22:6n-3, DHA). Commonly used VOs can be effect in comparison to progeny obtained from fish fed high fish meal rich in precursors of LC-PUFA, such as alpha-linolenic acid (18:3n-3, and fish oil (FM/FO) diets, with a reduction in growth of juveniles. ALA) and linoleic acid (18:2n-6, LA), and certain oils such as linseed Improved growth of progeny from the high fads2 broodstock combined oil (LO) even have a very high content of ALA, which is the primer with similar muscle fatty acid profiles is also an excellent option for substrate for the n-3 LC-PUFA. In the LC-PUFA biosynthesis tailoring and increasing the flesh n-3 LC-PUFA levels to meet the pathway, there are several elongation, desaturation and β-oxidation recommended dietary allowances for human consumption. steps, and in different species of fish, there are differences in the KEY WORDS: Nutritional adaptation, Offspring nutrition, Parental evolution of the synthesis capacity (Castro et al., 2016; Monroig et al., nutrition, Long-chain polyunsaturated fatty acids, Fatty acid 2018). Therefore, maximizing the capacity of these pathways in a desaturase, fads2, Epigenetics, Aquaculture, Nutritional given species is of interest to improve the utilization of VO sources in programming fish, given the limited availability of marine fish oils. Recently, studies have looked into the possibility of channelling INTRODUCTION specific metabolic pathways of fish either through broodstock Environmental factors such as nutritional interventions during early nutrition or through early nutritional conditioning (Engrola et al., developmental stages exert long-lasting metabolic and physiological 2018; Izquierdo et al., 2015; Panserat et al., 2019; Turkmen et al., changes in mammals (Burdge and Lillycrop, 2010) and other 2017a; Turkmen et al., 2017b; Vagner et al., 2007). Nutritional vertebrates, including fish (Izquierdo et al., 2015; Turkmen et al., programming presumes that early environmental clues such as diet 2017a; Turkmen et al., 2019a; Turkmen et al., 2017b; Xu et al., or specific nutrients provide the organism with the capacity to forecast environmental challenges in later life and give it the 1 opportunity to adjust its metabolism to better adapt it to the new Aquaculture Research Group (GIA), IU-ECOAQUA, Universidad de Las Palmas de Gran Canaria, Crta. Taliarte s/n, 35214 Telde, Spain. Department of Biology, environment, potentially improving health, reproduction and University of Alabama at Birmingham, Birmingham, AL 35294, USA. survival (Burdge and Lillycrop, 2010). Hence, this tool may have applications in the animal production sector, as shown in ruminants, *Author for correspondence ([email protected]) to channel the individuals to better utilization of key nutrients S.T., 0000-0003-0410-9161; M.J.Z., 0000-0003-1569-9152; H.X., 0000-0003- (Gotoh, 2015). In rodents, dietary fatty acid composition during 4425-0776; H.F.-P., 0000-0003-1410-8154; L.R., 0000-0003-4857-6693; pregnancy and lactation influences growth and glucose metabolism S.K., 0000-0001-7856-8374; M.I., 0000-0003-4297-210X in the offspring (Siemelink et al., 2002). Likewise, maternal Received 19 September 2019; Accepted 9 October 2020 phytoestrogens can affect gene expression and alter skin colour as Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 well as susceptibility to obesity in adulthood (Dolinoy et al., 2006). this sense, blood fatty acid composition or fads2 expression could be Among marine teleosts, gilthead seabream is a multi-batch spawner used as an important trait. Although there are genetic selection whose eggs largely depend on the continuous intake of nutrients by programmes in gilthead seabream such as the public Spanish breeding the broodstock during the spawning season when egg composition programme PROGENSA (Afonso et al., 2012), and other can be modified – to some extent – by the diets used (Fernandez commercial breeding programmes (Fernandes et al., 2017; Thorland Palacios et al., 2011). Indeed, previous research in gilthead et al., 2015) which include selection pressure on growth traits and an seabream has established that feeds supplied during the spawning absence of body deformities (García-Celdrán et al., 2015; Lee– season play a critical role in regulating lipid metabolism even in the Montero et al., 2015), up to now there has been no information about offspring (Izquierdo et al., 2015; Turkmen et al., 2017b). For the use of fads2 expression as a biological biomarker in fish. instance, expression of genes related to the fatty acid desaturation The present study aimed to determine the influence of using and elongation pathways, such as fatty acyl desaturase 2 ( fads2) and broodstock with high fads2 expression in combination with fatty acid elongase 6 (elovl6), was affected, as well as expression of conditioning feeding with a high VO diet on spawning quality and other genes involved in lipid metabolism, such as carnitine growth of offspring during larval development and juvenile stage palmitoyltransferase I (cpt1) or lipoprotein lipase (lpl) (Izquierdo when challenged with very low levels of marine fisheries-derived et al., 2015). Some stress-related genes were also found to be ingredients. Several parameters related to egg and larval quality, regulated in the offspring obtained from broodstock fed diets with growth and fatty acid profiles were studied to ascertain a potential different VO levels during early larval stages (Turkmen et al., nutritional programming effect in the offspring. 2019a). Low FM FO feed utilization can also be improved by early dietary interventions in Atlantic salmon (Salmo salar) (Clarkson MATERIALS AND METHODS et al., 2017), through the up-regulation of different genes including All the experiments described below were conducted according to those involved in oxidative phosphorylation, pyruvate metabolism, the European Union Directive (2010/63/EU) on the protection of tricarboxylic acid cycle, glycolysis or fatty acid metabolism (Vera animals for scientific purposes, at the facilities of Ecoaqua Institute, et al., 2017). That nutritional interventions by different fatty acids University of Las Palmas de Gran Canaria (Canary Islands, Spain). during periods of high developmental plasticity may also regulate dietary lipid utilization in later stages in fish was also shown in Sampling of blood and identification of parental groups European seabass (Vagner et al., 2007). In the gilthead seabream, A total of 70 gilthead seabream, Sparus aurata Linnaeus 1758, our own previous studies showed that replacement of different levels broodstock fish (42 females and 28 males, aged 2–4 years) were of FO (rich in n-3 series fatty acids with 20 or more carbon atoms, used for identifying individuals with high or low fads2 expression n-3 LC-PUFA) by VO (rich in C fatty acids) improved growth of for the current investigation. These individuals were fed a high VO offspring challenged with a high vegetable meal (VM) and VO diet diet (Table S1) at a daily feeding ration equal to 1% biomass at (Izquierdo et al., 2015). However, these studies did not show 08:00 h and 14:00 h, 6 days a week for 1 month. After the feeding whether this effect was related to the increase in C precursor or the period, whole blood was taken from the caudal vein of the brood fish reduction on n-3 LC-PUFA in the programming diet used during the for the identification of fads2 expression levels of the individuals. spawning period to condition the offspring. Prior to measurements, all fish were anaesthetized with 10 ppm Selective breeding provides the opportunity to improve the clove oil/methanol (1:1 v/v) in sea water. A sample of 2 ml blood economic production efficiency of aquatic livestock (Gjedrem et al., was taken with 2 ml sterile syringes (Terumo Europe NV, Leuven, 2012). In fish, critical analysis of data on the response to selection for Belgium) and transferred to 2.5 ml K3 EDTA tubes (L.P. Italiana, different species and traits shows that gain per generation can vary Milan, Italy). Whole-blood samples were kept on ice during between 4% and 40%, depending on the species and the chosen sampling and immediately centrifuged at 11,200 g, 4°C for 20 min. criterion (Chavanne et al., 2016; de Verdal et al., 2018; Gjedrem and Plasma was separated, and erythrocytes were snap frozen with liquid Rye, 2018; Janssen et al., 2017). Such selection programmes can be nitrogen and kept at −80°C until RNA extraction. RNA extraction also tailored to address the present needs in aquaculture such as and purification are explained more in detail below (see utilization of feeds that are less reliant on marine capture fishery- ‘Biochemistry and gene expression analyses’). Broodstock derived ingredients. Studies undertaken with rainbow trout showing the highest and the lowest fads2 expression from the 70 (Oncorhynchus mykiss)aswellasEuropeansea bass fish sampled were separated into two groups, HD and LD, (Dicentrarchus labrax) have shown the potential of selection for respectively. Twelve brood fish of each sex from the HD and 12 improved use of 100% VM and VO diets free of marine sources (Le from the LD fads2 expression group, with similar length and mass Boucher et al., 2012). One of the approaches in animal breeding (P>0.05, Table 1), were distributed into 12 experimental tanks in a programmes is to use biomarkers for a certain expected outcome by flow-through system with filtered seawater (mean±s.d. 37.0±0.5‰ identifying genes related to the desirable characteristics (Cassar-Malek salinity, 19.59–21.30°C) at a renewal rate of 100% per hour with et al., 2008), as was shown in rainbow trout (Le Boucher et al., 2013). proper aeration. In this sense, the fads2 gene which codifies the delta-6-desaturase enzyme (delta-6), a rate-limiting step of LC-PUFA biosynthesis, is a Spawning quality parameters very strong candidate as its regulation in response to VO is well Spawning quality was determined before and after feeding with the documented in a variety of fish species (Vagner and Santigosa, 2011), experimental diets (Table 2). At the beginning of the trial, the mean including gilthead seabream (Izquierdo et al., 2008). In mice, body mass for females and males was measured (Table 1). Fish from knockout of Fads2 and the absence of LC-PUFA in the diet the HD and LD groups were randomly assigned to experimental resulted in failure of reproduction, showing the key role of Fads2 tanks using a ratio of 1 female and 1 male per tank. After placing the among seven other desaturases (Scd1–5, Fads1, Fads3) in this species brood fish in the experimental tanks, fish were fed with commercial (Stoffel et al., 2008). Studies in humans show a very high correlation diets and spawning quality was monitored daily. Two isocaloric and (up to 70%) between red blood cell fatty acid composition of the isonitrogenous diets (Biomar, Aarhus, Denmark) were formulated parents and its inheritance in the offspring (Lemaitre et al., 2008). In to contain either high levels of FM and FO (diet F) or 30% FO and Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Table 1. Gilthead seabream broodstock mass, fads2 expression and proximate composition of eggs produced after the feeding experiments Broodstock Eggs fads2 expression Crude protein Crude lipid Ash Group Mass (kg) (fold-change) (% dry mass) (% dry mass) (% dry mass) FHD 2.1±0.2 3.9±2.6 69.8±2.7 22.4±5.1 16.3±1.4 FLD 1.9±0.2 0.3±0.1 63.3±2.9 23.3±2.5 19.4±3.6 VHD 2.0±0.9 7.0±3.7 69.3±0.8 26.0±1.6 17.8±0.5 VLD 1.8±0.1 0.5±0.2 68.2±3.2 26.9±0.2 13.3±0.6 Broodstock mass and fatty acyl desaturase 2 gene ( fads2) expression before the feeding experiment are means±s.d. (n=6). Broodstock were fed diets containing different fish oil (FO) and vegetable oil (VO) ratios for 30 days: FHD, high fads2 expression broodfish (HD) fed with 100% FO diet (F); FLD, low fads2 expression broodfish (LD) fed with 100% FO diet (F); VHD, high fads2 expression broodfish (HD) fed with 30% FO–70% VO diet (V); and VLD, low fads2 expression broodfish (LD) fed with 30% FO–70% VO diet (V). Proximate egg composition data are means±s.d. (n=3, one pool of eggs from all the spawn per broodstock tank). Different letters indicate a significant difference between the groups (P<0.01). Two-way ANOVA values were calculated for the effect of selection, diet and the interaction of selection and diet from mean±s.d. data (n=3). Selection had a significant effect on broodstock fads2 fold-change in expression; P<0.01); all other parameters were not significant (P>0.05). 70% VO (diet V; Table 2). Once equal spawning quality parameters The spawning quality parameters were determined using eggs were observed between the tanks during the acclimation period of collected daily from each tank at around 08:00 h and concentrated in 21 days (P>0.05, data not shown), tanks were randomly assigned to 5 l beakers. Immediately, eggs were transferred to the laboratory and one of the four experimental groups as follows: FHD, high fads2 aeration was supplied to ensure mixing of the eggs through the water expression broodfish (HD) fed with 100% FO diet (F); FLD, low column. Then, a 5 ml sample was taken using a graduated glass fads2 expression broodfish (LD) fed with 100% FO diet (F); VHD, pipette and transferred to a Bogorov chamber. Eggs were counted high fads2 expression broodfish (HD) fed with 30% FO–70% VO and observed under a binocular microscope (Leica Microsystems, diet (V); and VLD, low fads2 expression broodfish (LD) fed with Wetzlar, Germany) in five replicates to calculate the total number of 30% FO–70% VO diet (V). eggs and the percentage of fertilized eggs, and to determine the morphological characteristics. Egg viability rate was determined as the percentage of morphologically normal eggs at the morula stage, Table 2. Formulation, main ingredients and biochemical composition of described as transparent, perfectly spherical with clear, symmetrical the broodstock diets used during the spawning period early cleavage (Fernandez Palacios et al., 2011). After that, eggs were randomly transferred to 96-well ELISA microplates using a F diet V diet (30% (100% FO) FO–70% VO) micropipette (0.7 ml of seawater and one egg per well). Plates were observed under a binocular microscope to ensure that there was a Raw material (%) single fertilized egg in each well. These eggs were kept at a Meals from marine sources 50.0 50.0 Sunflower meal 13.2 13.2 controlled temperature of 23°C. By observing the egg from these Soybean meal 10.0 10.0 ELISA plates after 24 and 72 h under a binocular microscope, Fish oil 8.0 2.4 hatching rate and survival 3 days post-hatching (dph) were Linseed oil – 5.6 calculated as percentages. With these percentage values, the total Wheat 9.9 9.9 number of fertilized, viable and hatched eggs and larvae alive at Corn gluten meal 60 7.0 7.0 3 dph was calculated per kg female per spawn. Vitamin & mineral premix 1.0 1.0 Biochemical composition (% dry matter) Moisture 9.1 8.8 Larval rearing and growth Protein (crude) 56.3 56.1 After 1 month of feeding with the respective diets, eggs were collected Lipids (crude) 17.2 17.1 and distributed into 500 l tanks for mass production at a density of 100 Ash 8.6 8.5 −1 eggs l from each treatment group. All larvae were reared following −1 Gross energy (MJ kg ) 21.2 21.2 the same common rearing protocol, regardless of the origin of the Fatty acids (% of total fatty acids) spawn. Water renewal in the tanks was progressively increased from 16:1n-7 7.1 4.3 18:2n-6 5.6 9.9 10% to 40% per hour until 46 dph and the water was continuously −1 18:3n-3 0.9 16.3 aerated (125 ml min ). Larvae were reared under natural photoperiod 20:1n-7 12.4 6.8 and living phytoplankton [Nannochloropsis sp.; mean±s.d. 20:4n-6 0.4 0.3 3 −1 250(±100)×10 cells ml ] was added to the rearing tanks. From 3 20:5n-3 6.3 4.8 to 17 dph, larvae were fed twice a day with rotifers (Brachionus 22:1n-11 15.7 8.6 −1 plicatilis, 10 rotifer ml ) enriched with commercial emulsions (ORI- 22:6n-3 7.1 6.0 GREEN, Skretting, Norway). From 15 to 32 dph, Artemia sp. FO, fish oil; VO, vegetable oil. Contains Fishmeal NA LT 70 (Fishmeal SA 68, enriched with commercial emulsions (ORI-GREEN) was added to the Feed Service Bremen, Germany). 48 Hi Pro Solvent Extra (Svane Shipping, Kolding, Denmark). South American Fish Oil (LDN Fish Oil, Hedensted, rearing tanks 3 times a day. From 20 dph, larvae were fed commercial 4 5 Denmark). Ch. Daudruy (Dunkerque, France). Vitamin and mineral premix diets according to the suggested diet particle size by the manufacturer −1 provided the following vitamins (mg kg ): A 3.8, D 0.05, E 102.4, K3 9.8, B1 (Gemma Micro, Skretting, France). Water was continuously aerated 2.7, B2 8.3, B6 4.8, B12 0.25, B3 24.8, B5 17.2, folic acid 2.8, H 0.14, C 80; −1 (125 ml min ) attaining 6.1±0.4 ppm dissolved O (mean±s.d. ). −1 minerals (mg kg ): cobalt 0.94, iodine 0.7, selenium 0.2, iron 32.6, Average water temperature and pH along the trial were 21.1±0.4°C −1 manganese 12, copper 3.2, zinc 67; other (g kg ): taurine 2.45, methionine and 7.0±0.6, respectively. Water quality was monitored daily as 0.5, histidine 1.36, cholesterol 1.13 (DSM, Heerlen, The Netherlands; Evonik regards dissolved O and pH. At 3, 15 and 30 dph, growth was Industries, Essen, Germany; Deutsche Lanolin Gesellschaft, Frankfurt am 2 Main, Germany). determined by measuring the total length of 60 anaesthetized larvae Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 per treatment using a profile projector (V-12A, Nikon, Tokyo, Japan). Fish were kept in a flow-through system and had a natural photoperiod RNA extraction methods are detailed below (see ‘Biochemistry and (12 h light:12 h dark). Triplicate groups of fish initially coming from gene expression analyses’). At 46 dph, each experimental group was one of the four broodstock groups (FHD, FLD, VHD and VLD) were transferred to 10,000 l tanks in duplicate. reared with a pelleted low FM and low FO diet (Table 3) and were fed until apparent satiation twice a day at 08:00 h and 14:00 h, 6 days a Juvenile nutritional challenge test week over 60 days. Uneaten pellets were collected in a net by slowly Obtained offspring were kept in similar environmental conditions. opening the water outlet for 10 min after each meal, dried in an oven Fish were fed with commercial diets all through the grow-out period for 24 h and weighed to estimate net feed intake. The amount of feed until they reached 6 months of age, and were fed 3 times a day by given to each tank was recorded daily. Water temperature and hand until apparent satiation. Rearing water temperature and oxygen levels were monitored daily after the last feeding. photoperiod were natural and equal for all the experimental groups Average water temperature was 22.5±0.7°C, and dissolved oxygen (18.5–21.8°C, 11–13 h light). Triplicate groups of juvenile fish (mean −1 was 6.1±0.3 mg l during the experimental period. ±s.d. initial body mass of all groups: 23.3±0.7 g, n=12) from each All fish were anaesthetized with 10 ppm clove oil:methanol broodstock group were assigned to one of 12 tanks (500 l capacity) for (1:1 v/v) in sea water prior to measurements for the initial and final the juvenile feeding challenge experiment (n=75 per each treatment). samplings. Fish were fasted for 24 h then individually weighed. Growth was determined by measuring wet body mass after 24 h starvation at 30 and 60 days of the experiment. Prior to measurements, Table 3. Formulation, main ingredients and biochemical composition of all fish were anaesthetized with 10 ppm clove oil:methanol (1:1 v/v) in the diets used for juveniles during the nutritional challenge sea water. The experimental scheme is presented graphically in Fig. 1. Content Proximate composition Content Growth and feed utilization parameters were calculated using the following equations: feed conversion ratio (FCR)=(dry mass of Main ingredients (%) Biochemical composition (% dry matter) consumed feed)/(final biomass−initial biomass), and weight gain Fishmeal 5.0 Crude lipids 21.8 (%)=(final biomass−initial biomass)/initial biomass×100, where mass Fishmeal alternative 54.5 Crude protein 57.2 is in g. protein sources Rapeseed meal cake 11.3 Moisture 6.5 Biochemistry and gene expression analyses Wheat 6.9 Ash 6.7 2 −1 Lipid, protein, ash and moisture analysis of the samples was done as Fish oil 3.0 Gross energy (MJ kg ) 22.5 previously described (Turkmen et al., 2017b). For each 200 µl of Vegetable oil mix 13.0 Micronutrient mix 6.3 blood cells, 1 ml of TRI Reagent (Sigma-Aldrich, St Louis, MO, USA) was added into 2 ml Eppendorf tubes. To each tube, four Total fatty acids (%) Total fatty acids pieces of 1 mm diameter zirconium glass beads were added and (%) (continued) homogenized using TissueLyzer-II (Qiagen, Hilden, Germany) for 14:0 4.69 18:3n-4 0.04 −1 60 s with a frequency of 30 s ; 250 µl chloroform was added to 14:1n-5 0.17 18:3n-3 14.86 15:0 0.28 18:4n-3 0.91 homogenized samples, which were then centrifuged at 12,000 g for 16:0 13.67 20:0 0.21 15 min at 4°C for phase separation. The clear upper aqueous phase 16:1n-7 4.50 20:1n-9 0.37 containing RNA was mixed with 75% ethanol and transferred into 16:1n-5 0.09 20:1n-7 7.25 an RNeasy spin column to bind total RNA. Then, RNA was 16:2n-6 0.25 20:1n-5 0.37 extracted using an RNeasy Mini Kit (Qiagen) with the protocol 17:0 0.13 20:2n-6 0.16 supplied by the manufacturer. Real-time quantitative PCR was 16:3n-4 0.14 20:4n-6 0.31 16:3n-3 0.14 20:3n-3 0.09 performed in an iQ5 Multi-colour Real-Time PCR detection system 16:3n-1 0.04 20:4n-3 0.19 (Bio-Rad) using β-actin (acbt) as the housekeeping gene in a final 16:4n-3 0.22 20:5n-3 3.81 volume of 15 μl per reaction well and with 100 ng of total RNA 18:0 3.23 22:1n-11 9.35 reverse transcribed to cDNA. Samples, housekeeping gene, cDNA 18:1n-9 15.75 22:1n-9 1.00 template and reaction blanks were analysed in duplicate. Primer 18:1n-7 2.46 22:4n-6 0.04 efficiency was tested with serial dilutions of a cDNA pool (1:5, 18:1n-5 0.23 22:5n-6 0.03 1:10, 1:100 and 1:1000). Sequences of the primers used in this study 18:2n-6 9.84 22:5n-3 0.28 18:2n-4 0.05 22:6n-3 4.49 were: acbt (GenBank accession no. X89920) 5′–3′ (F) TCT GTC 18:3n-6 0.08 TGG ATC GGA GGC TC, (R) AAG CAT TTG CGG TGG ACG); *For a complete list of ingredients, see Torrecillas et al. (2017b) diet code 5FM/ and fads2 (GenBank accession no. AY055749) 5′–3′ (F) CGA 3FO. GAG CCA CAG CAG CAG GGA, (R) CGG CCT GCG CCT GAG 1 2 South American, Superprime (Feed Service, Bremen, Germany). Blood CAG TT). Gene, primer efficiency and blank samples were meal spray (Daka, Hedensted, Denmark), soya protein concentrates 60% analysed in 96-well PCR plates (Multiplate, Bio-Rad). Melting- (Svane Shipping), corn gluten meal 60 (Cargill, Schiphol, The Netherlands), curve analysis was performed, and amplification of a single product wheat gluten (Cargill). Linseed (2.6%) (Ch. Daudruy), rapeseed (5.2%) was confirmed after each run. Fold-change in expression of each (Emmelev, Otterup, Denmark) and palm oils (5.2%) (Cargill). Micronutrient ΔΔCT −1 gene was determined by the delta-delta CT method (2 ) (Livak mix (vitamin mix, 0.75%) supplied the following vitamins (mg kg ): A 3.8, D 0.05, E 102.4, K3 9.8, B1 2.7, B2 8.3, B6 4.8, B12 0.25, B3 24.8, B5 17.2, folic and Schmittgen, 2001). PCR efficiencies were similar, and no −1 acid 2.8, H 0.14, C 80; minerals (mg kg ): cobalt 0.94, iodine 0.7, selenium efficiency correction was required (Livak and Schmittgen, 2001; −1 0.2, iron 32.6, manganese 12, copper 3.2, zinc 67; other (g kg ): taurine 2.45, Schmittgen and Livak, 2008). methionine 0.5, histidine 1.36, cholesterol 1.13 (DSM; Evonik Industries; Deutsche Lanolin Gesellschaft); supplemented ingredients (5.49%); contains Statistical analysis lysine, methionine, monocalcium phosphate, choline, inositol, phospholipids Results are expressed as means±s.d. (n=3), unless otherwise (Vilomix, Mørke, Denmark), Evonik Industries, Pöhner (Hamburg, Germany); antioxidant (0.5%): BAROX BECP, Ethoxyquin (Vilomix); yttrium oxide (0.3%). stated in the tables and figures. The data were compared Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 1 month 1 month 2 months 1 month Before spawning Gene expression analysis Acclimation Nutritional interventions n=70 (42 females & 28 males) n=24 (12 females and 28 males) 1 male : 1 female each tank High fads2 FHD 100% F0 VHD 30% F0–70% V0 Low fads2 FLD 100% FO VLD 30% F0–70% VO Experimental diet Commercial diet Commercial diet Nutritional programming diet 5% FO–3% FM 100% FO or 30% FO–70% VO 0–3 days after hatching 3–45 days after hatching 3 months 2 months Hatching & mouth opening On-growing Nutritional challenge test FHD FHD VHD VHD FLD FLD VLD VLD Yolk sac absorption Artemia and rotifers Commercial diet Nutritional challenge diet enriched with commercial 5% FM–3% FO emulsifiers Fig. 1. Experimental design and sampling points. All gilthead seabream groups were tested in triplicate. Sampling points are shown as red dashed lines, and experimental periods when fish were fed with different diets are shown as green lines. Experimental diet used for fatty acyl desaturase 2 gene (fads2) selection is given in Table S1; nutritional programming diet is given in Table 2; and nutritional challenge diet used at the juvenile stage is given in Table 3. FO, fish oil; FM, fish meal; VO, vegetable oil; FHD, high fads2 expression broodfish (HD) fed with 100% FO diet (F); FLD, low fads2 expression broodfish (LD) fed with 100% FO diet (F); VHD, high fads2 expression broodfish (HD) fed with 30% FO–70% VO diet (V); and VLD, low fads2 expression broodfish (LD) fed with 30% FO–70% VO diet (V). statistically using analysis of variance (ANOVA), at a RESULTS significance level of 5%. All variables were checked for Broodstock fads2 expression level, spawning quality normality and homogeneity of variance using the Kolmogorov– and eggs Smirnoff and Levene tests, respectively (Sokal and Rohlf, 1969). Individual broodstock fish showed very different expression levels If significant differences were detected with ANOVA, means of fads2 gene after being fed the experimental (conditioning) diet. were compared by Student’s t-test. All data were analysed (VM and VO diet; Table S1). The average fold-change in gene using IBM SPSS v23.0.0.2 for Mac (IBM SPSS Inc., Chicago, expression was 7.1±13.6, with a maximum of 61.6 and a minimum IL, USA). A Pearson correlation test was performed to identify of 0.02. No relationship was found between fish mass and fads2 relationships between parameters using R (http://www. expression among individuals (P>0.05, R=0.0061). Mass and fads2 R-project.org/). expression of the broodstock fish are presented in Table 1. Feeding Time Feeding Time Blood collection Separation of HD and LD fish Seeding of obtained eggs Sampling of juveniles Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 During the first part of the spawning season, when broodstock significantly increased levels of ALA and LA in the eggs were fed with the same diet, there were no differences in total (P>0.05) (Table 4). Analysis of egg fatty acid composition number of eggs per kg female per spawn, fertilization rate, viability, (Table 4) showed that among saturated fatty acids, 14:0 and 17:0 hatching rate and survival of larvae at 3 dph, which on average were were lower in V diet groups (P<0.05), while the other saturated fatty 49,023±1195, 46,339±1862, 33,990±2513, 31,899±3092, 22,960± acids such as 15:0, 16:0, 18:0 and 20:0 were similar among all 2114 eggs/larvae (10 /kg female/spawn) (means±s.d., P>0.05), groups (P>0.05) (Table 4). Additionally, egg content of monoenoic respectively. After feeding the broodstock with either the F or the V fatty acids such as 14:1n-7, 14:1n-5, 16:1n-7, 18:1n-7, 18:1n-5, diet (Table 2) for 1 month, the total number of eggs produced and 20:1n-9, 20:1n-7, 20:1n-5 and 20:1n-11 was lower in V-diet groups survival of 3 dph larvae were lower in broodstock with low fads2 (Table 4). Also, ALA, the first substrate for delta-6 in the n-3 expression (FLD and VLD), regardless of the diet. The difference LC-PUFA biosynthesis pathway to EPA and DHA, was up to 4 between the two groups was 37% lower total number of eggs and times higher (P<0.05) in eggs obtained from V-diet groups 10.2% lower number of 3 dph larvae in low fads2 expression groups (Table 4). In contrast, egg content of LA, another substrate for (FLD and VLD) than in those with high fads2 expression (FHD and LC-PUFA biosynthesis and high in VOs, was similar among eggs VHD) (P<0.05) (Fig. 2). The same trend was observed for the other from different groups (P>0.05; Table 4). However, the products of spawning quality parameters including fertilized, viable or hatched n-3 LC-PUFA biosynthesis such as arachidonic acid (ARA) 20: eggs, but without significant differences among groups (P>0.05) 4n-6, EPA 20:5n-3 and DHA 22:6n-3 were similar among the eggs (Fig. 2). The percentage ratios of fertilized eggs after feeding of different experimental groups (P>0.05) (Table 4). broodstock with the F and V diet was 92.6±7.3, 91.8±7.6, 89.3±7.3 and 89.9±9.5 in FHD, FLD, VHD and VLD groups, respectively. Larval growth The proximate composition of these eggs, obtained after 1 month of Despite the fact that larvae were kept under similar conditions and broodstock feeding with the diets, was not significantly different fed with the same diets, growth was higher in FHD larvae in among the different broodstock groups (P>0.05) (Table 1). fads2 comparison with VHD larvae at 3 dph (P<0.05; Table 5). At expression levels, diet and the interaction between these parameters 15 dph, among the progeny obtained from broodstock fed the F did not affect the proximate composition of the eggs (P>0.05) diet, those from the FLD group had a greater length than those (Table 1). Irrespective of the fads2 expression levels of the from the FHD group (P<0.05). Between the larval groups from broodfish, replacement of FO by VO in the diet led to broodstock with low fads2 expression, VLD larvae were smaller in Groups 50 FHD FLD VHD VLD Number of eggs Fertilized eggs Viable eggs Hatched eggs 3 dph larvae Fig. 2. Spawning parameters of gilthead seabream broodstock fed with either 100% FO (F diet) or 70% VO–30% FO (V diet) for 1 month. Broodstock fish were separated according high or low fads2 expression levels in the blood as described in Fig. 1. Data are presented as means±s.d. (n=3); different letters denote differences between groups (P<0.05). 3 –1 No. of eggs/larvae (10 kg per female per spawn) Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Table 4. Fatty acid composition of eggs produced after the broodstock feeding experiments Two-way ANOVA % Total fatty acids FHD FLD VHD VLD S D S×D A,x B y 14:0 4.24±0.01 3.44±0.08 2.83±0.48 3.16±0.55 0.29 ** * A,x B y 14:1n-7 0.05±0.00 0.04±0.01 0.02±0.01 0.03±0.01 0.27 ** * A,x B y 14:1n-5 0.14±0.01 0.11±0.00 0.09±0.01 0.10±0.02 0.21 ** * 15:0 0.31±0.00 0.30±0.03 0.27±0.03 0.29±0.03 0.82 0.20 0.34 A,x B y 15:1n-5 0.04±0.00 0.03±0.00 0.03±0.00 0.03±0.00 0.08 0.13 * 16:0ISO 0.05±0.00 0.05±0.00 0.05±0.00 0.05±0.01 0.57 0.09 0.55 16:0 18.08±0.74 21.37±2.97 19.73±2.41 18.83±1.59 0.35 0.72 0.12 x x y y 16:1n-7 6.31±0.02 5.79±0.28 4.75±0.49 4.77±0.21 0.19 ** 0.16 16:1n-5 0.09±0.01 0.10±0.01 0.09±0.01 0.09±0.01 0.74 0.99 0.74 16:2n-6 0.01±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.57 0.63 0.67 A,x B y 16:2n-4 0.25±0.00 0.18±0.03 0.16±0.04 0.18±0.02 0.10 * * A,x B y 17:0 0.18±0.00 0.15±0.01 0.14±0.02 0.13±0.01 * ** 0.09 x y 16:3n-4 0.20±0.00 0.23±0.02 0.20±0.02 0.19±0.00 0.42 * * 16:3n-3 0.12±0.01 0.10±0.01 0.09±0.02 0.10±0.02 0.66 0.12 0.32 y x 16:3n-1 0.06±0.00 0.09±0.02 0.08±0.01 0.08±0.01 0.12 0.37 0.08 16:4n-3 0.07±0.03 0.07±0.02 0.06±0.01 0.06±0.00 0.87 0.45 0.77 16:4n-1 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.51 0.16 0.90 18:0 3.67±0.24 3.98±0.69 4.03±0.62 4.03±0.58 0.64 0.55 0.64 B A 18:1n-9 19.67±0.63 22.88±1.71 21.25±1.98 20.51±0.67 0.16 0.63 * x y 18:1n-7 3.24±0.16 2.95±0.10 2.75±0.26 2.71±0.19 0.18 ** 0.27 x y 18:1n-5 0.26±0.05 0.19±0.03 0.15±0.04 0.16±0.04 0.19 * 0.10 x y 18:2n-9 0.27±0.01 0.21±0.04 0.19±0.04 0.22±0.08 0.67 0.19 0.14 18:2n-6 (LA) 9.39±0.91 10.83±0.91 11.45±0.97 11.55±0.45 0.15 * 0.20 A B 18:2n-4 0.11±0.00 0.11±0.02 0.11±0.01 0.10±0.00 0.56 0.82 0.17 A B 18:3n-6 0.44±0.01 0.26±0.05 0.27±0.09 0.32±0.08 0.11 0.15 * x y 18:3n-4 0.12±0.01 0.11±0.01 0.10±0.00 0.10±0.02 0.21 * 0.32 18:3n-3 (ALA) 1.82±0.64 1.68±0.40 5.49±2.70 7.28±3.36 0.53 ** 0.46 18:3n-1 0.01±0.00 0.00±0.00 0.01±0.00 0.01±0.00 0.64 0.09 0.12 18:4n-3 0.92±0.05 0.49±0.24 0.50±0.28 0.58±0.20 0.18 0.21 0.07 A,x B y 18:4n-1 0.10±0.01 0.07±0.01 0.07±0.00 0.07±0.01 * ** * 20:0 0.09±0.01 0.10±0.04 0.11±0.04 0.08±0.01 0.62 0.97 0.34 A,x B y 20:1n-9 0.40±0.09 0.22±0.04 0.17±0.06 0.21±0.09 0.15 * * A,x B y 20:1n-7 3.16±0.68 1.52±0.36 1.12±0.36 1.36±0.54 * ** * A,x B y 20:1n-5 0.26±0.04 0.17±0.02 0.14±0.03 0.15±0.04 0.07 ** * 20:2n-9 0.08±0.01 0.08±0.01 0.07±0.02 0.07±0.04 0.61 0.44 1.00 20:2n-6 0.31±0.02 0.38±0.05 0.32±0.05 0.30±0.02 0.33 0.12 0.10 20:3n-9 0.03±0.00 0.02±0.00 0.02±0.00 0.02±0.00 0.13 0.08 0.12 20:3n-6 0.16±0.02 0.14±0.01 0.12±0.01 0.13±0.03 0.79 0.15 0.33 20:4n-6 0.61±0.02 0.62±0.06 0.63±0.04 0.61±0.10 0.99 0.88 0.70 y x 20:3n-3 0.16±0.01 0.17±0.01 0.27±0.07 0.29±0.07 0.73 ** 0.92 x y 20:4n-3 0.62±0.08 0.46±0.10 0.43±0.07 0.44±0.05 0.13 * 0.08 20:5n-3 5.54±0.65 3.93±1.28 4.00±1.12 4.09±0.39 0.19 0.24 0.15 A,y B x 22:1n-11 1.23±0.30 0.46±0.13 0.35±0.16 0.51±0.29 0.05 * ** 22:1n-9 0.32±0.10 0.20±0.04 0.15±0.05 0.16±0.10 0.28 * 0.19 22:4n-6 0.06±0.01 0.05±0.00 0.05±0.00 0.05±0.01 0.42 0.35 0.30 x y 22:5n-6 0.05±0.01 0.04±0.00 0.03±0.01 0.03±0.00 0.17 * 0.35 22:5n-3 2.04±0.13 1.94±0.20 1.91±0.37 1.83±0.64 0.70 0.62 0.96 22:6n-3 14.67±0.19 13.70±2.20 15.14±3.69 13.92±4.17 0.54 0.85 0.94 Eggs were obtained from gilthead seabream after feeding broodstock with diets containing different FO and VO ratios for 30 days (means±s.d., n=3, one pool of eggs from all the spawn per broodstock tank). Capital letters denote differences between selection groups fed with the same diet during the spawning period; lowercase letters denote differences among diet groups with similar fads2 expression. Two-way ANOVA values were calculated for the effect of selection (S), diet (D) and the interaction of selection and diet (S×D) (*P<0.05, **P<0.01; non-significant P-values are shown). comparison to FLD larvae (P<0.05). There were no differences in highest final mass (47.4±0.8 g) and had the highest relative gain in larval growth among any of the experimental groups at 30 dph mass (99.7±7.3%) (Fig. 3). The two-way ANOVA analysis showed (P>0.05; Table 5). that broodstock with high fads2 expression levels (FHD and VHD) had a positive effect on juvenile offspring gain in mass (P<0.001), a Nutritional challenge test with juveniles nutritional programming effect of the broodstock diet (P<0.01), and There was no significant difference (P>0.05) in the initial mass of a significant interaction between these two parameters (P>0.05) the 6 month old juveniles at the beginning of the nutritional (Fig. 3). Thus, the parents showing high fads2 levels improved the challenge test (FHD: 23.4±0.1, FLD: 22.1±0.1, VHD: 23.8±0.4 and gain in mass of the offspring, regardless of the nutritional history of VLD: 23.7±0.1g). However, after 60 days of feeding juveniles with the broodstock (F or V diet) (Fig. 3). Progeny obtained from the high VM and VO diet (Table 3), the FHD group reached the broodstock selected for high fads2 expression and fed with the F Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Table 5. Length of gilthead seabream larvae obtained from broodstock fed with either 100% FO (FHD, FLD) or 70% VO–30% FO (VHD, VLD) for 1 month Total length (mm) Two-way ANOVA Larval age (dph) FHD FLD VHD VLD S D S×D A B 3 3.41±0.01 3.38±0.19 3.23±0.09 3.29±0.10 * A B 15 4.66±0.39 5.02±0.26 4.43±0.55 4.53±0.14 * 30 7.49±0.26 7.26±0.32 7.05±0.42 7.31±0.24 Data presented as means±s.d. (n=3, average of 20 larvae from per tank). Capital letters denote significant differences between differentially fed broodstock groups during the spawning period with similar fads2 expression (P<0.05). Two-way ANOVA values were calculated for the effect of selection (S), diet (D) and the interaction of selection and diet (S×D) (*P<0.05). diet (FHD) showed significantly higher (P<0.01) growth than parameters (P<0.01) (Table 7). In addition, if LD groups were progeny obtained from broodstock selected for high fads2 compared, 18:0 was expressed at a lower level (P<0.05) in VLD expression and fed with the V diet (VHD) (Fig. 3). Gain in mass than in FLD juveniles (Table 7). Total monounsaturated fatty acids of the progeny obtained from broodstock selected for low fads2 were affected by the diet in LD groups and were higher in the VLD expression and fed with the F diet (FLD) or the V diet (VLD) were than in the FLD group (P<0.05). n-6 PUFA were changed in not significantly different (P>0.05) (Fig. 3). In summary, VO response to the broodstock diet (P<0.05) and the interaction inclusion in the broodstock diets, thus increasing the dietary 18C between broodstock diet and broodfish fads2 expression levels fatty acid levels, resulted in lower gain in mass in juvenile offspring (P<0.1). LA was higher in the VLD group if compared with the FLD of broodstock selected for high fads2 (P<0.05) (Fig. 3), but not in group (P<0.05); however, there was a clear tendency of LA those coming from low fads2 broodstock. There was no difference accumulation in progeny from V-diet fed broodstock than in those (P>0.05) in feed intake of the fish. Additionally, FCR in juveniles from the F-diet fed broodstock (Table 7). The same kind of from broodstock selected for high fads2 expression (FHD and VHD differences were observed in ALA, with a broodstock diet leading to groups) was significantly lower than that in low fads2 expression greater accumulation of ALA in the V-diet groups than in those groups (FLD and VLD groups) (P<0.05) regardless of the diet fed to coming from the F-diet broodstock, while the accumulation was their parents (Fig. 3). In contrast, gain in mass (%) values were significantly higher in the VLD than in the FLD group (P<0.05). significantly higher in progeny from broodstock showing low fads2 However, there were no differences (P>0.05) in EPA, DHA or total expression, regardless of the broodstock diet (P<0.05) (Fig. 3). n-3 PUFA content among the experimental groups (Table 7). In addition, there were no differences in total PUFA content of the Muscle biochemical composition and fatty acid profiles of progeny obtained from broodstock with different fads2 expression juvenile offspring after the nutritional challenge levels and fed with either the V or F diet (P>0.05). Muscle biochemical composition was similar among the experimental groups (P<0.05; Table 6). The type of broodstock DISCUSSION diet (F or V) as well as the fads2 expression levels of the parents and The importance of the fads2 gene for reproduction in mammals has their combination led to significant (P<0.05) differences in the been demonstrated in Fads2 −/− mouse, where males are not able to muscle fatty acid profiles of offspring; this was particularly relevant produce mature sperm and folliculogenesis is disrupted in females for total saturated, monounsaturated and n-6 PUFA levels, whereas (Stoffel et al., 2008). Knocking out the Fads2 gene inhibits the total n-3 PUFA contents were similar (Table 7). Total saturated fatty synthesis of LC-PUFA, and other desaturases (Scd1–5, Fads1, acid levels were higher (P<0.05) in the VLD group than in the VHD Fads3) are not able to compensate for the deletion of Fads2 in the group, while the VHD group had higher levels of saturated fatty biosynthesis of ARA, EPA and DHA. In the present study, the acids than the FHD group (P<0.05). Among the saturated fatty broodstock group with higher fads2 expression showed a better acids, 14:0 and 16:0 were higher in the VLD group than in the VHD reproductive performance than those with low fads2 expression, as and FLD groups (P<0.05), thus the two-way ANOVA showed a denoted by the greater total number of eggs produced per kg female significant effect of broodstock fads2 expression levels (P<0.05), per spawn. To the best of our knowledge, this is the first study broodstock diet (P<0.01) and the interaction between these showing the positive effects of broodstock fads2 expression levels Fig. 3. Gain in mass and food conversion Diet P=0.01 Diet P=0.01 ratio (FCR) after nutritional challenge of Selection P=0.001 Selection P=0.001 gilthead seabream juveniles from Interaction P=0.05 Interaction P=0.05 broodstock fed either the F or V diet. Juveniles were challenged after 60 days with 5% FM and 3% FO diets. Data are presented as means±s.d. (two-way ANOVA; n=3); different letters denote differences between groups (P<0.05). (means±s.d., n=3). FHD FLD VHD VLD FHD FLD VHD VLD Gain in mass (%) FCR Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Table 6. Biochemical composition of muscle tissue of 6 month old to these findings, the present study demonstrates the importance of gilthead seabream after the nutritional challenge the broodstock’s ability to express fads2 on reproductive success, in agreement with a study conducted in mammals (Stoffel et al., 2008). Moisture Crude protein Crude lipids Ash Group (%) (% dry mass) (% dry mass) (% dry mass) Feeding broodstock with the F diet resulted in a greater length of larvae at 3 dph among larvae obtained from parents showing high FHD 69.2±2.1 67.1±6.4 26.6±5.5 1.5±0.1 fads2 expression and greater growth in 15 dph larvae obtained from FLD 69.6±0.6 65.8±3.3 24.3±1.9 1.3±0.2 VHD 70.6±2.2 69.5±3.6 27.2±1.3 1.5±0.02 parents showing low fads2 expression. By 30 dph, larvae were able VLD 71.1±0.8 67.4±2.5 25.0±3.3 1.7±0.2 to compensate for early differences in growth, indicating that the common rearing protocols supplied sufficient amounts of nutrients Biochemical composition of muscle tissue after 2 months feeding on a very low FM (5%) and FO (3%) diet in 6 month old gilthead seabream originating from for growth. It has been shown in several species of fish that broodstock in the different fads2 expression–diet groups. Data are means±s.d. parental nutritional history and dietary interventions during early (n=3). Two-way ANOVA values were calculated for the effect of selection, diet ontogenesis can have significant impacts on offspring development, and the interaction of selection and diet from mean±s.d. data (n=3); there were somatic mass, metabolism and immune responses (Izquierdo et al., no significant effects (P>0.05). 2015; Morais et al., 2014; Turkmen et al., 2017b). In the present study, growth differences at 3 and 15 dph may be related to the on spawning quality in fish. The importance of LC-PUFA on fish genetic background of the eggs in relation to the broodstock reproductive success is well documented in a number of teleosts expression of fads2, as rearing with the same feeding protocol (Izquierdo et al., 2001; Watanabe and Vassallo-Agius, 2003), allowed the different larval groups to achieve similar growth including gilthead seabream (Fernandez Palacios et al., 2011; performance at 30 dph. Interestingly, when juvenile offspring were Fernández-Palacios et al., 1995; Izquierdo et al., 2001). In addition challenged with a low FM and low FO diet, those obtained from Table 7. Fatty acid composition of muscle of gilthead seabream fed the nutritional challenge diet −1 Fatty acid (g 100 g tissue) Two-way ANOVA FHD FLD VHD VLD S D D×S Saturated y B A,x 14:0 17.4±8.8 9.8±3.3 17.2±32 41.3±4.3 ** *** *** 15:0 8.3±5.7 1.7±0.6 3.2±18 4.1±0.3 * y B A,x 16:0 84.9±36.4 95.1±17 188.5±175 293.6±19.4 *** *** ** 17:0 7.6±5.4 2.9±1.9 3±15 3.1±1 y x 18:0 35.9±7.7 38±5.5 66.8±138 67.7±2.9 *** 20:0 8.8±3.6 4.3±0.4 5.3±0 5.1±0.7 y B,x A Σ Saturated 158.7±15.4 171.5±1.2 274.2±238 427.3±9.7 *** *** *** Monounsaturated A B 16:1n-9 7.7±5 0.4±0.2 0.8±4 1±0.1 ** * y B,x A 16:1n-7 17.1±5.2 25.1±4.6 42.7±43 71.1±9.6 *** *** * 18:1n-9 350.8±120.4 290.6±52.9 607.5±1335 582.7±199.8 *** y x 18:1n-7 27.1±0.6 28.3±5.1 48.9±118 50.6±2.3 *** 20:1n-9 8.7±4.4 2.9±0.8 3.5±0 3.2±0.7 20:1n-7 19.2±3.3 19.2±3.7 25±14 20.6±3.1 * 22:1n-11 37.8±27.2 15±1.2 14.7±11 9.3±3.3 22:1n-9 16.5±12.2 8±2.9 7±7 5.4±1.4 y x Σ Monounsaturated 504.8±135.6 383.8±92.6 766.8±1744 854.9±42.2 *** n-6 PUFA y x 18:2n-6 144.6±44.3 112.2±18.8 236±520 251.2±36.9 *** 18:3n-6 15.6±6.7 10.5±7 11.3±22 12.2±0.9 20:2n-6 14.8±7.3 4.3±1.2 6.2±3 5.8±0.4 * A B 20:3n-6 4.9±0.1 3.4±0 4.5±5 3.6±0.2 *** 20:4n-6 16±7.1 8.6±3.2 8.7±7 8.7±0.2 22:4n-6 19.5±12.7 3.3±1.5 2.5±7 3±1.7 ** 22:5n-6 9.6±5.9 4.4±0.7 4.3±3 4.9±1.3 y x Σn-6 PUFA 240±46.5 141.5±26.2 273.5±549 304.2±16.5 ** * n-3 PUFA y x 18:3n-3 88.4±16.1 58.4±11.3 136.9±283 144.1±42.6 *** 18:4n-3 13.8±5.6 9.2±1.4 15±36 15.2±1.9 A,x B y 20:3n-3 13.9±6.2 5±2.5 4.8±4 4.7±0.8 ** ** * 20:4n-3 10.6±4.6 8±2.6 9.7±8 8.9±1.2 20:5n-3 57.6±29.2 53.5±8.4 67.3±73 57.1±15.5 22:5n-3 39.3±17.5 26.3±6.3 30±9 23.3±4.4 22:6n-3 110.1±29.4 125±23.8 122.4±31 102.2±21.1 Σn-3 PUFA 370.1±119.6 325.9±14.6 386.1±360 355.4±18.6 Σ PUFA 662.1±184.6 475.9±112.2 673.8±957 658.5±39.4 Fatty acid composition of muscle tissue after 2 months feeding on a very low FM (5%) and FO (3%) diet in 6 month old gilthead seabream originating from broodstock in the different fads2 expression–diet groups. Note, total PUFA also includes 16:2, 16:3 and 16:4 fatty acids. Data are means±s.d. (n=3, one pool from five fishes per tank×3). Capital letters denote differences between broodstock selection groups fed with the same diet during the spawning period; lowercase letters denote significant differences among differentially fed broodstock groups during the spawning period with similar fads2 expression. Two-way ANOVA values were calculated for the effect of selection, diet and the interaction of selection and diet (*P<0.1, **P<0.05, ***P<0.01). Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 broodstock with high fads2 expression again showed a positive seabream organs, and ALA content in head kidney is related to effect on gain in mass, feed gain and FCR, although the values of increased plasma cortisol levels (Ganga et al., 2011). the last were slightly high as may be expected from a fish fed a In juveniles, replacement of FO by VO in broodstock diets also pelleted rather than a extruded diet (Izquierdo et al., 2003; Torno increased the ALA content in the muscle in comparison to that in et al., 2018). The use of fads2 gene expression as a selection juveniles obtained from broodstock fed FO, even though all the criterion in broodfish can indicate modifications in juvenile juveniles were fed the same low FM and low FO diet. VO inclusion offspring metabolism and improves their ability to cope with high in the broodstock diet also increased muscle saturated fatty acids, VM VO dietary levels. Recent studies in gilthead seabream show a particularly 16:0, and monounsaturated fatty acids, together with positive and significant correlation between fads2 expression levels 18:2n-6, while reducing other PUFA. These significant changes in in the peripheral blood cells and liver of broodstock (Ferosekhan the fatty acid profiles in juveniles obtained from broodstock fed et al., 2020). In mammals, a single nucleotide polymorphism in the different feeds, despite the fact that all juveniles were fed the same Fads2 gene occurs if mice are selected for low or high basal diet, indicate persistent changes in lipid metabolism by nutritional metabolism (Czajkowska et al., 2019). In fish, recent studies have programming initiated through broodstock nutrition. These results shown that one of the underlying mechanisms of these alterations in are in agreement with the previous findings that demonstrate that it metabolism appears to be caused by epigenetic modifications such is possible to nutritionally programme gilthead seabream offspring as methylation of the promoter region of fad2 in gilthead seabream by modifying the fatty acid profiles of the broodstock diet (Perera et al., 2019; Turkmen et al., 2019b). (Izquierdo et al., 2015; Turkmen et al., 2017b). However, in the We have previously demonstrated that it is possible to present study, FO replacement by LO negatively affected juvenile nutritionally programme gilthead seabream offspring by offspring growth and feed utilization, contrary to the improvement increasing ALA and LA, fatty acid precursors of LC-PUFA found in previous studies (Izquierdo et al., 2015; Turkmen et al., biosynthesis, and reducing EPA and DHA, products of the 2017b). A main difference between those studies and the present synthesis, in the broodstock diet (Izquierdo et al., 2015; Turkmen one is in the fatty acid profiles of the broodstock diets. et al., 2017b). However, the effect of increasing the dietary supply of Comparison of broodstock diets from those previous studies precursors without reducing the product fatty acids had not been (Izquierdo et al., 2015; Turkmen et al., 2017b) shows the ALA investigated. In the present study, the broodstock diets were content of the VO diet (V diet) was 20 times higher than in the FO diet formulated based on previous studies in which four different diets (F diet), and replacement of FO with VO caused a 40% decrease in n-3 with increasing levels of FO replacement by VO (0%, 60%, 80% LC-PUFA products. Feeding broodstock during the spawning period and 100% VO) were tested (Izquierdo et al., 2015; Turkmen et al., with a high ALA content diet in combination with the decrease in n-3 2017b). In these studies, 80% replacement of FO by VO had adverse LC-PUFA products altered the lipid metabolism of the progeny, which effects on spawning quality parameters, while fish fed diets with ledtoa20–30% increase of tissue n-3 LC-PUFA content and higher 60% replacement of FO performed equally well as those fed a 100% growth in 4 and 16 month old juveniles (Izquierdo et al., 2015; FO diet. In agreement with these findings, in the present study, a Turkmen et al., 2017b). However, in the present trial, because of the 70% replacement of FO by VO did not negatively affect spawning contribution of n-3 LC-PUFA of FM, although the level of ALA was quality and only slightly modified egg fatty acid profile. For 16 times higher in the V diet, LC-PUFA levels were only reduced by instance, FO replacement by VO in the broodstock diet led to an half in comparison to previous studies (Izquierdo et al., 2015; Turkmen increase in the egg content of saturated fatty acids, particularly 14:0 et al., 2017b). Comparison of offspring fatty acid composition shows n- and 16:0, direct products of lipid biosynthesis, in agreement with the 3 LC-PUFA content was not significantly different (∼5% change) and slightly higher lipid content found in these eggs. Regarding egg juveniles’ growth was also reduced. These results suggest the important LC-PUFA content, whereas FO replacement by VO led to a 15% role of LC-PUFA dietary levels for nutritional programming of reduction in DHA in the broodstock diet, DHA content in the egg gilthead seabream offspring through broodstock feeding. not only was not reduced but also was slightly increased by 2%. Indeed, dietary LC-PUFA are strong modulators of lipid DHA, oleic acid (18:1n-9) and 16:0 are the main fatty acids in metabolism in seabream and their ability to synthesize LC-PUFA various fish eggs and, together with EPA and ARA, are recognized (Izquierdo et al., 2008), as has also been seen in rodents (Gibson as the most important fatty acids during larval development et al., 2013). The results are in agreement with the long-lasting (Izquierdo, 1996). Additionally, whereas replacement of FO by effects of a reduction of both LC-PUFA precursors and products in VO in the broodstock diet lead to a 16 times increase in ALA in the first feeding diets for Atlantic salmon on lipid metabolism at the diet, ALA content in the eggs only increased 4 times. ALA is not a juvenile stage (Clarkson et al., 2017; Vera et al., 2017). major fatty acid component of fish eggs; thus, low retention of this In summary, this study confirms modifying the fatty acids in the fatty acid may be related to selective retention of the essential fatty broodstock diet causes metabolic changes in offspring of gilthead acids such as DHA. In vitro studies in pig oocytes showed that ALA seabream, and it also points to the importance of adequate supplement may enhance the nuclear maturation of oocyte and LC-PUFA levels in parental diets to enhance the offspring ability embryo development; however, excessive ALA could have a to synthesize LC-PUFA and promote juvenile growth. Additionally, negative influence by altering the oxidative status of the oocytes a major finding of the present study was that the broodstock’s ability (Lee et al., 2017). Dietary ALA is related to increased peroxidation to biosynthesize fatty acids as denoted by their fads2 expression risk. For instance, inclusion of LO, high in ALA, in place of FO in enhances reproductive performance. Similarly, broodstock fads2 the diet of juvenile gilthead seabream raises basal and post-acute expression levels affect offspring larval and juvenile growth rates, stress cortisol levels (Ganga et al., 2011). Moreover, excessive suggesting an advantage for offspring performance if fed with dietary ALA levels may cause the displacement of other n-3 PUFA a high VM VO diet. Further studies are being conducted to from phospholipids, particularly from the second position of the understand the underlying physiological and molecular phospholipids (Izquierdo, 2005; Izquierdo et al., 2000). Indeed, mechanisms involved in nutritional programming of gilthead increased FO replacement by LO leads to a deposition of ALA in seabream. Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Acknowledgements Gilthead Seabream, Sparus aurata Broodstock Fed a Low n-3 LC-PUFA Diet. Life The authors wish to thank Dr Monica Betancor for her suggestions on the analysis of 10, 117. doi:10.3390/life10070117 Ganga, R., Bell, J. G., Montero, D., Atalah, E., Vraskou, Y., Tort, L., Fernandez, fatty acids. A. and Izquierdo, M. S. (2011). Adrenocorticotrophic hormone-stimulated cortisol release by the head kidney inter-renal tissue from sea bream (Sparus aurata) fed Competing interests with linseed oil and soyabean oil. British Journal of Nutrition 105, 238-247. doi:10. The authors declare no competing or financial interests. 1017/S0007114510003430 Garcıa-C ́ eldrán, M., Ramis, G., Manchado, M., Estévez, A., Afonso, J., Marıa-́ Author contributions Dolores, E., Penalver, J. and Armero, E. (2015). Estimates of heritabilities and Conceptualization: S.T., M.I.; Methodology: S.T., M.J.Z., M.I.; Formal analysis: S.T., genetic correlations of growth and external skeletal deformities at different ages in M.J.Z., H.X., H.F.-P., L.R.; Investigation: S.T., H.X., H.F.-P., L.R.; Data curation: S.T.; a reared gilthead sea bream (Sparus aurata L.) population sourced from three Writing - original draft: S.T., M.I.; Writing - review & editing: M.J.Z., S.K., M.I.; broodstocks along the Spanish coasts. Aquaculture 445, 33-41. doi:10.1016/j. Visualization: S.T.; Supervision: S.K., M.I.; Project administration: M.J.Z., S.K., M.I.; aquaculture.2015.04.006 Funding acquisition: S.K., M.I. Gibson, R. A., Neumann, M. A., Lien, E. L., Boyd, K. A. and Tu, W. C. (2013). Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets Funding high in polyunsaturated fatty acids. Prostaglandins, Leukotrienes and Essential This study was (partially) funded under the EU funded project PerformFISH Fatty Acids 88, 139-146. doi:10.1016/j.plefa.2012.04.003 (Integrating Innovative Approaches for Competitive and Sustainable Performance Gjedrem, T. and Rye, M. (2018). Selection response in fish and shellfish: a review. Reviews in Aquaculture 10, 168-179. doi:10.1111/raq.12154 across the Mediterranean Aquaculture Value Chain; Horizon 2020 H2020-SFS- Gjedrem, T., Robinson, N. and Rye, M. (2012). The importance of selective 2016-2017; 27610). breeding in aquaculture to meet future demands for animal protein: A review. Aquaculture 350-353, 117-129. doi:10.1016/j.aquaculture.2012.04.008 Supplementary information Gotoh, T. (2015). Potential of the application of epigenetics in animal production. Supplementary information available online at Animal Production Science 55, 145-158. doi:10.1071/AN14467 https://jeb.biologists.org/lookup/doi/10.1242/jeb.214999.supplemental Izquierdo, M. S. (1996). Essential fatty acid requirements of cultured marine fish larvae. Aquaculture Nutrition 2, 183-191. doi:10.1111/j.1365-2095.1996.tb00058.x References Izquierdo, M. (2005). Essential fatty acid requirements in Mediterranean fish Afonso, J. M., Manchado, M., Estevez, A., Ramis, G., Lee, I., Ponce, M., Perez- species. Cah. Options Mediterr 63, 91-102. Sanchez, J., Armero, E., Navarro, A., Puertas, M. A. et al. (2012). Development Izquierdo, M., Socorro, J., Arantzamendi, L. and Hernández-Cruz, C. (2000). of a genetic program in gilthead seabream (Sparus aurata L.) between industry Recent advances in lipid nutrition in fish larvae. Fish Physiology and Biochemistry and research centers in Spain. In AQUA 2012. Prague, Czech Republic. 22, 97-107. doi:10.1023/A:1007810506259 Burdge, G. C. and Lillycrop, K. A. (2010). Nutrition, epigenetics, and Izquierdo, M. S., Fernandez-Palacios, H. and Tacon, A. G. J. (2001). Effect of developmental plasticity: implications for understanding human disease. Annual broodstock nutrition on reproductive performance of fish. Aquaculture 197, 25-42. review of nutrition 30, 315-339. doi:10.1146/annurev.nutr.012809.104751 doi:10.1016/S0044-8486(01)00581-6 Cassar-Malek, I., Picard, B., Bernard, C. and Hocquette, J.-F. (2008). Application Izquierdo, M. S., Obach, A., Arantzamendi, L., Montero, D., Robaina, L. and of gene expression studies in livestock production systems: a European Rosenlund, G. (2003). Dietary lipid sources for seabream and seabass: growth perspective. Australian Journal of Experimental Agriculture 48, 701-710. doi:10. performance, tissue composition and flesh quality. Aquaculture Nutrition 9, 1071/EA08018 397-407. doi:10.1046/j.1365-2095.2003.00270.x Castro, L. F., Tocher, D. R. and Monroig, O. (2016). Long-chain polyunsaturated Izquierdo, M. S., Montero, D., Robaina, L., Caballero, M. J., Rosenlund, G. and fatty acid biosynthesis in chordates: Insights into the evolution of Fads and Elovl Ginés, R. (2005). Alterations in fillet fatty acid profile and flesh quality in gilthead gene repertoire. Progress in Lipid Research 62, 25-40. doi:10.1016/j.plipres.2016. seabream (Sparus aurata) fed vegetable oils for a long term period. Recovery of 01.001 fatty acid profiles by fish oil feeding. Aquaculture 250, 431-444. Chavanne, H., Janssen, K., Hofherr, J., Contini, F., Haffray, P., Komen, H., Izquierdo, M. S., Robaina, L., Juárez-Carrillo, E., Oliva, V., Hernández-Cruz, C. Nielsen, E. E., Bargelloni, L. and Consortium, A. (2016). A comprehensive and Afonso, J. (2008). Regulation of growth, fatty acid composition and delta 6 survey on selective breeding programs and seed market in the European desaturase expression by dietary lipids in gilthead seabream larvae (Sparus aquaculture fish industry. Aquaculture International 24, 1287-1307. doi:10.1007/ aurata). Fish Physiology and Biochemistry 34, 117-127. doi:10.1007/s10695-007- s10499-016-9985-0 9152-7 Clarkson, M., Migaud, H., Metochis, C., Vera, L. M., Leeming, D., Tocher, D. R. Izquierdo, M. S., Turkmen, S., Montero, D., Zamorano, M. J., Afonso, J. M., and Taylor, J. F. (2017). Early nutritional intervention can improve utilisation of Karalazos, V. and Fernández-Palacios, H. (2015). Nutritional programming vegetable-based diets in diploid and triploid Atlantic salmon (Salmo salar L.). through broodstock diets to improve utilization of very low fishmeal and fish oil Br. J. Nutrition 118, 1-13. doi:10.1017/S0007114517001842 diets in gilthead sea bream. Aquaculture 449, 18-26. doi:10.1016/j.aquaculture. Czajkowska, M., Brzę k, P. and Dobrzyń,P. (2019). A novel polymorphism in the 2015.03.032 fatty acid desaturase 2 gene (Fads2): A possible role in the basal metabolic rate. Janssen, K., Chavanne, H., Berentsen, P. and Komen, H. (2017). Impact of PLoS ONE 14, e0213138. selective breeding on European aquaculture. Aquaculture 472, 8-16. doi:10.1016/ de Verdal, H., Komen, H., Quillet, E., Chatain, B., Allal, F., Benzie, J. A. H. and j.aquaculture.2016.03.012 Vandeputte, M. (2018). Improving feed efficiency in fish using selective breeding: Le Boucher, R., Dupont-Nivet, M., Vandeputte, M., Kerneïs, T., Goardon, L., a review. Reviews in Aquaculture 10, 833-851. doi:10.1111/raq.12202 Labbé, L., Chatain, B., Bothaire, M. J., Larroquet, L., Médale, F. et al. (2012). Dolinoy, D. C., Weidman, J. R., Waterland, R. A. and Jirtle, R. L. (2006). Maternal Selection for Adaptation to Dietary Shifts: Towards Sustainable Breeding of vy Genistein Alters Coat Color and Protects A Mouse Offspring from Obesity by Carnivorous Fish. PLoS ONE 7, e44898. doi:10.1371/journal.pone.0044898 Modifying the Fetal Epigenome. Environmental Health Perspectives 114, Le Boucher, R., Vandeputte, M., Dupont-Nivet, M., Quillet, E., Ruelle, F., 567-572. doi:10.1289/ehp.8700 Vergnet, A., Kaushik, S., Allamellou, J. M., Medale, F. and Chatain, B. (2013). Engrola, S., Aragao, C., Valente, L. M. and Conceiçao, L. E. (2018). Nutritional ̃ ̃ Genotype by diet interactions in European sea bass (Dicentrarchus labrax Modulation of Marine Fish Larvae Performance. In Emerging Issues in Fish L. 1756): nutritional challenge with totally plant-based diets. Journal of Animal Larvae Research (ed. M. Yúfera), pp. 209-228. Cham, Switzerland: Springer. Science 91, 44-56. Fernandes, T., Herlin, M., Belluga, M. D. L., Ballón, G., Martinez, P., Toro, M. A. Lee, J.-E., Yong, H., Kim, H.-Y., Lee, W.-H., Cheong, H.-T., Yang, B.-K. and Park, and Fernández, J. (2017). Estimation of genetic parameters for growth traits in a C.-K. (2017). Effect of Alpha-Linolenic Acid on Oocyte Maturation and Embryo hatchery population of gilthead sea bream (Sparus aurata L.). Aquaculture Development in Pigs. Development & Reproduction 21, 205-213. doi:10.12717/ International 25, 499-514. DR.2017.21.2.205 Fernandez Palacios, H., Norberg, B., Izquierdo, M. and Hamre, K. (2011). Effects Lee–Montero, I., Navarro, A., Negrıń –Báez, D., Zamorano, M., Berbel, C., of broodstock diet on eggs and larvae. In Larval Fish Nutrition (ed. J. G. Holt), pp. Sanchez, J., Garcıa–Celdran, M., Manchado, M., Estevez, A. and Armero, E. ́ ́ ́ 151-181. Oxford, UK: Wiley-Blackwell. (2015). Genetic parameters and genotype–environment interactions for skeleton Fernández-Palacios, H., Izquierdo, M. S., Robaina, L., Valencia, A., Salhi, M. deformities and growth traits at different ages on gilthead seabream (Sparus and Vergara, J. (1995). Effect of n−3 HUFA level in broodstock diets on egg aurata L.) in four S panish regions. Animal Genetics 46, 164-174. doi:10.1111/ quality of gilthead sea bream (Sparus aurata L.). Aquaculture 132, 325-337. age.12258 doi:10.1016/0044-8486(94)00345-O Lemaitre, R. N., Siscovick, D. S., Berry, E. M., Kark, J. D. and Friedlander, Y. Ferosekhan, S., Turkmen, S., Xu, H., Afonso, J. M., Zamorano, M. J., Kaushik, (2008). Familial aggregation of red blood cell membrane fatty acid composition: S. and Izquierdo, M. (2020). The Relationship between the Expression of Fatty the Kibbutzim Family Study. Metabolism 57, 662-668. doi:10.1016/j.metabol. Acyl Desaturase 2 (fads2) Gene in Peripheral Blood Cells (PBCs) and Liver in 2007.12.011 Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Lillycrop, K. A. and Burdge, G. C. (2018). Chapter 13 - Interactions Between Torrecillas, S., Robaina, L., Caballero, M. J., Montero, D., Calandra, G., Mompel, Polyunsaturated Fatty Acids and the Epigenome. In Polyunsaturated Fatty Acid D., Karalazos, V., Kaushik, S. and Izquierdo, M. S. (2017b). Combined Metabolism, pp. 225-239. AOCS Press. replacement of fishmeal and fish oil in European sea bass (Dicentrarchus labrax): Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data Production performance, tissue composition and liver morphology. Aquaculture using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25, 402-408. 474, 101-112. doi:10.1016/j.aquaculture.2017.03.031 doi:10.1006/meth.2001.1262 Turchini, G. M., Torstensen, B. E. and Ng, W.-K. (2009). Fish oil replacement in Monroig, O., Tocher, D. R. and Castro, L. F. C. (2018). Chapter 3 - Polyunsaturated finfish nutrition. Reviews in Aquaculture 1, 10-57. doi:10.1111/j.1753-5131.2008. Fatty Acid Biosynthesis and Metabolism in Fish A2 - Burdge, Graham C. In 01001.x Polyunsaturated Fatty Acid Metabolism, pp. 31-60. AOCS Press. Turkmen, S., Castro, P. L., Caballero, M. J., Hernández-Cruz, C. M., Saleh, R., Montero, D. and Izquierdo, M. (2010). Welfare and health of fish fed vegetable oils Zamorano, M. J., Regidor, J. and Izquierdo, M. (2017a). Nutritional stimuli of as alternative lipid sources to fish oil. In Fish oil replacement and alternative lipid gilthead seabream (Sparus aurata) larvae by dietary fatty acids: effects on larval sources in aquaculture feeds (eds. G. M. Turchini, W. K. Ng and D. R. Tocher), pp. performance, gene expression and neurogenesis. Aquaculture Research 48, 439-485. Boca Raton, Florida, USA: CRC Press. 202-213. doi:10.1111/are.12874 Morais, S., Mendes, A. C., Castanheira, M. F., Coutinho, J., Bandarra, N., Dias, Turkmen, S., Zamorano, M. J., Fernandez-Palacios, H., Hernandez-Cruz, C. M., J., Conceiçao, L. E. C. and Pousao-Ferreira, P. (2014). New formulated diets for ̃ ̃ Montero, D., Robaina, L. and Izquierdo, M. (2017b). Parental nutritional Solea senegalensis broodstock: Effects of parental nutrition on biosynthesis of programming and a reminder during juvenile stage affect growth, lipid metabolism long-chain polyunsaturated fatty acids and performance of early larval stages and and utilisation in later developmental stages of a marine teleost, the gilthead sea juvenile fish. Aquaculture 432, 374-382. doi:10.1016/j.aquaculture.2014.04.033 bream (Sparus aurata). British Journal of Nutrition 118, 500-512. doi:10.1017/ Panserat, S., Marandel, L., Seiliez, I. and Skiba-Cassy, S. (2019). New Insights on S0007114517002434 Intermediary Metabolism for a Better Understanding of Nutrition in Teleosts. Annu. Turkmen, S., Hernandez-Cruz, C. M., Zamorano, M. J., Fernandez-Palacios, H., Rev. Anim. Biosci. 7, 195-220. doi:10.1146/annurev-animal-020518-115250 Montero, D., Afonso, J. M. and Izquierdo, M. (2019a). Long-chain PUFA profiles Perera, E., Turkmen, S., Simo-Mirabet, P., Zamorano, M. J., Xu, H., Naya-Catala, in parental diets induce long-term effects on growth, fatty acid profiles, expression F., Izquierdo, M. and Perez-Sanchez, J. (2019). Stearoyl-CoA desaturase of fatty acid desaturase 2 and selected immune system-related genes in the (scd1a) is epigenetically regulated by broodstock nutrition in gilthead sea bream offspring of gilthead seabream. British Journal of Nutrition 122, 25-38. doi:10. (Sparus aurata). Epigenetics 15, 1-18. doi:10.1080/15592294.2019.1699982 Rosenlund, G., Corraze, G., Izquierdo, M. and Torstensen, B. E. (2010). The 1017/S0007114519000977 Turkmen, S., Perera, E., Zamorano, M. J., Simó-Mirabet, P., Xu, H., Pérez- effects of fish oil replacement on nutritional and organoleptic qualities of farmed fish. In Fish oil replacement and alternative lipid sources in aquaculture feeds Sánchez, J. and Izquierdo, M. (2019b). Effects of dietary lipid composition and (eds. G. M. Turchini, W. K. Ng and D. R. Tocher), pp. 487-522. Boca Raton, fatty acid desaturase 2 expression in broodstock gilthead sea bream on lipid Florida, USA: CRC Press. metabolism-related genes and methylation of the fads2 gene promoter in their Schmittgen, T. D. and Livak, K. J. (2008). Analyzing real-time PCR data by the offspring. International Journal of Molecular Sciences 20, 6250. doi:10.3390/ comparative CT method. Nature Protocols 3, 1101-1108. doi:10.1038/nprot. ijms20246250 2008.73 Vagner, M. and Santigosa, E. (2011). Characterization and modulation of gene Siemelink, M., Verhoef, A., Dormans, J. A., Span, P. N. and Piersma, A. H. expression and enzymatic activity of delta-6 desaturase in teleosts: A review. (2002). Dietary fatty acid composition during pregnancy and lactation in the rat Aquaculture 315, 131-143. doi:10.1016/j.aquaculture.2010.11.031 programs growth and glucose metabolism in the offspring. Diabetologia 45, Vagner, M., Zambonino Infante, J. L., Robin, J. H. and Person-Le Ruyet, J. 1397-1403. doi:10.1007/s00125-002-0918-2 (2007). Is it possible to influence European sea bass (Dicentrarchus labrax) Sokal, R. R. and Rohlf, F. J. (1969). The Principles and Practice of Statistics in juvenile metabolism by a nutritional conditioning during larval stage? Aquaculture Biological Research. WH Freeman and company. 267, 165-174. doi:10.1016/j.aquaculture.2007.01.031 Stoffel, W., Holz, B., Jenke, B., Binczek, E., Gunter, R., Kiss, C., Vera, L. M., Metochis, C., Taylor, J. F., Clarkson, M., Skjærven, K. H., Migaud, H. Karakesisoglou, L., Thevis, M., Weber, A.-A., Arnhold, S. et al. (2008). Δ6- and Tocher, D. R. (2017). Early nutritional programming affects liver desaturase (FADS2) deficiency unveils the role of ω3- and ω6-polyunsaturated transcriptome in diploid and triploid Atlantic salmon, Salmo salar. BMC fatty acids. The EMBO Journal 27, 2281-2292. doi:10.1038/emboj.2008.156 Genomics 18, 886. doi:10.1186/s12864-017-4264-7 Thorland, I., Kottaras, L., Refstie, T., Dimitroglou, A., Papaharisis, L. and Rye, Vestergren, A. S., Trattner, S., Pan, J., Johnsson, P., Kamal-Eldin, A., Brä nnä s, M. (2015). Response to Selection for Harvest Weight in a Family Based Selection E., Moazzami, A. A. and Pickova, J. (2012). The effect of combining linseed oil Program of Gilthead Seabream (Sparus aurata). In XII International Symposium and sesamin on the fatty acid composition in white muscle and on expression of on Genetics in Aquaculture, Santiago de Compostela, Spain. lipid-related genes in white muscle and liver of rainbow trout (Oncorhynchus Torno, C., Staats, S., Michl, S. C., De Pascual-Teresa, S., Izquierdo, M., mykiss). Aquaculture International 21, 843-859. doi:10.1007/s10499-012-9511-y Rimbach, G. and Schulz, C. (2018). Fatty Acid Composition and Fatty Acid Watanabe, T. and Vassallo-Agius, R. (2003). Broodstock nutrition research on Associated Gene-Expression in Gilthead Sea Bream (Sparus aurata) are Affected marine finfish in Japan. Aquaculture 227, 35-61. doi:10.1016/S0044- by Low-Fish Oil Diets, Dietary Resveratrol, and Holding Temperature. Marine 8486(03)00494-0 Drugs 16, 379. doi:10.3390/md16100379 Xu, H., Turkmen, S., Rimoldi, S., Terova, G., Zamorano, M. J., Afonso, J. M., Torrecillas, S., Mompel, D., Caballero, M. J., Montero, D., Merrifield, D., Rodiles, Sarih, S., Fernández-Palacios, H. and Izquierdo, M. (2019). Nutritional A., Robaina, L., Zamorano, M. J., Karalazos, V., Kaushik, S. et al. (2017a). intervention through dietary vegetable proteins and lipids to gilthead sea bream Effect of fishmeal and fish oil replacement by vegetable meals and oils on gut health of European sea bass (Dicentrarchus labrax). Aquaculture 468, 386-398. (Sparus aurata) broodstock affects the offspring utilization of a low fishmeal/fish oil doi:10.1016/j.aquaculture.2016.11.005 diet. Aquaculture 513, 734402. doi:10.1016/j.aquaculture.2019.734402 Journal of Experimental Biology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Biology The Company of Biologists http://www.deepdyve.com/lp/the-company-of-biologists/parental-lc-pufa-biosynthesis-capacity-and-nutritional-intervention-j6ZEspLcIO

Loading next page...

References (45)

Gjedrem (2012)
The importance of selective breeding in aquaculture to meet future demands for animal protein: A review
Aquaculture, 350-353
Ganga (2011)
Adrenocorticotrophic hormone-stimulated cortisol release by the head kidney inter-renal tissue from sea bream (Sparus aurata) fed with linseed oil and soyabean oil
British Journal of Nutrition, 105
Lee–Montero (2015)
Genetic parameters and genotype–environment interactions for skeleton deformities and growth traits at different ages on gilthead seabream (Sparus aurata L.) in four S panish regions
Animal Genetics, 46
Lillycrop (2018)
Chapter 13 - Interactions Between Polyunsaturated Fatty Acids and the Epigenome
Perera (2019)
Stearoyl-CoA desaturase (scd1a) is epigenetically regulated by broodstock nutrition in gilthead sea bream (Sparus aurata)
Epigenetics, 15
Morais (2014)
New formulated diets for Solea senegalensis broodstock: Effects of parental nutrition on biosynthesis of long-chain polyunsaturated fatty acids and performance of early larval stages and juvenile fish
Aquaculture, 432
Fernandez Palacios (2011)
Effects of broodstock diet on eggs and larvae
Izquierdo (2001)
Effect of broodstock nutrition on reproductive performance of fish
Aquaculture, 197
Dolinoy (2006)
Maternal Genistein Alters Coat Color and Protects Avy Mouse Offspring from Obesity by Modifying the Fetal Epigenome
Environmental Health Perspectives, 114
Clarkson (2017)
Early nutritional intervention can improve utilisation of vegetable-based diets in diploid and triploid Atlantic salmon (Salmo salar L.)
Br. J. Nutrition, 118
Gotoh (2015)
Potential of the application of epigenetics in animal production
Animal Production Science, 55
Monroig (2018)
Chapter 3 - Polyunsaturated Fatty Acid Biosynthesis and Metabolism in Fish A2 - Burdge, Graham C
Janssen (2017)
Impact of selective breeding on European aquaculture
Aquaculture, 472
Castro (2016)
Long-chain polyunsaturated fatty acid biosynthesis in chordates: Insights into the evolution of Fads and Elovl gene repertoire
Progress in Lipid Research, 62
Izquierdo (2015)
Nutritional programming through broodstock diets to improve utilization of very low fishmeal and fish oil diets in gilthead sea bream
Aquaculture, 449
Livak (2001)
Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method
Methods, 25
Cassar-Malek (2008)
Application of gene expression studies in livestock production systems: a European perspective
Australian Journal of Experimental Agriculture, 48
Le Boucher (2012)
Selection for Adaptation to Dietary Shifts: Towards Sustainable Breeding of Carnivorous Fish
PLoS ONE, 7
Burdge (2010)
Nutrition, epigenetics, and developmental plasticity: implications for understanding human disease
Annual review of nutrition, 30
Chavanne (2016)
A comprehensive survey on selective breeding programs and seed market in the European aquaculture fish industry
Aquaculture International, 24
Rosenlund (2010)
The effects of fish oil replacement on nutritional and organoleptic qualities of farmed fish
Engrola (2018)
Nutritional Modulation of Marine Fish Larvae Performance
Gjedrem (2018)
Selection response in fish and shellfish: a review
Reviews in Aquaculture, 10
Izquierdo (2005)
Alterations in fillet fatty acid profile and flesh quality in gilthead seabream (Sparus aurata) fed vegetable oils for a long term period. Recovery of fatty acid profiles by fish oil feeding
Aquaculture, 250
Lee (2017)
Effect of Alpha-Linolenic Acid on Oocyte Maturation and Embryo Development in Pigs
Development & Reproduction, 21
Izquierdo (1996)
Essential fatty acid requirements of cultured marine fish larvae
Aquaculture Nutrition, 2
Izquierdo (2000)
Recent advances in lipid nutrition in fish larvae
Fish Physiology and Biochemistry, 22
Czajkowska (2019)
A novel polymorphism in the fatty acid desaturase 2 gene (Fads2): A possible role in the basal metabolic rate
PLoS ONE, 14
Panserat (2019)
New Insights on Intermediary Metabolism for a Better Understanding of Nutrition in Teleosts
Annu. Rev. Anim. Biosci., 7
Fernández-Palacios (1995)
Effect of n−3 HUFA level in broodstock diets on egg quality of gilthead sea bream (Sparus aurata L.)
Aquaculture, 132
Siemelink (2002)
Dietary fatty acid composition during pregnancy and lactation in the rat programs growth and glucose metabolism in the offspring
Diabetologia, 45
Le Boucher (2013)
Genotype by diet interactions in European sea bass (Dicentrarchus labrax L. 1756): nutritional challenge with totally plant-based diets
Journal of Animal Science, 91
Montero (2010)
Welfare and health of fish fed vegetable oils as alternative lipid sources to fish oil
Yúfera M. (2018)
Emerging Issues in Fish Larvae Research
Gibson (2013)
Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets high in polyunsaturated fatty acids
Prostaglandins, Leukotrienes and Essential Fatty Acids, 88
Schmittgen (2008)
Analyzing real-time PCR data by the comparative CT method
Nature Protocols, 3
Lemaitre (2008)
Familial aggregation of red blood cell membrane fatty acid composition: the Kibbutzim Family Study
Metabolism, 57
García-Celdrán (2015)
Estimates of heritabilities and genetic correlations of growth and external skeletal deformities at different ages in a reared gilthead sea bream (Sparus aurata L.) population sourced from three broodstocks along the Spanish coasts
Aquaculture, 445
Ferosekhan (2020)
The Relationship between the Expression of Fatty Acyl Desaturase 2 (fads2) Gene in Peripheral Blood Cells (PBCs) and Liver in Gilthead Seabream, Sparus aurata Broodstock Fed a Low n-3 LC-PUFA Diet
Life, 10
Izquierdo (2003)
Dietary lipid sources for seabream and seabass: growth performance, tissue composition and flesh quality
Aquaculture Nutrition, 9
Izquierdo (2008)
Regulation of growth, fatty acid composition and delta 6 desaturase expression by dietary lipids in gilthead seabream larvae (Sparus aurata)
Fish Physiology and Biochemistry, 34
Afonso (2012)
Development of a genetic program in gilthead seabream (Sparus aurata L.) between industry and research centers in Spain
de Verdal (2018)
Improving feed efficiency in fish using selective breeding: a review
Reviews in Aquaculture, 10
Fernandes (2017)
Estimation of genetic parameters for growth traits in a hatchery population of gilthead sea bream (Sparus aurata L.)
Izquierdo (2005)
Essential fatty acid requirements in Mediterranean fish species
Cah. Options Mediterr, 63

Publisher: The Company of Biologists
Copyright: © 2021 The Company of Biologists. All rights reserved.
ISSN: 0022-0949
eISSN: 0022-0949
DOI: 10.1242/jeb.214999
Publisher site: See Article on Publisher Site

Abstract

© 2020. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 RESEARCH ARTICLE Parental LC-PUFA biosynthesis capacity and nutritional intervention with alpha-linolenic acid affect performance of Sparus aurata progeny 1,2, 1 1 1 1 Serhat Turkmen *, Maria J. Zamorano , Hanlin Xu , Hipólito Fernández-Palacios , Lidia Robaina , 1 1 Sadasivam Kaushik and Marisol Izquierdo ABSTRACT 2019). Polyunsaturated fatty acids (PUFA) play important roles in programming the metabolism of different organisms (Lillycrop and Environmental factors such as nutritional interventions during early Burdge, 2018). Gilthead seabream (Sparus aurata), a marine fish, is developmental stages affect and establish long-term metabolic an interesting animal model to study the effects of alterations of changes in all animals. Diet during the spawning period has a PUFA during the early stages as the yolk sac composition of the nutritional programming effect in offspring of gilthead seabream and offspring depends on the continuous uptake of nutrients and can be affects long-term metabolism. Studies showed modulation of genes modified to some extent through the diets supplied during the such as fads2, which is considered to be a rate-limiting step in the spawning period (Fernández-Palacios et al., 1995). In recent years, synthesis of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). there has been increasing interest in the replacement (either partial or However, it is still unknown whether this adaptation is related to the complete) of fishmeal (FM) and fish oil (FO) with more readily presence of precursors or to limitations in the pre-formed products, n-3 available plant protein sources and vegetable oils (VO) in diets used LC-PUFA, contained in the diets used during nutritional programming. for aquaculture (Turchini et al., 2009; Vestergren et al., 2012). This study investigated the combined effects of nutritional However, high substitution levels may negatively affect a range of programming on Sparus aurata through broodstock diets during the different parameters such as growth or health (Izquierdo et al., 2005; spawning period and in broodfish showing higher or lower fads2 Montero and Izquierdo, 2010; Rosenlund et al., 2010; Torrecillas expression levels in the blood after 1 month of feeding with a diet et al., 2017a; Torrecillas et al., 2017b). While marine oils are rich containing high levels of plant protein sources and vegetable oils (VM/ sources of n-3 long-chain polyunsaturated fatty acids (LC-PUFA), VO). Broodfish showing high fads2 expression had a noticeable oils extracted from conventional terrestrial oil seeds – namely VO – improvement in spawning quality parameters as well as in the growth of rarely have ≥20 carbon fatty acids and totally lack essential 6 month old offspring when challenged with a high VM/VO diet. fatty acids, such as eicosapentaenoic acid (20:5n-3, EPA) and Further, nutritional conditioning with 18:3n-3-rich diets had an adverse docosahexaenoic acid (22:6n-3, DHA). Commonly used VOs can be effect in comparison to progeny obtained from fish fed high fish meal rich in precursors of LC-PUFA, such as alpha-linolenic acid (18:3n-3, and fish oil (FM/FO) diets, with a reduction in growth of juveniles. ALA) and linoleic acid (18:2n-6, LA), and certain oils such as linseed Improved growth of progeny from the high fads2 broodstock combined oil (LO) even have a very high content of ALA, which is the primer with similar muscle fatty acid profiles is also an excellent option for substrate for the n-3 LC-PUFA. In the LC-PUFA biosynthesis tailoring and increasing the flesh n-3 LC-PUFA levels to meet the pathway, there are several elongation, desaturation and β-oxidation recommended dietary allowances for human consumption. steps, and in different species of fish, there are differences in the KEY WORDS: Nutritional adaptation, Offspring nutrition, Parental evolution of the synthesis capacity (Castro et al., 2016; Monroig et al., nutrition, Long-chain polyunsaturated fatty acids, Fatty acid 2018). Therefore, maximizing the capacity of these pathways in a desaturase, fads2, Epigenetics, Aquaculture, Nutritional given species is of interest to improve the utilization of VO sources in programming fish, given the limited availability of marine fish oils. Recently, studies have looked into the possibility of channelling INTRODUCTION specific metabolic pathways of fish either through broodstock Environmental factors such as nutritional interventions during early nutrition or through early nutritional conditioning (Engrola et al., developmental stages exert long-lasting metabolic and physiological 2018; Izquierdo et al., 2015; Panserat et al., 2019; Turkmen et al., changes in mammals (Burdge and Lillycrop, 2010) and other 2017a; Turkmen et al., 2017b; Vagner et al., 2007). Nutritional vertebrates, including fish (Izquierdo et al., 2015; Turkmen et al., programming presumes that early environmental clues such as diet 2017a; Turkmen et al., 2019a; Turkmen et al., 2017b; Xu et al., or specific nutrients provide the organism with the capacity to forecast environmental challenges in later life and give it the 1 opportunity to adjust its metabolism to better adapt it to the new Aquaculture Research Group (GIA), IU-ECOAQUA, Universidad de Las Palmas de Gran Canaria, Crta. Taliarte s/n, 35214 Telde, Spain. Department of Biology, environment, potentially improving health, reproduction and University of Alabama at Birmingham, Birmingham, AL 35294, USA. survival (Burdge and Lillycrop, 2010). Hence, this tool may have applications in the animal production sector, as shown in ruminants, *Author for correspondence ([email protected]) to channel the individuals to better utilization of key nutrients S.T., 0000-0003-0410-9161; M.J.Z., 0000-0003-1569-9152; H.X., 0000-0003- (Gotoh, 2015). In rodents, dietary fatty acid composition during 4425-0776; H.F.-P., 0000-0003-1410-8154; L.R., 0000-0003-4857-6693; pregnancy and lactation influences growth and glucose metabolism S.K., 0000-0001-7856-8374; M.I., 0000-0003-4297-210X in the offspring (Siemelink et al., 2002). Likewise, maternal Received 19 September 2019; Accepted 9 October 2020 phytoestrogens can affect gene expression and alter skin colour as Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 well as susceptibility to obesity in adulthood (Dolinoy et al., 2006). this sense, blood fatty acid composition or fads2 expression could be Among marine teleosts, gilthead seabream is a multi-batch spawner used as an important trait. Although there are genetic selection whose eggs largely depend on the continuous intake of nutrients by programmes in gilthead seabream such as the public Spanish breeding the broodstock during the spawning season when egg composition programme PROGENSA (Afonso et al., 2012), and other can be modified – to some extent – by the diets used (Fernandez commercial breeding programmes (Fernandes et al., 2017; Thorland Palacios et al., 2011). Indeed, previous research in gilthead et al., 2015) which include selection pressure on growth traits and an seabream has established that feeds supplied during the spawning absence of body deformities (García-Celdrán et al., 2015; Lee– season play a critical role in regulating lipid metabolism even in the Montero et al., 2015), up to now there has been no information about offspring (Izquierdo et al., 2015; Turkmen et al., 2017b). For the use of fads2 expression as a biological biomarker in fish. instance, expression of genes related to the fatty acid desaturation The present study aimed to determine the influence of using and elongation pathways, such as fatty acyl desaturase 2 ( fads2) and broodstock with high fads2 expression in combination with fatty acid elongase 6 (elovl6), was affected, as well as expression of conditioning feeding with a high VO diet on spawning quality and other genes involved in lipid metabolism, such as carnitine growth of offspring during larval development and juvenile stage palmitoyltransferase I (cpt1) or lipoprotein lipase (lpl) (Izquierdo when challenged with very low levels of marine fisheries-derived et al., 2015). Some stress-related genes were also found to be ingredients. Several parameters related to egg and larval quality, regulated in the offspring obtained from broodstock fed diets with growth and fatty acid profiles were studied to ascertain a potential different VO levels during early larval stages (Turkmen et al., nutritional programming effect in the offspring. 2019a). Low FM FO feed utilization can also be improved by early dietary interventions in Atlantic salmon (Salmo salar) (Clarkson MATERIALS AND METHODS et al., 2017), through the up-regulation of different genes including All the experiments described below were conducted according to those involved in oxidative phosphorylation, pyruvate metabolism, the European Union Directive (2010/63/EU) on the protection of tricarboxylic acid cycle, glycolysis or fatty acid metabolism (Vera animals for scientific purposes, at the facilities of Ecoaqua Institute, et al., 2017). That nutritional interventions by different fatty acids University of Las Palmas de Gran Canaria (Canary Islands, Spain). during periods of high developmental plasticity may also regulate dietary lipid utilization in later stages in fish was also shown in Sampling of blood and identification of parental groups European seabass (Vagner et al., 2007). In the gilthead seabream, A total of 70 gilthead seabream, Sparus aurata Linnaeus 1758, our own previous studies showed that replacement of different levels broodstock fish (42 females and 28 males, aged 2–4 years) were of FO (rich in n-3 series fatty acids with 20 or more carbon atoms, used for identifying individuals with high or low fads2 expression n-3 LC-PUFA) by VO (rich in C fatty acids) improved growth of for the current investigation. These individuals were fed a high VO offspring challenged with a high vegetable meal (VM) and VO diet diet (Table S1) at a daily feeding ration equal to 1% biomass at (Izquierdo et al., 2015). However, these studies did not show 08:00 h and 14:00 h, 6 days a week for 1 month. After the feeding whether this effect was related to the increase in C precursor or the period, whole blood was taken from the caudal vein of the brood fish reduction on n-3 LC-PUFA in the programming diet used during the for the identification of fads2 expression levels of the individuals. spawning period to condition the offspring. Prior to measurements, all fish were anaesthetized with 10 ppm Selective breeding provides the opportunity to improve the clove oil/methanol (1:1 v/v) in sea water. A sample of 2 ml blood economic production efficiency of aquatic livestock (Gjedrem et al., was taken with 2 ml sterile syringes (Terumo Europe NV, Leuven, 2012). In fish, critical analysis of data on the response to selection for Belgium) and transferred to 2.5 ml K3 EDTA tubes (L.P. Italiana, different species and traits shows that gain per generation can vary Milan, Italy). Whole-blood samples were kept on ice during between 4% and 40%, depending on the species and the chosen sampling and immediately centrifuged at 11,200 g, 4°C for 20 min. criterion (Chavanne et al., 2016; de Verdal et al., 2018; Gjedrem and Plasma was separated, and erythrocytes were snap frozen with liquid Rye, 2018; Janssen et al., 2017). Such selection programmes can be nitrogen and kept at −80°C until RNA extraction. RNA extraction also tailored to address the present needs in aquaculture such as and purification are explained more in detail below (see utilization of feeds that are less reliant on marine capture fishery- ‘Biochemistry and gene expression analyses’). Broodstock derived ingredients. Studies undertaken with rainbow trout showing the highest and the lowest fads2 expression from the 70 (Oncorhynchus mykiss)aswellasEuropeansea bass fish sampled were separated into two groups, HD and LD, (Dicentrarchus labrax) have shown the potential of selection for respectively. Twelve brood fish of each sex from the HD and 12 improved use of 100% VM and VO diets free of marine sources (Le from the LD fads2 expression group, with similar length and mass Boucher et al., 2012). One of the approaches in animal breeding (P>0.05, Table 1), were distributed into 12 experimental tanks in a programmes is to use biomarkers for a certain expected outcome by flow-through system with filtered seawater (mean±s.d. 37.0±0.5‰ identifying genes related to the desirable characteristics (Cassar-Malek salinity, 19.59–21.30°C) at a renewal rate of 100% per hour with et al., 2008), as was shown in rainbow trout (Le Boucher et al., 2013). proper aeration. In this sense, the fads2 gene which codifies the delta-6-desaturase enzyme (delta-6), a rate-limiting step of LC-PUFA biosynthesis, is a Spawning quality parameters very strong candidate as its regulation in response to VO is well Spawning quality was determined before and after feeding with the documented in a variety of fish species (Vagner and Santigosa, 2011), experimental diets (Table 2). At the beginning of the trial, the mean including gilthead seabream (Izquierdo et al., 2008). In mice, body mass for females and males was measured (Table 1). Fish from knockout of Fads2 and the absence of LC-PUFA in the diet the HD and LD groups were randomly assigned to experimental resulted in failure of reproduction, showing the key role of Fads2 tanks using a ratio of 1 female and 1 male per tank. After placing the among seven other desaturases (Scd1–5, Fads1, Fads3) in this species brood fish in the experimental tanks, fish were fed with commercial (Stoffel et al., 2008). Studies in humans show a very high correlation diets and spawning quality was monitored daily. Two isocaloric and (up to 70%) between red blood cell fatty acid composition of the isonitrogenous diets (Biomar, Aarhus, Denmark) were formulated parents and its inheritance in the offspring (Lemaitre et al., 2008). In to contain either high levels of FM and FO (diet F) or 30% FO and Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Table 1. Gilthead seabream broodstock mass, fads2 expression and proximate composition of eggs produced after the feeding experiments Broodstock Eggs fads2 expression Crude protein Crude lipid Ash Group Mass (kg) (fold-change) (% dry mass) (% dry mass) (% dry mass) FHD 2.1±0.2 3.9±2.6 69.8±2.7 22.4±5.1 16.3±1.4 FLD 1.9±0.2 0.3±0.1 63.3±2.9 23.3±2.5 19.4±3.6 VHD 2.0±0.9 7.0±3.7 69.3±0.8 26.0±1.6 17.8±0.5 VLD 1.8±0.1 0.5±0.2 68.2±3.2 26.9±0.2 13.3±0.6 Broodstock mass and fatty acyl desaturase 2 gene ( fads2) expression before the feeding experiment are means±s.d. (n=6). Broodstock were fed diets containing different fish oil (FO) and vegetable oil (VO) ratios for 30 days: FHD, high fads2 expression broodfish (HD) fed with 100% FO diet (F); FLD, low fads2 expression broodfish (LD) fed with 100% FO diet (F); VHD, high fads2 expression broodfish (HD) fed with 30% FO–70% VO diet (V); and VLD, low fads2 expression broodfish (LD) fed with 30% FO–70% VO diet (V). Proximate egg composition data are means±s.d. (n=3, one pool of eggs from all the spawn per broodstock tank). Different letters indicate a significant difference between the groups (P<0.01). Two-way ANOVA values were calculated for the effect of selection, diet and the interaction of selection and diet from mean±s.d. data (n=3). Selection had a significant effect on broodstock fads2 fold-change in expression; P<0.01); all other parameters were not significant (P>0.05). 70% VO (diet V; Table 2). Once equal spawning quality parameters The spawning quality parameters were determined using eggs were observed between the tanks during the acclimation period of collected daily from each tank at around 08:00 h and concentrated in 21 days (P>0.05, data not shown), tanks were randomly assigned to 5 l beakers. Immediately, eggs were transferred to the laboratory and one of the four experimental groups as follows: FHD, high fads2 aeration was supplied to ensure mixing of the eggs through the water expression broodfish (HD) fed with 100% FO diet (F); FLD, low column. Then, a 5 ml sample was taken using a graduated glass fads2 expression broodfish (LD) fed with 100% FO diet (F); VHD, pipette and transferred to a Bogorov chamber. Eggs were counted high fads2 expression broodfish (HD) fed with 30% FO–70% VO and observed under a binocular microscope (Leica Microsystems, diet (V); and VLD, low fads2 expression broodfish (LD) fed with Wetzlar, Germany) in five replicates to calculate the total number of 30% FO–70% VO diet (V). eggs and the percentage of fertilized eggs, and to determine the morphological characteristics. Egg viability rate was determined as the percentage of morphologically normal eggs at the morula stage, Table 2. Formulation, main ingredients and biochemical composition of described as transparent, perfectly spherical with clear, symmetrical the broodstock diets used during the spawning period early cleavage (Fernandez Palacios et al., 2011). After that, eggs were randomly transferred to 96-well ELISA microplates using a F diet V diet (30% (100% FO) FO–70% VO) micropipette (0.7 ml of seawater and one egg per well). Plates were observed under a binocular microscope to ensure that there was a Raw material (%) single fertilized egg in each well. These eggs were kept at a Meals from marine sources 50.0 50.0 Sunflower meal 13.2 13.2 controlled temperature of 23°C. By observing the egg from these Soybean meal 10.0 10.0 ELISA plates after 24 and 72 h under a binocular microscope, Fish oil 8.0 2.4 hatching rate and survival 3 days post-hatching (dph) were Linseed oil – 5.6 calculated as percentages. With these percentage values, the total Wheat 9.9 9.9 number of fertilized, viable and hatched eggs and larvae alive at Corn gluten meal 60 7.0 7.0 3 dph was calculated per kg female per spawn. Vitamin & mineral premix 1.0 1.0 Biochemical composition (% dry matter) Moisture 9.1 8.8 Larval rearing and growth Protein (crude) 56.3 56.1 After 1 month of feeding with the respective diets, eggs were collected Lipids (crude) 17.2 17.1 and distributed into 500 l tanks for mass production at a density of 100 Ash 8.6 8.5 −1 eggs l from each treatment group. All larvae were reared following −1 Gross energy (MJ kg ) 21.2 21.2 the same common rearing protocol, regardless of the origin of the Fatty acids (% of total fatty acids) spawn. Water renewal in the tanks was progressively increased from 16:1n-7 7.1 4.3 18:2n-6 5.6 9.9 10% to 40% per hour until 46 dph and the water was continuously −1 18:3n-3 0.9 16.3 aerated (125 ml min ). Larvae were reared under natural photoperiod 20:1n-7 12.4 6.8 and living phytoplankton [Nannochloropsis sp.; mean±s.d. 20:4n-6 0.4 0.3 3 −1 250(±100)×10 cells ml ] was added to the rearing tanks. From 3 20:5n-3 6.3 4.8 to 17 dph, larvae were fed twice a day with rotifers (Brachionus 22:1n-11 15.7 8.6 −1 plicatilis, 10 rotifer ml ) enriched with commercial emulsions (ORI- 22:6n-3 7.1 6.0 GREEN, Skretting, Norway). From 15 to 32 dph, Artemia sp. FO, fish oil; VO, vegetable oil. Contains Fishmeal NA LT 70 (Fishmeal SA 68, enriched with commercial emulsions (ORI-GREEN) was added to the Feed Service Bremen, Germany). 48 Hi Pro Solvent Extra (Svane Shipping, Kolding, Denmark). South American Fish Oil (LDN Fish Oil, Hedensted, rearing tanks 3 times a day. From 20 dph, larvae were fed commercial 4 5 Denmark). Ch. Daudruy (Dunkerque, France). Vitamin and mineral premix diets according to the suggested diet particle size by the manufacturer −1 provided the following vitamins (mg kg ): A 3.8, D 0.05, E 102.4, K3 9.8, B1 (Gemma Micro, Skretting, France). Water was continuously aerated 2.7, B2 8.3, B6 4.8, B12 0.25, B3 24.8, B5 17.2, folic acid 2.8, H 0.14, C 80; −1 (125 ml min ) attaining 6.1±0.4 ppm dissolved O (mean±s.d. ). −1 minerals (mg kg ): cobalt 0.94, iodine 0.7, selenium 0.2, iron 32.6, Average water temperature and pH along the trial were 21.1±0.4°C −1 manganese 12, copper 3.2, zinc 67; other (g kg ): taurine 2.45, methionine and 7.0±0.6, respectively. Water quality was monitored daily as 0.5, histidine 1.36, cholesterol 1.13 (DSM, Heerlen, The Netherlands; Evonik regards dissolved O and pH. At 3, 15 and 30 dph, growth was Industries, Essen, Germany; Deutsche Lanolin Gesellschaft, Frankfurt am 2 Main, Germany). determined by measuring the total length of 60 anaesthetized larvae Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 per treatment using a profile projector (V-12A, Nikon, Tokyo, Japan). Fish were kept in a flow-through system and had a natural photoperiod RNA extraction methods are detailed below (see ‘Biochemistry and (12 h light:12 h dark). Triplicate groups of fish initially coming from gene expression analyses’). At 46 dph, each experimental group was one of the four broodstock groups (FHD, FLD, VHD and VLD) were transferred to 10,000 l tanks in duplicate. reared with a pelleted low FM and low FO diet (Table 3) and were fed until apparent satiation twice a day at 08:00 h and 14:00 h, 6 days a Juvenile nutritional challenge test week over 60 days. Uneaten pellets were collected in a net by slowly Obtained offspring were kept in similar environmental conditions. opening the water outlet for 10 min after each meal, dried in an oven Fish were fed with commercial diets all through the grow-out period for 24 h and weighed to estimate net feed intake. The amount of feed until they reached 6 months of age, and were fed 3 times a day by given to each tank was recorded daily. Water temperature and hand until apparent satiation. Rearing water temperature and oxygen levels were monitored daily after the last feeding. photoperiod were natural and equal for all the experimental groups Average water temperature was 22.5±0.7°C, and dissolved oxygen (18.5–21.8°C, 11–13 h light). Triplicate groups of juvenile fish (mean −1 was 6.1±0.3 mg l during the experimental period. ±s.d. initial body mass of all groups: 23.3±0.7 g, n=12) from each All fish were anaesthetized with 10 ppm clove oil:methanol broodstock group were assigned to one of 12 tanks (500 l capacity) for (1:1 v/v) in sea water prior to measurements for the initial and final the juvenile feeding challenge experiment (n=75 per each treatment). samplings. Fish were fasted for 24 h then individually weighed. Growth was determined by measuring wet body mass after 24 h starvation at 30 and 60 days of the experiment. Prior to measurements, Table 3. Formulation, main ingredients and biochemical composition of all fish were anaesthetized with 10 ppm clove oil:methanol (1:1 v/v) in the diets used for juveniles during the nutritional challenge sea water. The experimental scheme is presented graphically in Fig. 1. Content Proximate composition Content Growth and feed utilization parameters were calculated using the following equations: feed conversion ratio (FCR)=(dry mass of Main ingredients (%) Biochemical composition (% dry matter) consumed feed)/(final biomass−initial biomass), and weight gain Fishmeal 5.0 Crude lipids 21.8 (%)=(final biomass−initial biomass)/initial biomass×100, where mass Fishmeal alternative 54.5 Crude protein 57.2 is in g. protein sources Rapeseed meal cake 11.3 Moisture 6.5 Biochemistry and gene expression analyses Wheat 6.9 Ash 6.7 2 −1 Lipid, protein, ash and moisture analysis of the samples was done as Fish oil 3.0 Gross energy (MJ kg ) 22.5 previously described (Turkmen et al., 2017b). For each 200 µl of Vegetable oil mix 13.0 Micronutrient mix 6.3 blood cells, 1 ml of TRI Reagent (Sigma-Aldrich, St Louis, MO, USA) was added into 2 ml Eppendorf tubes. To each tube, four Total fatty acids (%) Total fatty acids pieces of 1 mm diameter zirconium glass beads were added and (%) (continued) homogenized using TissueLyzer-II (Qiagen, Hilden, Germany) for 14:0 4.69 18:3n-4 0.04 −1 60 s with a frequency of 30 s ; 250 µl chloroform was added to 14:1n-5 0.17 18:3n-3 14.86 15:0 0.28 18:4n-3 0.91 homogenized samples, which were then centrifuged at 12,000 g for 16:0 13.67 20:0 0.21 15 min at 4°C for phase separation. The clear upper aqueous phase 16:1n-7 4.50 20:1n-9 0.37 containing RNA was mixed with 75% ethanol and transferred into 16:1n-5 0.09 20:1n-7 7.25 an RNeasy spin column to bind total RNA. Then, RNA was 16:2n-6 0.25 20:1n-5 0.37 extracted using an RNeasy Mini Kit (Qiagen) with the protocol 17:0 0.13 20:2n-6 0.16 supplied by the manufacturer. Real-time quantitative PCR was 16:3n-4 0.14 20:4n-6 0.31 16:3n-3 0.14 20:3n-3 0.09 performed in an iQ5 Multi-colour Real-Time PCR detection system 16:3n-1 0.04 20:4n-3 0.19 (Bio-Rad) using β-actin (acbt) as the housekeeping gene in a final 16:4n-3 0.22 20:5n-3 3.81 volume of 15 μl per reaction well and with 100 ng of total RNA 18:0 3.23 22:1n-11 9.35 reverse transcribed to cDNA. Samples, housekeeping gene, cDNA 18:1n-9 15.75 22:1n-9 1.00 template and reaction blanks were analysed in duplicate. Primer 18:1n-7 2.46 22:4n-6 0.04 efficiency was tested with serial dilutions of a cDNA pool (1:5, 18:1n-5 0.23 22:5n-6 0.03 1:10, 1:100 and 1:1000). Sequences of the primers used in this study 18:2n-6 9.84 22:5n-3 0.28 18:2n-4 0.05 22:6n-3 4.49 were: acbt (GenBank accession no. X89920) 5′–3′ (F) TCT GTC 18:3n-6 0.08 TGG ATC GGA GGC TC, (R) AAG CAT TTG CGG TGG ACG); *For a complete list of ingredients, see Torrecillas et al. (2017b) diet code 5FM/ and fads2 (GenBank accession no. AY055749) 5′–3′ (F) CGA 3FO. GAG CCA CAG CAG CAG GGA, (R) CGG CCT GCG CCT GAG 1 2 South American, Superprime (Feed Service, Bremen, Germany). Blood CAG TT). Gene, primer efficiency and blank samples were meal spray (Daka, Hedensted, Denmark), soya protein concentrates 60% analysed in 96-well PCR plates (Multiplate, Bio-Rad). Melting- (Svane Shipping), corn gluten meal 60 (Cargill, Schiphol, The Netherlands), curve analysis was performed, and amplification of a single product wheat gluten (Cargill). Linseed (2.6%) (Ch. Daudruy), rapeseed (5.2%) was confirmed after each run. Fold-change in expression of each (Emmelev, Otterup, Denmark) and palm oils (5.2%) (Cargill). Micronutrient ΔΔCT −1 gene was determined by the delta-delta CT method (2 ) (Livak mix (vitamin mix, 0.75%) supplied the following vitamins (mg kg ): A 3.8, D 0.05, E 102.4, K3 9.8, B1 2.7, B2 8.3, B6 4.8, B12 0.25, B3 24.8, B5 17.2, folic and Schmittgen, 2001). PCR efficiencies were similar, and no −1 acid 2.8, H 0.14, C 80; minerals (mg kg ): cobalt 0.94, iodine 0.7, selenium efficiency correction was required (Livak and Schmittgen, 2001; −1 0.2, iron 32.6, manganese 12, copper 3.2, zinc 67; other (g kg ): taurine 2.45, Schmittgen and Livak, 2008). methionine 0.5, histidine 1.36, cholesterol 1.13 (DSM; Evonik Industries; Deutsche Lanolin Gesellschaft); supplemented ingredients (5.49%); contains Statistical analysis lysine, methionine, monocalcium phosphate, choline, inositol, phospholipids Results are expressed as means±s.d. (n=3), unless otherwise (Vilomix, Mørke, Denmark), Evonik Industries, Pöhner (Hamburg, Germany); antioxidant (0.5%): BAROX BECP, Ethoxyquin (Vilomix); yttrium oxide (0.3%). stated in the tables and figures. The data were compared Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 1 month 1 month 2 months 1 month Before spawning Gene expression analysis Acclimation Nutritional interventions n=70 (42 females & 28 males) n=24 (12 females and 28 males) 1 male : 1 female each tank High fads2 FHD 100% F0 VHD 30% F0–70% V0 Low fads2 FLD 100% FO VLD 30% F0–70% VO Experimental diet Commercial diet Commercial diet Nutritional programming diet 5% FO–3% FM 100% FO or 30% FO–70% VO 0–3 days after hatching 3–45 days after hatching 3 months 2 months Hatching & mouth opening On-growing Nutritional challenge test FHD FHD VHD VHD FLD FLD VLD VLD Yolk sac absorption Artemia and rotifers Commercial diet Nutritional challenge diet enriched with commercial 5% FM–3% FO emulsifiers Fig. 1. Experimental design and sampling points. All gilthead seabream groups were tested in triplicate. Sampling points are shown as red dashed lines, and experimental periods when fish were fed with different diets are shown as green lines. Experimental diet used for fatty acyl desaturase 2 gene (fads2) selection is given in Table S1; nutritional programming diet is given in Table 2; and nutritional challenge diet used at the juvenile stage is given in Table 3. FO, fish oil; FM, fish meal; VO, vegetable oil; FHD, high fads2 expression broodfish (HD) fed with 100% FO diet (F); FLD, low fads2 expression broodfish (LD) fed with 100% FO diet (F); VHD, high fads2 expression broodfish (HD) fed with 30% FO–70% VO diet (V); and VLD, low fads2 expression broodfish (LD) fed with 30% FO–70% VO diet (V). statistically using analysis of variance (ANOVA), at a RESULTS significance level of 5%. All variables were checked for Broodstock fads2 expression level, spawning quality normality and homogeneity of variance using the Kolmogorov– and eggs Smirnoff and Levene tests, respectively (Sokal and Rohlf, 1969). Individual broodstock fish showed very different expression levels If significant differences were detected with ANOVA, means of fads2 gene after being fed the experimental (conditioning) diet. were compared by Student’s t-test. All data were analysed (VM and VO diet; Table S1). The average fold-change in gene using IBM SPSS v23.0.0.2 for Mac (IBM SPSS Inc., Chicago, expression was 7.1±13.6, with a maximum of 61.6 and a minimum IL, USA). A Pearson correlation test was performed to identify of 0.02. No relationship was found between fish mass and fads2 relationships between parameters using R (http://www. expression among individuals (P>0.05, R=0.0061). Mass and fads2 R-project.org/). expression of the broodstock fish are presented in Table 1. Feeding Time Feeding Time Blood collection Separation of HD and LD fish Seeding of obtained eggs Sampling of juveniles Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 During the first part of the spawning season, when broodstock significantly increased levels of ALA and LA in the eggs were fed with the same diet, there were no differences in total (P>0.05) (Table 4). Analysis of egg fatty acid composition number of eggs per kg female per spawn, fertilization rate, viability, (Table 4) showed that among saturated fatty acids, 14:0 and 17:0 hatching rate and survival of larvae at 3 dph, which on average were were lower in V diet groups (P<0.05), while the other saturated fatty 49,023±1195, 46,339±1862, 33,990±2513, 31,899±3092, 22,960± acids such as 15:0, 16:0, 18:0 and 20:0 were similar among all 2114 eggs/larvae (10 /kg female/spawn) (means±s.d., P>0.05), groups (P>0.05) (Table 4). Additionally, egg content of monoenoic respectively. After feeding the broodstock with either the F or the V fatty acids such as 14:1n-7, 14:1n-5, 16:1n-7, 18:1n-7, 18:1n-5, diet (Table 2) for 1 month, the total number of eggs produced and 20:1n-9, 20:1n-7, 20:1n-5 and 20:1n-11 was lower in V-diet groups survival of 3 dph larvae were lower in broodstock with low fads2 (Table 4). Also, ALA, the first substrate for delta-6 in the n-3 expression (FLD and VLD), regardless of the diet. The difference LC-PUFA biosynthesis pathway to EPA and DHA, was up to 4 between the two groups was 37% lower total number of eggs and times higher (P<0.05) in eggs obtained from V-diet groups 10.2% lower number of 3 dph larvae in low fads2 expression groups (Table 4). In contrast, egg content of LA, another substrate for (FLD and VLD) than in those with high fads2 expression (FHD and LC-PUFA biosynthesis and high in VOs, was similar among eggs VHD) (P<0.05) (Fig. 2). The same trend was observed for the other from different groups (P>0.05; Table 4). However, the products of spawning quality parameters including fertilized, viable or hatched n-3 LC-PUFA biosynthesis such as arachidonic acid (ARA) 20: eggs, but without significant differences among groups (P>0.05) 4n-6, EPA 20:5n-3 and DHA 22:6n-3 were similar among the eggs (Fig. 2). The percentage ratios of fertilized eggs after feeding of different experimental groups (P>0.05) (Table 4). broodstock with the F and V diet was 92.6±7.3, 91.8±7.6, 89.3±7.3 and 89.9±9.5 in FHD, FLD, VHD and VLD groups, respectively. Larval growth The proximate composition of these eggs, obtained after 1 month of Despite the fact that larvae were kept under similar conditions and broodstock feeding with the diets, was not significantly different fed with the same diets, growth was higher in FHD larvae in among the different broodstock groups (P>0.05) (Table 1). fads2 comparison with VHD larvae at 3 dph (P<0.05; Table 5). At expression levels, diet and the interaction between these parameters 15 dph, among the progeny obtained from broodstock fed the F did not affect the proximate composition of the eggs (P>0.05) diet, those from the FLD group had a greater length than those (Table 1). Irrespective of the fads2 expression levels of the from the FHD group (P<0.05). Between the larval groups from broodfish, replacement of FO by VO in the diet led to broodstock with low fads2 expression, VLD larvae were smaller in Groups 50 FHD FLD VHD VLD Number of eggs Fertilized eggs Viable eggs Hatched eggs 3 dph larvae Fig. 2. Spawning parameters of gilthead seabream broodstock fed with either 100% FO (F diet) or 70% VO–30% FO (V diet) for 1 month. Broodstock fish were separated according high or low fads2 expression levels in the blood as described in Fig. 1. Data are presented as means±s.d. (n=3); different letters denote differences between groups (P<0.05). 3 –1 No. of eggs/larvae (10 kg per female per spawn) Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Table 4. Fatty acid composition of eggs produced after the broodstock feeding experiments Two-way ANOVA % Total fatty acids FHD FLD VHD VLD S D S×D A,x B y 14:0 4.24±0.01 3.44±0.08 2.83±0.48 3.16±0.55 0.29 ** * A,x B y 14:1n-7 0.05±0.00 0.04±0.01 0.02±0.01 0.03±0.01 0.27 ** * A,x B y 14:1n-5 0.14±0.01 0.11±0.00 0.09±0.01 0.10±0.02 0.21 ** * 15:0 0.31±0.00 0.30±0.03 0.27±0.03 0.29±0.03 0.82 0.20 0.34 A,x B y 15:1n-5 0.04±0.00 0.03±0.00 0.03±0.00 0.03±0.00 0.08 0.13 * 16:0ISO 0.05±0.00 0.05±0.00 0.05±0.00 0.05±0.01 0.57 0.09 0.55 16:0 18.08±0.74 21.37±2.97 19.73±2.41 18.83±1.59 0.35 0.72 0.12 x x y y 16:1n-7 6.31±0.02 5.79±0.28 4.75±0.49 4.77±0.21 0.19 ** 0.16 16:1n-5 0.09±0.01 0.10±0.01 0.09±0.01 0.09±0.01 0.74 0.99 0.74 16:2n-6 0.01±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.57 0.63 0.67 A,x B y 16:2n-4 0.25±0.00 0.18±0.03 0.16±0.04 0.18±0.02 0.10 * * A,x B y 17:0 0.18±0.00 0.15±0.01 0.14±0.02 0.13±0.01 * ** 0.09 x y 16:3n-4 0.20±0.00 0.23±0.02 0.20±0.02 0.19±0.00 0.42 * * 16:3n-3 0.12±0.01 0.10±0.01 0.09±0.02 0.10±0.02 0.66 0.12 0.32 y x 16:3n-1 0.06±0.00 0.09±0.02 0.08±0.01 0.08±0.01 0.12 0.37 0.08 16:4n-3 0.07±0.03 0.07±0.02 0.06±0.01 0.06±0.00 0.87 0.45 0.77 16:4n-1 0.01±0.00 0.01±0.00 0.01±0.00 0.01±0.00 0.51 0.16 0.90 18:0 3.67±0.24 3.98±0.69 4.03±0.62 4.03±0.58 0.64 0.55 0.64 B A 18:1n-9 19.67±0.63 22.88±1.71 21.25±1.98 20.51±0.67 0.16 0.63 * x y 18:1n-7 3.24±0.16 2.95±0.10 2.75±0.26 2.71±0.19 0.18 ** 0.27 x y 18:1n-5 0.26±0.05 0.19±0.03 0.15±0.04 0.16±0.04 0.19 * 0.10 x y 18:2n-9 0.27±0.01 0.21±0.04 0.19±0.04 0.22±0.08 0.67 0.19 0.14 18:2n-6 (LA) 9.39±0.91 10.83±0.91 11.45±0.97 11.55±0.45 0.15 * 0.20 A B 18:2n-4 0.11±0.00 0.11±0.02 0.11±0.01 0.10±0.00 0.56 0.82 0.17 A B 18:3n-6 0.44±0.01 0.26±0.05 0.27±0.09 0.32±0.08 0.11 0.15 * x y 18:3n-4 0.12±0.01 0.11±0.01 0.10±0.00 0.10±0.02 0.21 * 0.32 18:3n-3 (ALA) 1.82±0.64 1.68±0.40 5.49±2.70 7.28±3.36 0.53 ** 0.46 18:3n-1 0.01±0.00 0.00±0.00 0.01±0.00 0.01±0.00 0.64 0.09 0.12 18:4n-3 0.92±0.05 0.49±0.24 0.50±0.28 0.58±0.20 0.18 0.21 0.07 A,x B y 18:4n-1 0.10±0.01 0.07±0.01 0.07±0.00 0.07±0.01 * ** * 20:0 0.09±0.01 0.10±0.04 0.11±0.04 0.08±0.01 0.62 0.97 0.34 A,x B y 20:1n-9 0.40±0.09 0.22±0.04 0.17±0.06 0.21±0.09 0.15 * * A,x B y 20:1n-7 3.16±0.68 1.52±0.36 1.12±0.36 1.36±0.54 * ** * A,x B y 20:1n-5 0.26±0.04 0.17±0.02 0.14±0.03 0.15±0.04 0.07 ** * 20:2n-9 0.08±0.01 0.08±0.01 0.07±0.02 0.07±0.04 0.61 0.44 1.00 20:2n-6 0.31±0.02 0.38±0.05 0.32±0.05 0.30±0.02 0.33 0.12 0.10 20:3n-9 0.03±0.00 0.02±0.00 0.02±0.00 0.02±0.00 0.13 0.08 0.12 20:3n-6 0.16±0.02 0.14±0.01 0.12±0.01 0.13±0.03 0.79 0.15 0.33 20:4n-6 0.61±0.02 0.62±0.06 0.63±0.04 0.61±0.10 0.99 0.88 0.70 y x 20:3n-3 0.16±0.01 0.17±0.01 0.27±0.07 0.29±0.07 0.73 ** 0.92 x y 20:4n-3 0.62±0.08 0.46±0.10 0.43±0.07 0.44±0.05 0.13 * 0.08 20:5n-3 5.54±0.65 3.93±1.28 4.00±1.12 4.09±0.39 0.19 0.24 0.15 A,y B x 22:1n-11 1.23±0.30 0.46±0.13 0.35±0.16 0.51±0.29 0.05 * ** 22:1n-9 0.32±0.10 0.20±0.04 0.15±0.05 0.16±0.10 0.28 * 0.19 22:4n-6 0.06±0.01 0.05±0.00 0.05±0.00 0.05±0.01 0.42 0.35 0.30 x y 22:5n-6 0.05±0.01 0.04±0.00 0.03±0.01 0.03±0.00 0.17 * 0.35 22:5n-3 2.04±0.13 1.94±0.20 1.91±0.37 1.83±0.64 0.70 0.62 0.96 22:6n-3 14.67±0.19 13.70±2.20 15.14±3.69 13.92±4.17 0.54 0.85 0.94 Eggs were obtained from gilthead seabream after feeding broodstock with diets containing different FO and VO ratios for 30 days (means±s.d., n=3, one pool of eggs from all the spawn per broodstock tank). Capital letters denote differences between selection groups fed with the same diet during the spawning period; lowercase letters denote differences among diet groups with similar fads2 expression. Two-way ANOVA values were calculated for the effect of selection (S), diet (D) and the interaction of selection and diet (S×D) (*P<0.05, **P<0.01; non-significant P-values are shown). comparison to FLD larvae (P<0.05). There were no differences in highest final mass (47.4±0.8 g) and had the highest relative gain in larval growth among any of the experimental groups at 30 dph mass (99.7±7.3%) (Fig. 3). The two-way ANOVA analysis showed (P>0.05; Table 5). that broodstock with high fads2 expression levels (FHD and VHD) had a positive effect on juvenile offspring gain in mass (P<0.001), a Nutritional challenge test with juveniles nutritional programming effect of the broodstock diet (P<0.01), and There was no significant difference (P>0.05) in the initial mass of a significant interaction between these two parameters (P>0.05) the 6 month old juveniles at the beginning of the nutritional (Fig. 3). Thus, the parents showing high fads2 levels improved the challenge test (FHD: 23.4±0.1, FLD: 22.1±0.1, VHD: 23.8±0.4 and gain in mass of the offspring, regardless of the nutritional history of VLD: 23.7±0.1g). However, after 60 days of feeding juveniles with the broodstock (F or V diet) (Fig. 3). Progeny obtained from the high VM and VO diet (Table 3), the FHD group reached the broodstock selected for high fads2 expression and fed with the F Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Table 5. Length of gilthead seabream larvae obtained from broodstock fed with either 100% FO (FHD, FLD) or 70% VO–30% FO (VHD, VLD) for 1 month Total length (mm) Two-way ANOVA Larval age (dph) FHD FLD VHD VLD S D S×D A B 3 3.41±0.01 3.38±0.19 3.23±0.09 3.29±0.10 * A B 15 4.66±0.39 5.02±0.26 4.43±0.55 4.53±0.14 * 30 7.49±0.26 7.26±0.32 7.05±0.42 7.31±0.24 Data presented as means±s.d. (n=3, average of 20 larvae from per tank). Capital letters denote significant differences between differentially fed broodstock groups during the spawning period with similar fads2 expression (P<0.05). Two-way ANOVA values were calculated for the effect of selection (S), diet (D) and the interaction of selection and diet (S×D) (*P<0.05). diet (FHD) showed significantly higher (P<0.01) growth than parameters (P<0.01) (Table 7). In addition, if LD groups were progeny obtained from broodstock selected for high fads2 compared, 18:0 was expressed at a lower level (P<0.05) in VLD expression and fed with the V diet (VHD) (Fig. 3). Gain in mass than in FLD juveniles (Table 7). Total monounsaturated fatty acids of the progeny obtained from broodstock selected for low fads2 were affected by the diet in LD groups and were higher in the VLD expression and fed with the F diet (FLD) or the V diet (VLD) were than in the FLD group (P<0.05). n-6 PUFA were changed in not significantly different (P>0.05) (Fig. 3). In summary, VO response to the broodstock diet (P<0.05) and the interaction inclusion in the broodstock diets, thus increasing the dietary 18C between broodstock diet and broodfish fads2 expression levels fatty acid levels, resulted in lower gain in mass in juvenile offspring (P<0.1). LA was higher in the VLD group if compared with the FLD of broodstock selected for high fads2 (P<0.05) (Fig. 3), but not in group (P<0.05); however, there was a clear tendency of LA those coming from low fads2 broodstock. There was no difference accumulation in progeny from V-diet fed broodstock than in those (P>0.05) in feed intake of the fish. Additionally, FCR in juveniles from the F-diet fed broodstock (Table 7). The same kind of from broodstock selected for high fads2 expression (FHD and VHD differences were observed in ALA, with a broodstock diet leading to groups) was significantly lower than that in low fads2 expression greater accumulation of ALA in the V-diet groups than in those groups (FLD and VLD groups) (P<0.05) regardless of the diet fed to coming from the F-diet broodstock, while the accumulation was their parents (Fig. 3). In contrast, gain in mass (%) values were significantly higher in the VLD than in the FLD group (P<0.05). significantly higher in progeny from broodstock showing low fads2 However, there were no differences (P>0.05) in EPA, DHA or total expression, regardless of the broodstock diet (P<0.05) (Fig. 3). n-3 PUFA content among the experimental groups (Table 7). In addition, there were no differences in total PUFA content of the Muscle biochemical composition and fatty acid profiles of progeny obtained from broodstock with different fads2 expression juvenile offspring after the nutritional challenge levels and fed with either the V or F diet (P>0.05). Muscle biochemical composition was similar among the experimental groups (P<0.05; Table 6). The type of broodstock DISCUSSION diet (F or V) as well as the fads2 expression levels of the parents and The importance of the fads2 gene for reproduction in mammals has their combination led to significant (P<0.05) differences in the been demonstrated in Fads2 −/− mouse, where males are not able to muscle fatty acid profiles of offspring; this was particularly relevant produce mature sperm and folliculogenesis is disrupted in females for total saturated, monounsaturated and n-6 PUFA levels, whereas (Stoffel et al., 2008). Knocking out the Fads2 gene inhibits the total n-3 PUFA contents were similar (Table 7). Total saturated fatty synthesis of LC-PUFA, and other desaturases (Scd1–5, Fads1, acid levels were higher (P<0.05) in the VLD group than in the VHD Fads3) are not able to compensate for the deletion of Fads2 in the group, while the VHD group had higher levels of saturated fatty biosynthesis of ARA, EPA and DHA. In the present study, the acids than the FHD group (P<0.05). Among the saturated fatty broodstock group with higher fads2 expression showed a better acids, 14:0 and 16:0 were higher in the VLD group than in the VHD reproductive performance than those with low fads2 expression, as and FLD groups (P<0.05), thus the two-way ANOVA showed a denoted by the greater total number of eggs produced per kg female significant effect of broodstock fads2 expression levels (P<0.05), per spawn. To the best of our knowledge, this is the first study broodstock diet (P<0.01) and the interaction between these showing the positive effects of broodstock fads2 expression levels Fig. 3. Gain in mass and food conversion Diet P=0.01 Diet P=0.01 ratio (FCR) after nutritional challenge of Selection P=0.001 Selection P=0.001 gilthead seabream juveniles from Interaction P=0.05 Interaction P=0.05 broodstock fed either the F or V diet. Juveniles were challenged after 60 days with 5% FM and 3% FO diets. Data are presented as means±s.d. (two-way ANOVA; n=3); different letters denote differences between groups (P<0.05). (means±s.d., n=3). FHD FLD VHD VLD FHD FLD VHD VLD Gain in mass (%) FCR Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Table 6. Biochemical composition of muscle tissue of 6 month old to these findings, the present study demonstrates the importance of gilthead seabream after the nutritional challenge the broodstock’s ability to express fads2 on reproductive success, in agreement with a study conducted in mammals (Stoffel et al., 2008). Moisture Crude protein Crude lipids Ash Group (%) (% dry mass) (% dry mass) (% dry mass) Feeding broodstock with the F diet resulted in a greater length of larvae at 3 dph among larvae obtained from parents showing high FHD 69.2±2.1 67.1±6.4 26.6±5.5 1.5±0.1 fads2 expression and greater growth in 15 dph larvae obtained from FLD 69.6±0.6 65.8±3.3 24.3±1.9 1.3±0.2 VHD 70.6±2.2 69.5±3.6 27.2±1.3 1.5±0.02 parents showing low fads2 expression. By 30 dph, larvae were able VLD 71.1±0.8 67.4±2.5 25.0±3.3 1.7±0.2 to compensate for early differences in growth, indicating that the common rearing protocols supplied sufficient amounts of nutrients Biochemical composition of muscle tissue after 2 months feeding on a very low FM (5%) and FO (3%) diet in 6 month old gilthead seabream originating from for growth. It has been shown in several species of fish that broodstock in the different fads2 expression–diet groups. Data are means±s.d. parental nutritional history and dietary interventions during early (n=3). Two-way ANOVA values were calculated for the effect of selection, diet ontogenesis can have significant impacts on offspring development, and the interaction of selection and diet from mean±s.d. data (n=3); there were somatic mass, metabolism and immune responses (Izquierdo et al., no significant effects (P>0.05). 2015; Morais et al., 2014; Turkmen et al., 2017b). In the present study, growth differences at 3 and 15 dph may be related to the on spawning quality in fish. The importance of LC-PUFA on fish genetic background of the eggs in relation to the broodstock reproductive success is well documented in a number of teleosts expression of fads2, as rearing with the same feeding protocol (Izquierdo et al., 2001; Watanabe and Vassallo-Agius, 2003), allowed the different larval groups to achieve similar growth including gilthead seabream (Fernandez Palacios et al., 2011; performance at 30 dph. Interestingly, when juvenile offspring were Fernández-Palacios et al., 1995; Izquierdo et al., 2001). In addition challenged with a low FM and low FO diet, those obtained from Table 7. Fatty acid composition of muscle of gilthead seabream fed the nutritional challenge diet −1 Fatty acid (g 100 g tissue) Two-way ANOVA FHD FLD VHD VLD S D D×S Saturated y B A,x 14:0 17.4±8.8 9.8±3.3 17.2±32 41.3±4.3 ** *** *** 15:0 8.3±5.7 1.7±0.6 3.2±18 4.1±0.3 * y B A,x 16:0 84.9±36.4 95.1±17 188.5±175 293.6±19.4 *** *** ** 17:0 7.6±5.4 2.9±1.9 3±15 3.1±1 y x 18:0 35.9±7.7 38±5.5 66.8±138 67.7±2.9 *** 20:0 8.8±3.6 4.3±0.4 5.3±0 5.1±0.7 y B,x A Σ Saturated 158.7±15.4 171.5±1.2 274.2±238 427.3±9.7 *** *** *** Monounsaturated A B 16:1n-9 7.7±5 0.4±0.2 0.8±4 1±0.1 ** * y B,x A 16:1n-7 17.1±5.2 25.1±4.6 42.7±43 71.1±9.6 *** *** * 18:1n-9 350.8±120.4 290.6±52.9 607.5±1335 582.7±199.8 *** y x 18:1n-7 27.1±0.6 28.3±5.1 48.9±118 50.6±2.3 *** 20:1n-9 8.7±4.4 2.9±0.8 3.5±0 3.2±0.7 20:1n-7 19.2±3.3 19.2±3.7 25±14 20.6±3.1 * 22:1n-11 37.8±27.2 15±1.2 14.7±11 9.3±3.3 22:1n-9 16.5±12.2 8±2.9 7±7 5.4±1.4 y x Σ Monounsaturated 504.8±135.6 383.8±92.6 766.8±1744 854.9±42.2 *** n-6 PUFA y x 18:2n-6 144.6±44.3 112.2±18.8 236±520 251.2±36.9 *** 18:3n-6 15.6±6.7 10.5±7 11.3±22 12.2±0.9 20:2n-6 14.8±7.3 4.3±1.2 6.2±3 5.8±0.4 * A B 20:3n-6 4.9±0.1 3.4±0 4.5±5 3.6±0.2 *** 20:4n-6 16±7.1 8.6±3.2 8.7±7 8.7±0.2 22:4n-6 19.5±12.7 3.3±1.5 2.5±7 3±1.7 ** 22:5n-6 9.6±5.9 4.4±0.7 4.3±3 4.9±1.3 y x Σn-6 PUFA 240±46.5 141.5±26.2 273.5±549 304.2±16.5 ** * n-3 PUFA y x 18:3n-3 88.4±16.1 58.4±11.3 136.9±283 144.1±42.6 *** 18:4n-3 13.8±5.6 9.2±1.4 15±36 15.2±1.9 A,x B y 20:3n-3 13.9±6.2 5±2.5 4.8±4 4.7±0.8 ** ** * 20:4n-3 10.6±4.6 8±2.6 9.7±8 8.9±1.2 20:5n-3 57.6±29.2 53.5±8.4 67.3±73 57.1±15.5 22:5n-3 39.3±17.5 26.3±6.3 30±9 23.3±4.4 22:6n-3 110.1±29.4 125±23.8 122.4±31 102.2±21.1 Σn-3 PUFA 370.1±119.6 325.9±14.6 386.1±360 355.4±18.6 Σ PUFA 662.1±184.6 475.9±112.2 673.8±957 658.5±39.4 Fatty acid composition of muscle tissue after 2 months feeding on a very low FM (5%) and FO (3%) diet in 6 month old gilthead seabream originating from broodstock in the different fads2 expression–diet groups. Note, total PUFA also includes 16:2, 16:3 and 16:4 fatty acids. Data are means±s.d. (n=3, one pool from five fishes per tank×3). Capital letters denote differences between broodstock selection groups fed with the same diet during the spawning period; lowercase letters denote significant differences among differentially fed broodstock groups during the spawning period with similar fads2 expression. Two-way ANOVA values were calculated for the effect of selection, diet and the interaction of selection and diet (*P<0.1, **P<0.05, ***P<0.01). Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 broodstock with high fads2 expression again showed a positive seabream organs, and ALA content in head kidney is related to effect on gain in mass, feed gain and FCR, although the values of increased plasma cortisol levels (Ganga et al., 2011). the last were slightly high as may be expected from a fish fed a In juveniles, replacement of FO by VO in broodstock diets also pelleted rather than a extruded diet (Izquierdo et al., 2003; Torno increased the ALA content in the muscle in comparison to that in et al., 2018). The use of fads2 gene expression as a selection juveniles obtained from broodstock fed FO, even though all the criterion in broodfish can indicate modifications in juvenile juveniles were fed the same low FM and low FO diet. VO inclusion offspring metabolism and improves their ability to cope with high in the broodstock diet also increased muscle saturated fatty acids, VM VO dietary levels. Recent studies in gilthead seabream show a particularly 16:0, and monounsaturated fatty acids, together with positive and significant correlation between fads2 expression levels 18:2n-6, while reducing other PUFA. These significant changes in in the peripheral blood cells and liver of broodstock (Ferosekhan the fatty acid profiles in juveniles obtained from broodstock fed et al., 2020). In mammals, a single nucleotide polymorphism in the different feeds, despite the fact that all juveniles were fed the same Fads2 gene occurs if mice are selected for low or high basal diet, indicate persistent changes in lipid metabolism by nutritional metabolism (Czajkowska et al., 2019). In fish, recent studies have programming initiated through broodstock nutrition. These results shown that one of the underlying mechanisms of these alterations in are in agreement with the previous findings that demonstrate that it metabolism appears to be caused by epigenetic modifications such is possible to nutritionally programme gilthead seabream offspring as methylation of the promoter region of fad2 in gilthead seabream by modifying the fatty acid profiles of the broodstock diet (Perera et al., 2019; Turkmen et al., 2019b). (Izquierdo et al., 2015; Turkmen et al., 2017b). However, in the We have previously demonstrated that it is possible to present study, FO replacement by LO negatively affected juvenile nutritionally programme gilthead seabream offspring by offspring growth and feed utilization, contrary to the improvement increasing ALA and LA, fatty acid precursors of LC-PUFA found in previous studies (Izquierdo et al., 2015; Turkmen et al., biosynthesis, and reducing EPA and DHA, products of the 2017b). A main difference between those studies and the present synthesis, in the broodstock diet (Izquierdo et al., 2015; Turkmen one is in the fatty acid profiles of the broodstock diets. et al., 2017b). However, the effect of increasing the dietary supply of Comparison of broodstock diets from those previous studies precursors without reducing the product fatty acids had not been (Izquierdo et al., 2015; Turkmen et al., 2017b) shows the ALA investigated. In the present study, the broodstock diets were content of the VO diet (V diet) was 20 times higher than in the FO diet formulated based on previous studies in which four different diets (F diet), and replacement of FO with VO caused a 40% decrease in n-3 with increasing levels of FO replacement by VO (0%, 60%, 80% LC-PUFA products. Feeding broodstock during the spawning period and 100% VO) were tested (Izquierdo et al., 2015; Turkmen et al., with a high ALA content diet in combination with the decrease in n-3 2017b). In these studies, 80% replacement of FO by VO had adverse LC-PUFA products altered the lipid metabolism of the progeny, which effects on spawning quality parameters, while fish fed diets with ledtoa20–30% increase of tissue n-3 LC-PUFA content and higher 60% replacement of FO performed equally well as those fed a 100% growth in 4 and 16 month old juveniles (Izquierdo et al., 2015; FO diet. In agreement with these findings, in the present study, a Turkmen et al., 2017b). However, in the present trial, because of the 70% replacement of FO by VO did not negatively affect spawning contribution of n-3 LC-PUFA of FM, although the level of ALA was quality and only slightly modified egg fatty acid profile. For 16 times higher in the V diet, LC-PUFA levels were only reduced by instance, FO replacement by VO in the broodstock diet led to an half in comparison to previous studies (Izquierdo et al., 2015; Turkmen increase in the egg content of saturated fatty acids, particularly 14:0 et al., 2017b). Comparison of offspring fatty acid composition shows n- and 16:0, direct products of lipid biosynthesis, in agreement with the 3 LC-PUFA content was not significantly different (∼5% change) and slightly higher lipid content found in these eggs. Regarding egg juveniles’ growth was also reduced. These results suggest the important LC-PUFA content, whereas FO replacement by VO led to a 15% role of LC-PUFA dietary levels for nutritional programming of reduction in DHA in the broodstock diet, DHA content in the egg gilthead seabream offspring through broodstock feeding. not only was not reduced but also was slightly increased by 2%. Indeed, dietary LC-PUFA are strong modulators of lipid DHA, oleic acid (18:1n-9) and 16:0 are the main fatty acids in metabolism in seabream and their ability to synthesize LC-PUFA various fish eggs and, together with EPA and ARA, are recognized (Izquierdo et al., 2008), as has also been seen in rodents (Gibson as the most important fatty acids during larval development et al., 2013). The results are in agreement with the long-lasting (Izquierdo, 1996). Additionally, whereas replacement of FO by effects of a reduction of both LC-PUFA precursors and products in VO in the broodstock diet lead to a 16 times increase in ALA in the first feeding diets for Atlantic salmon on lipid metabolism at the diet, ALA content in the eggs only increased 4 times. ALA is not a juvenile stage (Clarkson et al., 2017; Vera et al., 2017). major fatty acid component of fish eggs; thus, low retention of this In summary, this study confirms modifying the fatty acids in the fatty acid may be related to selective retention of the essential fatty broodstock diet causes metabolic changes in offspring of gilthead acids such as DHA. In vitro studies in pig oocytes showed that ALA seabream, and it also points to the importance of adequate supplement may enhance the nuclear maturation of oocyte and LC-PUFA levels in parental diets to enhance the offspring ability embryo development; however, excessive ALA could have a to synthesize LC-PUFA and promote juvenile growth. Additionally, negative influence by altering the oxidative status of the oocytes a major finding of the present study was that the broodstock’s ability (Lee et al., 2017). Dietary ALA is related to increased peroxidation to biosynthesize fatty acids as denoted by their fads2 expression risk. For instance, inclusion of LO, high in ALA, in place of FO in enhances reproductive performance. Similarly, broodstock fads2 the diet of juvenile gilthead seabream raises basal and post-acute expression levels affect offspring larval and juvenile growth rates, stress cortisol levels (Ganga et al., 2011). Moreover, excessive suggesting an advantage for offspring performance if fed with dietary ALA levels may cause the displacement of other n-3 PUFA a high VM VO diet. Further studies are being conducted to from phospholipids, particularly from the second position of the understand the underlying physiological and molecular phospholipids (Izquierdo, 2005; Izquierdo et al., 2000). Indeed, mechanisms involved in nutritional programming of gilthead increased FO replacement by LO leads to a deposition of ALA in seabream. Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Acknowledgements Gilthead Seabream, Sparus aurata Broodstock Fed a Low n-3 LC-PUFA Diet. Life The authors wish to thank Dr Monica Betancor for her suggestions on the analysis of 10, 117. doi:10.3390/life10070117 Ganga, R., Bell, J. G., Montero, D., Atalah, E., Vraskou, Y., Tort, L., Fernandez, fatty acids. A. and Izquierdo, M. S. (2011). Adrenocorticotrophic hormone-stimulated cortisol release by the head kidney inter-renal tissue from sea bream (Sparus aurata) fed Competing interests with linseed oil and soyabean oil. British Journal of Nutrition 105, 238-247. doi:10. The authors declare no competing or financial interests. 1017/S0007114510003430 Garcıa-C ́ eldrán, M., Ramis, G., Manchado, M., Estévez, A., Afonso, J., Marıa-́ Author contributions Dolores, E., Penalver, J. and Armero, E. (2015). Estimates of heritabilities and Conceptualization: S.T., M.I.; Methodology: S.T., M.J.Z., M.I.; Formal analysis: S.T., genetic correlations of growth and external skeletal deformities at different ages in M.J.Z., H.X., H.F.-P., L.R.; Investigation: S.T., H.X., H.F.-P., L.R.; Data curation: S.T.; a reared gilthead sea bream (Sparus aurata L.) population sourced from three Writing - original draft: S.T., M.I.; Writing - review & editing: M.J.Z., S.K., M.I.; broodstocks along the Spanish coasts. Aquaculture 445, 33-41. doi:10.1016/j. Visualization: S.T.; Supervision: S.K., M.I.; Project administration: M.J.Z., S.K., M.I.; aquaculture.2015.04.006 Funding acquisition: S.K., M.I. Gibson, R. A., Neumann, M. A., Lien, E. L., Boyd, K. A. and Tu, W. C. (2013). Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets Funding high in polyunsaturated fatty acids. Prostaglandins, Leukotrienes and Essential This study was (partially) funded under the EU funded project PerformFISH Fatty Acids 88, 139-146. doi:10.1016/j.plefa.2012.04.003 (Integrating Innovative Approaches for Competitive and Sustainable Performance Gjedrem, T. and Rye, M. (2018). Selection response in fish and shellfish: a review. Reviews in Aquaculture 10, 168-179. doi:10.1111/raq.12154 across the Mediterranean Aquaculture Value Chain; Horizon 2020 H2020-SFS- Gjedrem, T., Robinson, N. and Rye, M. (2012). The importance of selective 2016-2017; 27610). breeding in aquaculture to meet future demands for animal protein: A review. Aquaculture 350-353, 117-129. doi:10.1016/j.aquaculture.2012.04.008 Supplementary information Gotoh, T. (2015). Potential of the application of epigenetics in animal production. Supplementary information available online at Animal Production Science 55, 145-158. doi:10.1071/AN14467 https://jeb.biologists.org/lookup/doi/10.1242/jeb.214999.supplemental Izquierdo, M. S. (1996). Essential fatty acid requirements of cultured marine fish larvae. Aquaculture Nutrition 2, 183-191. doi:10.1111/j.1365-2095.1996.tb00058.x References Izquierdo, M. (2005). Essential fatty acid requirements in Mediterranean fish Afonso, J. M., Manchado, M., Estevez, A., Ramis, G., Lee, I., Ponce, M., Perez- species. Cah. Options Mediterr 63, 91-102. Sanchez, J., Armero, E., Navarro, A., Puertas, M. A. et al. (2012). Development Izquierdo, M., Socorro, J., Arantzamendi, L. and Hernández-Cruz, C. (2000). of a genetic program in gilthead seabream (Sparus aurata L.) between industry Recent advances in lipid nutrition in fish larvae. Fish Physiology and Biochemistry and research centers in Spain. In AQUA 2012. Prague, Czech Republic. 22, 97-107. doi:10.1023/A:1007810506259 Burdge, G. C. and Lillycrop, K. A. (2010). Nutrition, epigenetics, and Izquierdo, M. S., Fernandez-Palacios, H. and Tacon, A. G. J. (2001). Effect of developmental plasticity: implications for understanding human disease. Annual broodstock nutrition on reproductive performance of fish. Aquaculture 197, 25-42. review of nutrition 30, 315-339. doi:10.1146/annurev.nutr.012809.104751 doi:10.1016/S0044-8486(01)00581-6 Cassar-Malek, I., Picard, B., Bernard, C. and Hocquette, J.-F. (2008). Application Izquierdo, M. S., Obach, A., Arantzamendi, L., Montero, D., Robaina, L. and of gene expression studies in livestock production systems: a European Rosenlund, G. (2003). Dietary lipid sources for seabream and seabass: growth perspective. Australian Journal of Experimental Agriculture 48, 701-710. doi:10. performance, tissue composition and flesh quality. Aquaculture Nutrition 9, 1071/EA08018 397-407. doi:10.1046/j.1365-2095.2003.00270.x Castro, L. F., Tocher, D. R. and Monroig, O. (2016). Long-chain polyunsaturated Izquierdo, M. S., Montero, D., Robaina, L., Caballero, M. J., Rosenlund, G. and fatty acid biosynthesis in chordates: Insights into the evolution of Fads and Elovl Ginés, R. (2005). Alterations in fillet fatty acid profile and flesh quality in gilthead gene repertoire. Progress in Lipid Research 62, 25-40. doi:10.1016/j.plipres.2016. seabream (Sparus aurata) fed vegetable oils for a long term period. Recovery of 01.001 fatty acid profiles by fish oil feeding. Aquaculture 250, 431-444. Chavanne, H., Janssen, K., Hofherr, J., Contini, F., Haffray, P., Komen, H., Izquierdo, M. S., Robaina, L., Juárez-Carrillo, E., Oliva, V., Hernández-Cruz, C. Nielsen, E. E., Bargelloni, L. and Consortium, A. (2016). A comprehensive and Afonso, J. (2008). Regulation of growth, fatty acid composition and delta 6 survey on selective breeding programs and seed market in the European desaturase expression by dietary lipids in gilthead seabream larvae (Sparus aquaculture fish industry. Aquaculture International 24, 1287-1307. doi:10.1007/ aurata). Fish Physiology and Biochemistry 34, 117-127. doi:10.1007/s10695-007- s10499-016-9985-0 9152-7 Clarkson, M., Migaud, H., Metochis, C., Vera, L. M., Leeming, D., Tocher, D. R. Izquierdo, M. S., Turkmen, S., Montero, D., Zamorano, M. J., Afonso, J. M., and Taylor, J. F. (2017). Early nutritional intervention can improve utilisation of Karalazos, V. and Fernández-Palacios, H. (2015). Nutritional programming vegetable-based diets in diploid and triploid Atlantic salmon (Salmo salar L.). through broodstock diets to improve utilization of very low fishmeal and fish oil Br. J. Nutrition 118, 1-13. doi:10.1017/S0007114517001842 diets in gilthead sea bream. Aquaculture 449, 18-26. doi:10.1016/j.aquaculture. Czajkowska, M., Brzę k, P. and Dobrzyń,P. (2019). A novel polymorphism in the 2015.03.032 fatty acid desaturase 2 gene (Fads2): A possible role in the basal metabolic rate. Janssen, K., Chavanne, H., Berentsen, P. and Komen, H. (2017). Impact of PLoS ONE 14, e0213138. selective breeding on European aquaculture. Aquaculture 472, 8-16. doi:10.1016/ de Verdal, H., Komen, H., Quillet, E., Chatain, B., Allal, F., Benzie, J. A. H. and j.aquaculture.2016.03.012 Vandeputte, M. (2018). Improving feed efficiency in fish using selective breeding: Le Boucher, R., Dupont-Nivet, M., Vandeputte, M., Kerneïs, T., Goardon, L., a review. Reviews in Aquaculture 10, 833-851. doi:10.1111/raq.12202 Labbé, L., Chatain, B., Bothaire, M. J., Larroquet, L., Médale, F. et al. (2012). Dolinoy, D. C., Weidman, J. R., Waterland, R. A. and Jirtle, R. L. (2006). Maternal Selection for Adaptation to Dietary Shifts: Towards Sustainable Breeding of vy Genistein Alters Coat Color and Protects A Mouse Offspring from Obesity by Carnivorous Fish. PLoS ONE 7, e44898. doi:10.1371/journal.pone.0044898 Modifying the Fetal Epigenome. Environmental Health Perspectives 114, Le Boucher, R., Vandeputte, M., Dupont-Nivet, M., Quillet, E., Ruelle, F., 567-572. doi:10.1289/ehp.8700 Vergnet, A., Kaushik, S., Allamellou, J. M., Medale, F. and Chatain, B. (2013). Engrola, S., Aragao, C., Valente, L. M. and Conceiçao, L. E. (2018). Nutritional ̃ ̃ Genotype by diet interactions in European sea bass (Dicentrarchus labrax Modulation of Marine Fish Larvae Performance. In Emerging Issues in Fish L. 1756): nutritional challenge with totally plant-based diets. Journal of Animal Larvae Research (ed. M. Yúfera), pp. 209-228. Cham, Switzerland: Springer. Science 91, 44-56. Fernandes, T., Herlin, M., Belluga, M. D. L., Ballón, G., Martinez, P., Toro, M. A. Lee, J.-E., Yong, H., Kim, H.-Y., Lee, W.-H., Cheong, H.-T., Yang, B.-K. and Park, and Fernández, J. (2017). Estimation of genetic parameters for growth traits in a C.-K. (2017). Effect of Alpha-Linolenic Acid on Oocyte Maturation and Embryo hatchery population of gilthead sea bream (Sparus aurata L.). Aquaculture Development in Pigs. Development & Reproduction 21, 205-213. doi:10.12717/ International 25, 499-514. DR.2017.21.2.205 Fernandez Palacios, H., Norberg, B., Izquierdo, M. and Hamre, K. (2011). Effects Lee–Montero, I., Navarro, A., Negrıń –Báez, D., Zamorano, M., Berbel, C., of broodstock diet on eggs and larvae. In Larval Fish Nutrition (ed. J. G. Holt), pp. Sanchez, J., Garcıa–Celdran, M., Manchado, M., Estevez, A. and Armero, E. ́ ́ ́ 151-181. Oxford, UK: Wiley-Blackwell. (2015). Genetic parameters and genotype–environment interactions for skeleton Fernández-Palacios, H., Izquierdo, M. S., Robaina, L., Valencia, A., Salhi, M. deformities and growth traits at different ages on gilthead seabream (Sparus and Vergara, J. (1995). Effect of n−3 HUFA level in broodstock diets on egg aurata L.) in four S panish regions. Animal Genetics 46, 164-174. doi:10.1111/ quality of gilthead sea bream (Sparus aurata L.). Aquaculture 132, 325-337. age.12258 doi:10.1016/0044-8486(94)00345-O Lemaitre, R. N., Siscovick, D. S., Berry, E. M., Kark, J. D. and Friedlander, Y. Ferosekhan, S., Turkmen, S., Xu, H., Afonso, J. M., Zamorano, M. J., Kaushik, (2008). Familial aggregation of red blood cell membrane fatty acid composition: S. and Izquierdo, M. (2020). The Relationship between the Expression of Fatty the Kibbutzim Family Study. Metabolism 57, 662-668. doi:10.1016/j.metabol. Acyl Desaturase 2 (fads2) Gene in Peripheral Blood Cells (PBCs) and Liver in 2007.12.011 Journal of Experimental Biology RESEARCH ARTICLE Journal of Experimental Biology (2020) 223, jeb214999. doi:10.1242/jeb.214999 Lillycrop, K. A. and Burdge, G. C. (2018). Chapter 13 - Interactions Between Torrecillas, S., Robaina, L., Caballero, M. J., Montero, D., Calandra, G., Mompel, Polyunsaturated Fatty Acids and the Epigenome. In Polyunsaturated Fatty Acid D., Karalazos, V., Kaushik, S. and Izquierdo, M. S. (2017b). Combined Metabolism, pp. 225-239. AOCS Press. replacement of fishmeal and fish oil in European sea bass (Dicentrarchus labrax): Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data Production performance, tissue composition and liver morphology. Aquaculture using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25, 402-408. 474, 101-112. doi:10.1016/j.aquaculture.2017.03.031 doi:10.1006/meth.2001.1262 Turchini, G. M., Torstensen, B. E. and Ng, W.-K. (2009). Fish oil replacement in Monroig, O., Tocher, D. R. and Castro, L. F. C. (2018). Chapter 3 - Polyunsaturated finfish nutrition. Reviews in Aquaculture 1, 10-57. doi:10.1111/j.1753-5131.2008. Fatty Acid Biosynthesis and Metabolism in Fish A2 - Burdge, Graham C. In 01001.x Polyunsaturated Fatty Acid Metabolism, pp. 31-60. AOCS Press. Turkmen, S., Castro, P. L., Caballero, M. J., Hernández-Cruz, C. M., Saleh, R., Montero, D. and Izquierdo, M. (2010). Welfare and health of fish fed vegetable oils Zamorano, M. J., Regidor, J. and Izquierdo, M. (2017a). Nutritional stimuli of as alternative lipid sources to fish oil. In Fish oil replacement and alternative lipid gilthead seabream (Sparus aurata) larvae by dietary fatty acids: effects on larval sources in aquaculture feeds (eds. G. M. Turchini, W. K. Ng and D. R. Tocher), pp. performance, gene expression and neurogenesis. Aquaculture Research 48, 439-485. Boca Raton, Florida, USA: CRC Press. 202-213. doi:10.1111/are.12874 Morais, S., Mendes, A. C., Castanheira, M. F., Coutinho, J., Bandarra, N., Dias, Turkmen, S., Zamorano, M. J., Fernandez-Palacios, H., Hernandez-Cruz, C. M., J., Conceiçao, L. E. C. and Pousao-Ferreira, P. (2014). New formulated diets for ̃ ̃ Montero, D., Robaina, L. and Izquierdo, M. (2017b). Parental nutritional Solea senegalensis broodstock: Effects of parental nutrition on biosynthesis of programming and a reminder during juvenile stage affect growth, lipid metabolism long-chain polyunsaturated fatty acids and performance of early larval stages and and utilisation in later developmental stages of a marine teleost, the gilthead sea juvenile fish. Aquaculture 432, 374-382. doi:10.1016/j.aquaculture.2014.04.033 bream (Sparus aurata). British Journal of Nutrition 118, 500-512. doi:10.1017/ Panserat, S., Marandel, L., Seiliez, I. and Skiba-Cassy, S. (2019). New Insights on S0007114517002434 Intermediary Metabolism for a Better Understanding of Nutrition in Teleosts. Annu. Turkmen, S., Hernandez-Cruz, C. M., Zamorano, M. J., Fernandez-Palacios, H., Rev. Anim. Biosci. 7, 195-220. doi:10.1146/annurev-animal-020518-115250 Montero, D., Afonso, J. M. and Izquierdo, M. (2019a). Long-chain PUFA profiles Perera, E., Turkmen, S., Simo-Mirabet, P., Zamorano, M. J., Xu, H., Naya-Catala, in parental diets induce long-term effects on growth, fatty acid profiles, expression F., Izquierdo, M. and Perez-Sanchez, J. (2019). Stearoyl-CoA desaturase of fatty acid desaturase 2 and selected immune system-related genes in the (scd1a) is epigenetically regulated by broodstock nutrition in gilthead sea bream offspring of gilthead seabream. British Journal of Nutrition 122, 25-38. doi:10. (Sparus aurata). Epigenetics 15, 1-18. doi:10.1080/15592294.2019.1699982 Rosenlund, G., Corraze, G., Izquierdo, M. and Torstensen, B. E. (2010). The 1017/S0007114519000977 Turkmen, S., Perera, E., Zamorano, M. J., Simó-Mirabet, P., Xu, H., Pérez- effects of fish oil replacement on nutritional and organoleptic qualities of farmed fish. In Fish oil replacement and alternative lipid sources in aquaculture feeds Sánchez, J. and Izquierdo, M. (2019b). Effects of dietary lipid composition and (eds. G. M. Turchini, W. K. Ng and D. R. Tocher), pp. 487-522. Boca Raton, fatty acid desaturase 2 expression in broodstock gilthead sea bream on lipid Florida, USA: CRC Press. metabolism-related genes and methylation of the fads2 gene promoter in their Schmittgen, T. D. and Livak, K. J. (2008). Analyzing real-time PCR data by the offspring. International Journal of Molecular Sciences 20, 6250. doi:10.3390/ comparative CT method. Nature Protocols 3, 1101-1108. doi:10.1038/nprot. ijms20246250 2008.73 Vagner, M. and Santigosa, E. (2011). Characterization and modulation of gene Siemelink, M., Verhoef, A., Dormans, J. A., Span, P. N. and Piersma, A. H. expression and enzymatic activity of delta-6 desaturase in teleosts: A review. (2002). Dietary fatty acid composition during pregnancy and lactation in the rat Aquaculture 315, 131-143. doi:10.1016/j.aquaculture.2010.11.031 programs growth and glucose metabolism in the offspring. Diabetologia 45, Vagner, M., Zambonino Infante, J. L., Robin, J. H. and Person-Le Ruyet, J. 1397-1403. doi:10.1007/s00125-002-0918-2 (2007). Is it possible to influence European sea bass (Dicentrarchus labrax) Sokal, R. R. and Rohlf, F. J. (1969). The Principles and Practice of Statistics in juvenile metabolism by a nutritional conditioning during larval stage? Aquaculture Biological Research. WH Freeman and company. 267, 165-174. doi:10.1016/j.aquaculture.2007.01.031 Stoffel, W., Holz, B., Jenke, B., Binczek, E., Gunter, R., Kiss, C., Vera, L. M., Metochis, C., Taylor, J. F., Clarkson, M., Skjærven, K. H., Migaud, H. Karakesisoglou, L., Thevis, M., Weber, A.-A., Arnhold, S. et al. (2008). Δ6- and Tocher, D. R. (2017). Early nutritional programming affects liver desaturase (FADS2) deficiency unveils the role of ω3- and ω6-polyunsaturated transcriptome in diploid and triploid Atlantic salmon, Salmo salar. BMC fatty acids. The EMBO Journal 27, 2281-2292. doi:10.1038/emboj.2008.156 Genomics 18, 886. doi:10.1186/s12864-017-4264-7 Thorland, I., Kottaras, L., Refstie, T., Dimitroglou, A., Papaharisis, L. and Rye, Vestergren, A. S., Trattner, S., Pan, J., Johnsson, P., Kamal-Eldin, A., Brä nnä s, M. (2015). Response to Selection for Harvest Weight in a Family Based Selection E., Moazzami, A. A. and Pickova, J. (2012). The effect of combining linseed oil Program of Gilthead Seabream (Sparus aurata). In XII International Symposium and sesamin on the fatty acid composition in white muscle and on expression of on Genetics in Aquaculture, Santiago de Compostela, Spain. lipid-related genes in white muscle and liver of rainbow trout (Oncorhynchus Torno, C., Staats, S., Michl, S. C., De Pascual-Teresa, S., Izquierdo, M., mykiss). Aquaculture International 21, 843-859. doi:10.1007/s10499-012-9511-y Rimbach, G. and Schulz, C. (2018). Fatty Acid Composition and Fatty Acid Watanabe, T. and Vassallo-Agius, R. (2003). Broodstock nutrition research on Associated Gene-Expression in Gilthead Sea Bream (Sparus aurata) are Affected marine finfish in Japan. Aquaculture 227, 35-61. doi:10.1016/S0044- by Low-Fish Oil Diets, Dietary Resveratrol, and Holding Temperature. Marine 8486(03)00494-0 Drugs 16, 379. doi:10.3390/md16100379 Xu, H., Turkmen, S., Rimoldi, S., Terova, G., Zamorano, M. J., Afonso, J. M., Torrecillas, S., Mompel, D., Caballero, M. J., Montero, D., Merrifield, D., Rodiles, Sarih, S., Fernández-Palacios, H. and Izquierdo, M. (2019). Nutritional A., Robaina, L., Zamorano, M. J., Karalazos, V., Kaushik, S. et al. (2017a). intervention through dietary vegetable proteins and lipids to gilthead sea bream Effect of fishmeal and fish oil replacement by vegetable meals and oils on gut health of European sea bass (Dicentrarchus labrax). Aquaculture 468, 386-398. (Sparus aurata) broodstock affects the offspring utilization of a low fishmeal/fish oil doi:10.1016/j.aquaculture.2016.11.005 diet. Aquaculture 513, 734402. doi:10.1016/j.aquaculture.2019.734402 Journal of Experimental Biology

Journal

Journal of Experimental Biology – The Company of Biologists

Published: Dec 1, 2020

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Parental LC-PUFA biosynthesis capacity and nutritional intervention with alpha-linolenic acid affect performance of Sparus aurata progeny

Parental LC-PUFA biosynthesis capacity and nutritional intervention with alpha-linolenic acid affect performance of Sparus aurata progeny

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Parental LC-PUFA biosynthesis capacity and nutritional intervention with alpha-linolenic acid affect performance of Sparus aurata progeny

Parental LC-PUFA biosynthesis capacity and nutritional intervention with alpha-linolenic acid affect performance of Sparus aurata progeny

References (45)

Abstract

Journal

Recommended Articles

There are no references for this article.

Our policy towards the use of cookies