Cloning,characterization and transcription analysis of fatty acyl desaturase (Δ6Fad‐like) and elongase (Elovl4‐like,Elove5‐like) during embryos development of Strongylocentrotus intermedius

Strongylocentrotus intermedius has high nutritional value because it is rich in proteins, amino acids and long‐chain polyunsaturated fatty acids (LC‐PUFA). LC‐PUFA are essential nutrients that not only determine the nutritional value of sea urchins but also guarantee normal growth and reproduction performance. To better understand the molecular basis of LC‐PUFA biosynthesis in S. intermedius, the Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes were cloned and fatty acid compositions during the early developmental stages of sea urchins were detected. The full‐length of Δ6Fad‐like was 2,199 bp, with a putative open reading frame of 1,248 bp encoding a polypeptide of 415 amino acid (AA). The Elovl4‐like and Elovl5‐like genes encoded 310 and 234 AA, respectively, which exhibited all of the characteristics of the Elovl family, such as a histidine box motif and putative transmembrane‐spanning domains. Tissue distribution analysis revealed that Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes were expressed at the highest levels in the gonads and intestine, and the expression levels gradually increased in embryos during development. These results can help to understand the role of the Δ6Fad‐like, Elovl4‐like and Elovl5‐like genes in the different developmental stages of the sea urchin and to clarify the biosynthetic pathways of LC‐PUFA during the development of the sea urchin and provide a theoretical basis for improving the quality and breeding of excellent traits in sea urchins.     

Aurantiochytrium sp. meal as DHA source in Nile tilapia diet,part II: Body fatty acid retention and muscle fatty acid profile

Nile tilapia juveniles (8.35 ± 0.80 g) were fed on four levels (0.0%; 0.5%; 1.0%; 2.0%, 4.0%) of Aurantiochytrium sp. meal (ALL‐G‐RICH?), a source of docosahexaenoic acid (DHA). The 1% Aurantiochytrium sp. meal diet was compared to a control diet, which contained the same amount of DHA as cod liver oil (CLO) at 1.7% diet. Groups of 25 fish were stocked in 100 L tanks and fed twice daily until apparent satiation, for 57 days, at 28°C. Increasing dietary Aurantiochytrium sp. meal reduced the body retention of DHA and n‐3 polyunsaturated fatty acids (n‐3 PUFA) but increased the body retention of alpha‐linolenic (α‐LNA), linoleic (LOA) and n‐6 polyunsaturated fatty acids (n‐6 PUFA). Fatty acid profile in tilapia muscle was affected by increasing dietary inclusions of Aurantiochytrium sp. meal, with an increase in DHA, α‐LNA, n‐3 PUFA and n‐3 long chain‐polyunsaturated fatty acids (n‐3 LC‐PUFA) but a decrease in monounsaturated fatty acids (MUFA), n‐6 PUFA and n‐6 long‐chain polyunsaturated fatty acids (n‐6 LC‐PUFA). There was a larger body retention of DHA, α‐LNA, LOA, n‐3 PUFA and n‐6 PUFA fatty acids and a higher percentage of DHA, n‐3 PUFA and n‐3 LC‐PUFA in muscle fatty acid profile in fish fed on CLO diets than in those fed on 1% Aurantiochytrium sp. Therefore, Aurantiochytrium sp. meal is an alternative source of DHA for Nile tilapia diets.     

Long‐chain polyunsaturated fatty acid metabolism in carnivorous marine teleosts: Insight into the profile of endogenous biosynthesis in golden pompano Trachinotus ovatus

Golden pompano Trachinotus ovatus is an important farmed carnivorous marine teleost. Although some enzymes for long‐chain polyunsaturated fatty acid (LC‐PUFA) biosynthesis have been identified, the ability of T. ovatus for endogenous biosynthesis is unknown. Here, we evaluated in vivo LC‐PUFA synthesis in a 56‐day culture experiment using six diets (D1–D6) formulated with linseed and soybean oils to produce dietary linolenic/linoleic acid (ALA/LA) ratios ranging from 0.14 to 2.20. The control diet (D0) used fish oil as lipid source. The results showed that, compared with the corresponding indices of fish fed D0, the weight gain rate and specific growth rate as well as the contents of eicosapentaenoic (EPA) and docosahexaenoic acids in tissues (liver, muscle, brain and eye) of D1–D6 groups were significantly lower (p < .05). These data suggested that T. ovatus could not synthesize LC‐PUFA from C₁₈ PUFA or such ability was very low. However, tissue levels of 20:4n‐3 in fish fed diets D1–D6 were higher than that of D0 fish (p < .05), and positively correlated with dietary ALA/LA ratio, while levels of EPA showed no difference among the D1–D6 groups. These results indicated that Δ5 desaturation, required for the conversion of 20:4n‐3 to EPA, may be lacking or very low, suggesting incomplete LC‐PUFA biosynthesis ability in T. ovatus.     

Diet,size and location as determinants of n‐3 long‐chain polyunsaturated fatty acid content in farmed Atlantic Salmon (Salmo salar)

We explored how currently manufactured feeds, under real‐world conditions and across geographically distinct locations, promoted flesh n‐3 long‐chain polyunsaturated fatty acid (LC‐PUFA, i.e. 20:5n‐3 + 22:6n‐3) levels in various life stages of farmed Atlantic Salmon (Salmo salar). Potential effects on flesh LC‐PUFA included: (1) diet and fish weight at one Canadian east coast farm, (2) diet and farm location across six east coast farms, and (3) diet and farm location between east and west coast farms. For objectives 1 and 2, salmon were fed a currently manufactured feed (labelled as feeds A, B or C) and harvested at 1, 3 and 5 kg. LC‐PUFA levels in 5 kg (harvest size) fish were then compared to previously published values for west coast farmed Atlantic Salmon (Obj. 3). Combined results revealed that variability in LC‐PUFA levels was better explained by diet than by fish weight or farm location. Fish size, however, was also important for two reasons. First, feeding a high LC‐PUFA diet early in life appeared important for ensuring high LC‐PUFA levels at harvest size. Second, salmon flesh LC‐PUFA levels increased with fish size, but only when dietary LC‐PUFA was provided above an apparent threshold value (~3000 mg per 100 g or 10% of total fatty acids) that likely promoted LC‐PUFA incorporation and storage. Overall, our comparison makes new recommendations for feed manufacturers and demonstrates that farmed Atlantic Salmon reared under real‐world conditions on currently available salmon feeds were good sources of n‐3 LC‐PUFA to consumers.     

Characterization of a γ‐aminobutyrate type A receptor‐associated protein gene,which is involved in the response of Portunus trituberculatus to CO2‐induced ocean acidification

The γ‐aminobutyrate type A receptor‐associated protein (GABARAP) is a ubiquitin‐like modifier implicated in membrane trafficking and fusion events involving the γ‐aminobutyrate type A receptor, autophagy and apoptosis. In this study, the gene encoding GABARAP was cloned from swimming crab Portunus trituberculatus (PtGABARAP) based on the expression sequence tag (EST). The full‐length cDNA of 664 bp includes a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 223 bp with a poly(A) tail, and an open reading frame (ORF) of 354 bp encoding a polypeptide of 117 amino acids with a predicted molecular weight of 13.96 kDa. The deduced amino acid sequence shares high similarity (93%–100%) with GABARAPs from other species and includes a conserved Atg8 domain. In a phylogenetic analysis PtGABARAP clustered with GABARAPs from other species, and more widely with other GABARAP family proteins. The impact of elevated ocean acidification (OA) on P. trituberculatus behaviours was investigated, and real‐time RT‐PCR revealed that PtGABARAP expression was up‐regulated after OA exposure. Ocean acidification also caused crabs anxiety‐like behaviours, like the shoal average speed increase, preference for dark environment (scototaxis) and fast exploration. The results indicated that GABARAP might be involved in the interactions of GABA_A receptors and elevated‐CO₂ seawater.     

Dietary n‐3 LC‐PUFAs affect lipoprotein lipase (LPL) and fatty acid synthase (FAS) activities and mRNA expression during vitellogenesis and ovarian fatty acid composition of female silver pomfret (Pampus argenteus) broodstock

We studied the effects of dietary n‐3 LC‐PUFAs on the activities and mRNA expression levels of tissue lipoprotein lipase (LPL) and fatty acid synthase (FAS) during vitellogenesis and ovarian fatty acid composition in female silver pomfret broodstock. Broodstock were fed one of four experimental diets for 185 days: FO (100% fish oil), FSO (70% fish oil + 30% soybean oil), SFO (30% fish oil + 70% soybean oil) or SO (100% soybean oil). The results revealed that hepatic LPL and FAS and ovarian FAS activities and mRNA expression levels significantly increased at vitellogenesis and postvitellogenesis relative to previtellogenesis, with no significant differences between these two stages, except for hepatic LPL mRNA expression. Dietary n‐3 LC‐PUFAs decreased tissue FAS and increased LPL activities and mRNA expression levels. The ovarian concentrations of 20:4n‐6 (ARA), 20:5n‐3 (EPA), 22:6n‐3 (DHA) and n‐3 LC‐PUFAs were directly influenced by n‐3 LC‐PUFA levels. Total n‐3 LC‐PUFA concentrations in SO were 57% lower than those in FO, while 18:2n‐6 concentrations in SO were 4.7 ×  higher than those in FO. These results revealed that high dietary n‐3 LC‐PUFAs levels significantly affected tissue lipid metabolism in female silver pomfret broodstock during vitellogenesis by upregulating LPL and downregulating FAS.     

Effect of dietary L‐carnitine supplementation on growth performance and lipid metabolism in Rhynchocypris lagowski Dybowski fed oxidized fish oil

Lipid content of a diet is very susceptible to oxidation, which has many negative effects on farmed animals. Therefore, this study studied the protective effect of L‐carnitine (LC) on fish body stimulated by oxidized fish oil (OFO) from lipid metabolism. Lipid content of the diet was replaced by OFO in 0, 100 and 400 meq/kg. L‐carnitine was added to the diet in two levels, 500 and 1,000 mg/kg, giving a total of seven experimental diets. A total of 735 healthy Rhynchocypris lagowski Dybowski with an initial weight of 4.48 ± 0.14 g after 2‐week adaptation randomly divided into 15 glass aquariums. Fish were fed satiated three times daily. After 8 weeks, biometry was done to evaluate growth performance, and hepatopancreas and muscle samples were taken for biochemical analysis. The result showed that feeding with OFO had negative growth. However, in fish received both OFO and LC, growth indices improved slightly (p > .05). Feeding with OFO and LC, the content of EPA, DHA and PUFA in the muscle of R. lagowski was significantly higher than that in the control group (p < .05), which reached the maximum value in the OFO100 + LC500 group. The content of SFA, MUFA, ∑n‐6 and PUFA in hepatopancreas increased significantly (p < .05), and the content of SFA reached the maximum in OFO100 + LC500 group. Feeding with OFO increased hepatopancreas total cholesterol, triacylglycerol, HDL/LDL ratio, FAS and ACCα that involved in lipid synthesis enzymes, while reduced HL and HSLα enzyme activity and gene expression that associated with lipid decomposition. Dietary LC moderated the effects of OFO on lipid metabolism. According to the result of the present study, it can be argued that feeding of R. lagowski with OFO has negative effects on growth performance and lipid metabolism, whereas LC dosages used in this study have increased the oxidation rate of fatty acids in the hepatopancreas of R. lagowski and improved the accumulation of fat in hepatopancreas cells induced by oxidized fish oil.     

Effects of dietary n‐3 long‐chain unsaturated fatty acid on growth performance,lipid deposition,hepatic fatty acid composition and health‐related serum enzyme activity of juvenile Japanese seabass Lateolabrax japonicus

Studies were conducted to investigate the effects of dietary n‐3 long‐chain polyunsaturated fatty acid (n‐3 LC‐PUFA) on growth performance, lipid deposition, hepatic fatty acid composition and serum enzyme activities of juvenile Japanese seabass Lateolabrax japonicus (initial mean weight 29.2 ± 1.34 g). Triplicate groups of 30 Japanese seabass were fed with six diets containing grade levels of n‐3 LC‐PUFA (1.30, 2.98, 5.64, 10.31, 14.51, 24.13 g kg^–1 of dry weight) to apparent satiation twice daily for 9 weeks. The specific growth rate (SGR) was the highest in 10.31 g kg^–1 dietary n‐3 LC‐PUFA group. Crude lipid content of the fish decreased significantly with increasing dietary n‐3 LC‐PUFA. Meanwhile, the hepatic lipid content increased significantly in the 24.13 g kg^–1 group. Hepatic n‐3 LC‐PUFA content of total fatty acids was closely correlated with that in diet. No significant difference was observed in serum alanine transaminase (ALT) and aspartate aminotransferase (AST) activities. Moderate n‐3 LC‐PUFA level (10.31 g kg^–1 of dry weight) in the diet was beneficial to enhance the activity of lysozyme in serum. Based on SGR, the optimum dietary n‐3 LC‐PUFA content was estimated to be around 10.94 g kg^–1 of dry weight by second‐order polynomial regression method.     

Effects of Dietary DHA/EPA Ratios on Fatty Acid Composition,Lipid Metabolism‐related Enzyme Activity,and Gene Expression of Juvenile Grass Carp,Ctenopharyngodon idellus

To determine the effects of docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) ratios on grass carp, Ctenopharyngodon idellus, a 38‐d feeding trial was conducted using six isonitrogenous and isoenergetic semi‐purified diets containing constant n‐3 long‐chain polyunsaturated fatty acid (LC‐PUFA) (0.5% of dry matter), but varying ratios of DHA to EPA and a control diet (no n‐3 LC‐PUFA was included). The results revealed higher final weight and specific growth rate in the DHA/EPA 0.21 group. The n‐3 LC‐PUFA content increased in the CK (control) groups compared with that in the control diet. Lipoprotein lipase (LPL) activity increased in the treatment groups. Malate dehydrogenase showed lower activity in the DHA/EPA 1.08 group, as well as to the change in the level of glucose‐6‐phosphate dehydrogenase (G6PDH). The gene expressions of LPL increased in the treatment groups and that of peroxisome proliferator‐activated receptor α gene showed higher expressions in DHA/EPA 1.08, 0.49, and 0.21 groups. However, no remarkable differences were found among the six groups in the peroxisome proliferator‐activated receptor γ gene expression. Our findings indicated that dietary n‐3 LC‐PUFA affected fatty acid composition and lipid metabolism of grass carp. Further, fish achieved the best effect in decreasing the lipid accumulation when dietary DHA/EPA ratio was not greater than 1.     

First step of non‐fish meal,non‐fish oil diet development for red seabream, (Pagrus major), with plant protein sources and microalgae Schizochytrium sp

A feeding experiment was conducted to develop non‐fish meal and non‐fish oil diet for red seabream by using plant protein source and Schizochytrium meal which is rich in 22:6n‐3 (DHA). Three iso‐nitrogenous and iso‐lipidic experimental diets were prepared (CP 41.2% ± 0.4%, CL 16.4% ± 1%). Control diet contained both fish meal (40%) and fish oil (6%). In the second diet, fish meal was replaced by plant meals (soy protein concentrate, soybean meal, corn gluten meal) [FO]. In the third diet, fish meal and fish oil were replaced by algae meal (Schizochytrium sp. powder) and plant proteins [AO]. Duplicated groups of juvenile red seabream (8.8 g ± 1.5) were fed the experimental diets for 12 weeks to near satiation. There was no statistical difference among treatment in specific growth rate. Feed conversion ratio of AO diet group was higher than that of control. In wet basis, whole body protein level was significantly higher in AO diet than FO group while lipid content was lower than control group. In fatty acid profile, AO group had significantly lower 18:4n‐3, 20:4n‐3, 22:5n‐3 and 20:5n‐3 (EPA) level, but significantly higher 18:3n‐3 and DHA level than the other two diet fed fish. The results might suggest that further developments in microalgae diet offer a promising lipid source of n‐3 PUFA as essential fatty acid on marine fish. And it showed possibility to develop non‐fish meal and non‐fish oil feed for marine aquaculture fish by using microalgae.     

Replacement of fish oil with a DHA‐rich Schizochytrium meal on growth performance,activities of digestive enzyme and fatty acid profile of Pacific white shrimp (Litopenaeus vannamei) larvae

This trial was conducted to evaluate the effects of replacing dietary fish oil with Schizochytrium meal for Pacific white shrimp (Litopenaeus vannamei) larvae (initial body weight 4.21 ± 0.10 mg). Six test microdiets were formulated using Schizochytrium meal to replace 0 g/kg, 250 g/kg, 500 g/kg, 750 g/kg, 1000 g/kg or 1500 g/kg fish oil DHA. No significant differences were observed in survival, growth, final body length and activities of digestive enzyme among shrimp fed different diets (p > .05). No significant differences were observed in C20:5n‐3 (EPA) in muscle samples (p > .05). C18:3n‐3 and C20:4n‐6 in muscle increased as Schizochytrium meal replacement level increased (p < .05). No significant differences were observed in C22:6n‐3 (DHA) and n‐3 fatty acids among shrimp fed diets that algae meal replaced 0 g/kg ‐ 1000 g/kg of fish oil. Shrimp fed diet R150 had higher DHA content than other groups and had higher n‐3 fatty acids than that of shrimp fed diets R50, R75 and R100 (p < .05). C18:2n‐6, PUFA and n‐6 fatty acids in muscle increased, while n‐3/n‐6 ratio decreased with increasing algae meal replacement level from 0 g/kg to 1000 g/kg (p < .05). In conclusion, Schizochytrium meal could replace 1500 g/kg fish oil DHA in the microdiets without negatively affecting shrimp larvae survival, growth and activities of digestive enzyme.     

Effects of dietary alpha‐linolenic acids on growth performance,lipid metabolism and antioxidant responses of juvenile Russian sturgeon Acipenser gueldenstaedtii

Five isonitrogenous diets were formulated with graded alpha‐linolenic acid (LNA) levels (0, 5, 10, 15 and 20 g/kg) to investigate LNA requirement of juvenile Russian sturgeon Acipenser gueldenstaedtii. Weight gain and specific growth rate of fish fed LNA5 and LNA10 were significantly higher than those in other groups, while the feed conversion ratio of these two groups was lower than others. Dietary LNA increased n‐3 polyunsaturated fatty acid and n‐3/n‐6 ratio, but decreased saturated fatty acid contents in the liver. DHA in the fish tissue also increased with the increased dietary LNA. The superoxide dismutase activity was highest in fish fed LNA5. Fish fed LNA10 showed the highest catalase activity and the highest malondialdehyde content. A 459‐bp fragment of Δ6 fatty acid desaturase and a 474‐bp fragment of elongases of very long chain fatty acids 5 were cloned and analysed. The expressions of these two genes were higher in fish fed LNA15 and LNA20. The highest hepatic lipase activity occurred in fish fed LNA 20, and the malate dehydrogenase activity peaked in the LNA5 group. Based on SGR and FCR, the range of optimum dietary LNA concentration for juvenile Russian sturgeon is recommended at 6.85–10.69 g/kg.     

Nutritional stimuli of gilthead seabream (Sparus aurata) larvae by dietary fatty acids: effects on larval performance,gene expression and neurogenesis

The concept of nutritional programming raises the interesting possibility of directing specific metabolic pathways or functions in juvenile fish, for example, to improve the use of substitutes to fishmeal and oil, and hence to promote sustainability in aquaculture. The aim of the study was to determine effects of early nutritional stimuli of gilthead seabream larvae and check if nutritional programming of gilthead sea bream is possible between 16 days post hatching (dph) and 26 dph. A trial was conducted to determine the effects of early nutritional stimuli of gilthead seabream larvae. Five experimental microdiets (pellet size <250 μm) were formulated containing five different proportions of a marine lipid source rich in long‐chain polyunsaturated fatty acids (LC‐PUFA) and two vegetable lipid sources rich in linolenic and linoleic acids. The results of this study demonstrate that dietary n‐3 LC‐PUFA levels increased larval growth and survival affecting Δ6 desaturase gen (fads2) expression and retinal neurons density. However, the high mortalities obtained along on‐growing in fish fed low n‐3 LC‐PUFA at 16 dph constrained the feasibility of nutritional programming of gilthead seabream during this late developmental window and needs to be further investigated.     

Molecular cloning,characterization, tissue expression and nutritional regulation of O‐GlcNAc transferase gene in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

O‐GlcNAc transferase gene (OGT) was considered as the sole rate‐limiting enzyme in the O‐GlcNAc modification. In the present study, the OGT gene of hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was cloned and characterized, and its expression in response to dietary carbohydrate level and acute glucose treatment was investigated. The full‐length of OGT (GenBank accession no. KY656469 ) was 4,063 bp, including a 302 bp 5′untranslated terminal region (UTR), a 3,165 bp coding region that encoded 1,054 amino acids residues and a 596 bp 3′ UTR. The highly conservation of OGT gene between fish and mammals was also observed through multiple sequences alignment and phylogenetic analysis. O‐GlcNAc transferase gene was ubiquitously expressed in all detected tissues with highest expressions in brain and liver, to a lesser degree, in eye, heart, kidney and intestine. The increasing dietary carbohydrate from 8.02% to 16.08% had no significant effect on the mRNA expression of OGT. However, the expression of OGT was slightly elevated at 6 hr post‐glucose injection, and the elevation became significant at 24 hr time‐point. These data may enhance our understanding on the nutritional regulation of OGT and O‐GlcNAc modification in fish species.     

Effects of varying dietary docosahexaenoic acid levels on growth,proximate composition and tissue fatty acid profile of juvenile silver pomfrets,Pampus argenteus (Euphrasen, 1788)

This study investigated the effects of varying dietary levels of decosahexaenoic acid (DHA) on growth performance, proximate composition and whole body fatty acid profiles of juvenile silver pomfret, Pampus argenteus. Triplicate groups of fish (30.55 ± 0.08 g) were fed diets containing 5.2%, 9.31% and 13.38% DHA (% of total fatty acids) or 0.85%, 1.52% and 2.18% DHA on dry diet weight for diets 1, 2 and 3 respectively. Survival was not affected by dietary DHA levels. The growth performance and feed utilization parameters of fish fed diets 2 and 3 were significantly (P < 0.05) higher than those fed diet 1, although these parameters in diets 2 and 3 did not differ significantly (P > 0.05). Whole body lipid and fatty acid profiles were influenced by dietary DHA levels. Significantly higher n‐3 fatty acids particularly DHA, DHA:EPA(eicosapentaenoic acid) ratios and n‐3:n‐6 ratios were observed in fish fed diets 2 and 3 compared to those fed diet 1. Better growth performance and higher whole body DHA:EPA (2.31, 2.29) ratios and n‐3:n‐6 ratios (2.17, 2.12) observed in fish fed diets 2 and 3, respectively, suggests that silver pomfret juveniles have a higher requirement for n‐3 fatty acids, notably DHA for optimum growth and survival.     

Dietary ratios of n‐3/n‐6 fatty acids do not affect growth of Nile tilapia at optimal temperatures (28°C) nor at temperatures that simulate the onset of winter (22°C)

Nile tilapia (Oreochromis niloticus) juveniles were fed diets containing 13 g/kg total polyunsaturated fatty acids (PUFAs) at different n‐3/n‐6 dietary ratios (0.2, 0.5, 0.8, 1.3 and 2.9) for 56 days, at 28°C. Subsequently, fish were submitted to a winter‐onset simulation (22°C) for 33 days. PUFA n‐3/n‐6 dietary ratios did not affect fish growth at either temperature. At 28°C, tilapia body fat composition increased with decreasing dietary PUFA n‐3/n‐6. Winter‐onset simulation significantly changed feed intake. The lowest dietary n‐3/n‐6 ratio resulted in the highest feed intake. At both temperatures, body concentrations of α‐linolenic acid, docosahexaenoic acid, eicosatrienoic acid and docosapentaenoic acid decreased as dietary n‐3/n‐6 decreased. Body concentrations of eicosapentaenoic acid (EPA, 20:5 n‐3) increased with decreasing concentrations of dietary EPA. The n‐6 fatty acids with the highest concentrations in tilapia bodies were linoleic acid and arachidonic acid (ARA, 20:4 n‐6). At 28°C, SREBP1 gene expression was upregulated in tilapia fed the lowest n‐3/n‐6 diet compared to tilapia fed the highest n‐3/n‐6 ratio diet. Our results demonstrate that a dietary PUFA of 13 g/kg, regardless of the n‐3/n‐6 ratio, can promote weight gains of 2.65 g/fish per day at 28°C and 2.35 g/fish per day at 22°C.