Recombinant expression and comparative bioactivity of tongue sole insulin‐like growth factor (IGF)‐1 and IGF‐2 in Pichia pastoris

Insulin‐like growth factor (IGF) plays a key role in the complex system that regulates bony fish growth, differentiation, and reproduction. In the current study, recombinant tongue sole IGF‐1 and IGF‐2 were obtained using the Pichia pastoris expression system and their comparative bioactivities were investigated. Tricine–SDS–PAGE and western blot analysis showed that the recombinant tongue sole IGFs were secreted into the culture medium and had a molecular weight of 8.7 kDa. The optimal incubation time and pH for recombinant expression of IGFs were 36 hr and 5.0 respectively. Functional analysis demonstrated that both recombinant tongue sole IGF‐1 and IGF‐2 significantly promoted cell proliferation of MFC‐7 in vitro. In addition, the recombinant tongue sole IGF‐1 and IGF‐2 proteins could suppress hepatic mRNA levels of igf‐1 and igf‐2 in vitro, which showed that they have similar physiological functions. Taken together, the biologically active recombinant tongue sole IGF‐I and IGF‐II proteins will allow us to further investigate their physiological roles in growth regulation of this species. Furthermore, the present results also hinted at the potential application of these two recombinant IGF‐I and IGF‐II proteins into the tongue sole farming industry.     

Compensatory growth,feed intake and body composition of Labeo rohita fingerlings following feed deprivation

Ten‐week study was performed on Labeo rohita fingerlings (av. wt. 3.75 ± 0.06 g) to investigate the nature of compensatory growth. Fingerlings were deprived of food for 0 (control), 1 (D1), 2 (D2) or 3 (D3) weeks and refed to satiation for 5 weeks. The feed deprivation was carried out in D3 group on week 3, 4 and 5; D2 group on week 4 and 5; D1 group on week 5, and refeeding of all the groups were started from week 6 onwards. The D1 and D2 groups caught up in body weight with that of control fish within 2 weeks and 4 weeks of refeeding, respectively, but the D3 group had significantly (P < 0.05) lower body weight than the control after 5 weeks of refeeding. Higher growth efficiency was observed in all the feed deprived groups in the 1st week of refeeding only. Feed intake in D1 group became similar with the control after 5 weeks of refeeding, but the D2 and D3 groups were still hyperphagic when the experiment terminated. Thus, compensatory growth was due to hyperphagia and improved growth efficiency. In conclusion, complete compensatory growth was observed in L. rohita fingerlings after feed deprivation of 1 and 2 weeks.     

Cichlasoma dimerus responds to refeeding with a partial compensatory growth associated with an increment of the feed conversion efficiency and a rapid recovery of GH/IGFs axis

Many fish species display compensatory growth (CG), a phenomenon by which fasted fish grow faster during refeeding. However, most studies use a group‐housed fish approach that could be problematic in social fish when interaction between individuals is not considered or eliminated. Additionally, the growth hormone (GH)/insulin‐like growth factors’ (IGF‐1 and IGF‐2) axis is implicated in postnatal growth in vertebrates, but its relevance in CG is not fully understood. Thus, the aim of this work was to determine whether CG occurs in a social fish, Cichlasoma dimerus, using an individually held fish approach and secondly, to evaluate the GH/IGFs expression profile during refeeding by 3 days and 3 weeks. C. dimerus showed partial CG. The feed conversion efficiency (FCE) was higher in three‐day‐refed fish, which presented higher GH plasma and mRNA levels than controls but shown no differences in liver and muscle GH receptors (GHR1 and GHR2) and IGFs mRNA levels. Surprisingly, three‐week‐refed fish exhibited GHR1 and IGF‐2 increments, but a reduction in GHR2 expression in muscle. These results show a strong association between GH levels, growth rate and FCE during refeeding, and a long‐lasting effect of refeeding on muscular expression of GHRs and IGF‐2.     

Dietary lipid levels affect lipoprotein clearance,fatty acid transport,lipogenesis and lipolysis at the transcriptional level in muscle and adipose tissue of large yellow croaker (Larimichthys crocea)

To investigate the effect of dietary lipid levels on lipid metabolism in muscle and adipose tissue of large yellow croaker (Larimichthys crocea), fish (average body weight 150.0 g) were fed to one of three diets with a low (6%), moderate (12%, the control diet) or high (18%) crude lipid level for 10 weeks. The expression of genes related to lipoprotein clearance (lpl, ldlr, lrp1 and srbi), fatty acid transport (cd36, fatp1, fabp3 and fabp11), lipogenesis (fas and dgat2) and lipolysis (atgl, cpt1 and aco) in muscle and adipose tissue was analysed. In muscle of fish fed the low‐lipid diet, the expression of lpl, lrp1, fas, dgat2 and atgl was significantly downregulated, whereas fabp3 and fabp11 were significantly upregulated compared with those fed the control diet. In muscle of fish fed the high‐lipid diet, the expression of lpl, ldlr, lrp1, cd36, fabp3, fas and dgat2 was significantly decreased, while fabp11 expression was significantly increased compared with those fed the control diet. Compared with the control group, only a significant downregulation of fabp3 expression in adipose tissue was observed in the low‐lipid group. In the high‐lipid group compared with the control group, the expression of ldlr, fabp11, dgat2, atgl and aco in adipose tissue was significantly upregulated, whereas cpt1 expression in adipose tissue was significantly downregulated. These results indicated that the expression of the selected key genes related to lipid metabolism was tissue‐specific regulation by dietary lipid levels in large yellow croaker.     

Effects of Estradiol and Testosterone on the Expression of Growth‐related Genes in Female and Male Nile Tilapia,Oreochromis niloticus

Nile tilapia exhibits strong sexual growth dimorphism. The potential role of sex steroid hormones in sexual growth dimorphism is not fully understood. We investigated the effects of estradiol (E₂) and testosterone (T) on growth rate, plasma sex hormones, and expression of growth hormone (GH)‐insulin‐like growth factor (IGF) axis genes and muscle regulatory factor (MRF) genes in female and male Nile tilapia. The results revealed that serum concentrations of E₂ and T were significantly higher after correlative injection (P < 0.05). Compared to male fish, female fish had lower growth rates. E₂ increased growth performance in females with no significant effects on males, whereas T significantly increased growth performance in males, with no significant effects on females. In females, E₂ significantly increased expression of ghr1, ghr2, igf1, and igf2, while T decreased igf2 and increased ghr1 and ghr2 expression. In males, T increased expression of igf1, igf2, ghr1, and ghr2, and E₂ decreased expression of igf1, ghr1, and ghr2. Additionally, E₂ and T enhanced the expression of MRF genes (myod1, myod2, myog, and myf5) in female and male fish, respectively. The results suggest that sex steroid hormones play a role in sexual growth dimorphism by regulating the expression of GH‐IGF axis and MRF genes.     

Effects of total replacement of fish oil by pork lard or rapeseed oil and recovery by a fish oil finishing diet on growth,health and fish quality of gibel carp (Carassius auratus gibelio)

An 84‐day growth trial was designed to investigate effects of dietary replacements fish oil with pork lard (PL) or rapeseed oil (RO) on growth and quality of gibel carp (Carassius auratus gibelio var. CAS III) (initial body weight: 158.2 ± 0.2 g), and responses of the fish refed fish oil (FO) diet. Three isonitrogenous (crude protein: 30%) and isolipid (crude lipid: 10%) diets were formulated containing 7.73% FO, PL or RO. Five experimental treatments including FO group (FO), PL group (PL), RO group (RO), group fed PL for 42 days and refed FO for 42 days (PL+rFO), RO and refed FO group (RO+rFO) was tested. At the end of first 42 days, the fish fed PL and RO had higher mortality than that of the control (P > 0.05). At the end of whole experiment, fish fed PL and RO showed higher plasma cortisol than FO fish (P < 0.05). RO+rFO fish showed higher lysozyme activity than RO fish (P < 0.05). Fish growth and feed utilization, composition of whole body and muscle, free amino acids, texture, off‐flavour substances or sensory attributes were not affected by dietary treatments (P > 0.05). PL and RO diet decreased muscle EPA, DHA and n‐3/n‐6 ratio (P < 0.05), while FO‐refeeding had recovery effect. It can be concluded that the replacement of FO by PL and RO does not affect the growth, feed utilization or fish tasting quality in gibel carp. Fish muscle fatty acids modified by dietary PL and RO can be recovered by refeeding with FO diet.     

Impact of starvation on digestive enzymes activities and plasma metabolites in Siberian sturgeon (Acipenser baerii,Brandt, 1869)

To increase the current knowledge about the relationship between nutritional status and the digestive capacity of Siberian sturgeon (Acipenser baerii), we addressed the effect of starvation‐refeeding and macronutrient composition on growth parameters and key digestive enzyme activities in A. baerii. Acipenser baerii juveniles were fed four different diets for 3 weeks, then starved for 2 weeks and allowed to refed for 5 weeks with the same diets. Another group of fish were fed 10 weeks with the corresponding diets. Among 10‐week fed fish, high‐protein diets promoted higher body weight values, while the lowest specific growth rate was observed in fish fed a low‐protein, medium‐carbohydrate, high‐lipid diet (p < .05). At the end of the experiment, in fish refed for 5 weeks following a feeding‐starvation cycle and in 10‐week fed animals, the higher levels of blood glucose, triglycerides and cholesterol were found in fish fed low‐protein diets (p < .05). In all treatments, 2 weeks of starvation decreased α‐amylase activity in the intestine (p < .05), while 4 days of refeeding increased lipase (p > .05) and α‐amylase activity in the intestine as well as pepsin in the stomach (p < .05). Our findings suggest that A. baerii maintains a high capacity to digest proteins and lipids after 2 weeks of starvation and that α‐amylase can be used as an indicator of the nutritional status in fish submitted to starvation‐refeeding cycles. Indeed, refeeding with high‐protein and CHO:L ratio diets after starvation could improve the growth rate of A. baerii in culture.     

Effects of dietary chlorogenic acid on growth,flesh quality and serum biochemical indices of grass carp (Ctenopharyngodon idella)

The study was to investigate effects of dietary chlorogenic acid (CGA) on growth performance, flesh quality and serum biochemical indices of grass carp (95.1 ± 0.3 g) (Ctenopharyngodon idella) fed seven different diets, including control diet, Eucommia ulmoides (EU)‐supplemented diet (20 g kg^–1) and CGA‐supplemented diets containing 100, 200, 400, 600 and 800 mg/kg CGA. Contents of collagen and alkaline‐insoluble collagen in muscle and skin were significantly increased by dietary CGA and EU (p < .05). Total essential amino acids (TEAA) and total amino acids (TAA) in muscle of grass carp fed EU diet or 400, 600 and 800 mg/kg CGA diet were significantly higher than those of fish fed control diet and 100 and 200 mg/kg CGA diet (p < .05). Fish fed 200–800 mg/kg CGA showed significantly lower muscle crude lipid content than EU, control and 100 mg/kg CGA groups (p < .05). Fish fed CGA‐supplemented diets (100–800 mg/kg) had significantly higher muscle fibre density and lower muscle fibre diameter than control group (p < .05). In conclusion, supplementation of CGA improved flesh quality of grass carp, and supplemental level of CGA for improving flesh quality and growth was estimated to be 400 mg/kg diet.     

Physiological Response to Dolops carvalhoi (Crustacea: Branchiura) Infection by Pacu,Piaractus mesopotamicus,Subjected to Short Cycles of Food Restriction and Refeeding

Feeding strategies that reduce feed and promote compensatory growth could be an interesting tool to reduce costs in the fish production. However, fish health must be monitored to evaluate if their physiological response to adverse conditions, such as parasite infection, does not become compromised. A 12‐wk growth trial was conducted to determine the physiological responses of pacu, Piaractus mesopotamicus, that were subjected to different fasting/refeeding cycles and infected with the Dolops carvalhoi. The schemes were: (i) control group fish (FD), (ii) food‐restricted and controlled refeeding group (FR/Rc), and (iii) food‐restricted and refeeding to satiation group (FR/Rs). After 84 d, the fish were exposed to D. carvalhoi for 30 h. The fish subjected to food restriction did not exhibit compensatory growth. Cortisol levels decreased in all groups within 30 h after infection. Glucose levels increased 6 h after the D. carvalhoi in the FR/Rs and 30 h after infection in the FD. In all of the fish groups, the hematocrit values were reduced after infection, and it was associated with a reduction in the mean corpuscular volume and erythrocytes. At 30 h after infection, the number of erythroblasts increased. The use of the feeding schemes does not indicate a failure of the pacu physiological responses.     

Fish protein hydrolysate in diets of turbot affects muscle fibre morphometry,and the expression of muscle growth‐related genes

The study was conducted to investigate the effects of fish protein hydrolysate (FPH) in diets for turbot on growth performance, muscle fibre morphometry, and the expression of muscle growth‐related genes. A control diet (FPH0) contained 0 g/kg FPH, and four experimental diets were formulated replacing fishmeal with FPH at levels of 45 (FPH4.5), 120 (FPH12), 180 (FPH18) and 300 (FPH30) g/kg. Fish fed the FPH12 and FPH18 diets had higher specific growth rate (SGR) than fish fed the FPH0 diet (p < .05), and a quadratic regression was found between SGR and dietary FPH level (p = .001, R² = .677). Cross‐section area (CSA) and the length of sarcomere in the FPH12 group increased compared with the control group (p < .05), and a quadratic regression was observed between CSA and dietary FPH level (p = .006, R² = .574) and between sarcomere length and dietary FPH level (p = .018, R² = .788). An appropriate level of FPH down‐regulated myostatin 2 gene expression and up‐regulated proliferating cell nuclear antigen gene expression, while the expression of myogenic regulatory factors was not affected by dietary treatments (p > .05). To conclude, an appropriate level of FPH may improve muscle growth by regulating the expression of muscle growth‐related genes, and muscle microstructure and ultrastructure.     

Compensatory growth and digestive enzyme activity of Litopenaeus vannamei submitted to feeding restriction in a biofloc system

Feeding restriction is a strategy in shrimp farming management that may promote compensatory growth after feeding is re‐established. This study aims to evaluate the effects of two feeding restriction regimens on the compensatory growth and digestive enzymes activity of Litopenaeus vannamei reared in biofloc system. Juvenile shrimp (0.46 ± 0.18 g) were stocked (320 individuals/m³) in 310 L tanks. The experiment comprised two phases: (a) Feeding Restriction (30 days) when shrimp were submitted to three feeding regimes, Control (fed daily), R1F1 (repetitively fasted one day and fed one day) and R2F1 (repetitively fasted 2 days and fed 1 day); and (b) Refeeding (28 days) when shrimp were fed daily. In the restriction phase, shrimp growth and digestive enzyme activities were reduced in R2F1 and R1F1. However, during the refeeding phase, enzyme activities and feed conversion improve significantly in R2F1 and R1F1. Control group attained higher final weight, but its final biomass was similar to R1F1. Litopenaeus vannamei exhibited partial compensatory growth, probably due to improved feed conversion efficiency driven by increased enzyme activity. It is possible to reduce feeding by 50% (R1F1) in biofloc systems for 28 days, without compromising the biomass produced at the end of a 30‐day refeeding period.     

The differences in postprandial free amino acid concentrations and the gene expression of PepT1 and amino acid transporters after fishmeal partial replacement by meat and bone meal in juvenile turbot (Scophthalmus maximus L.)

Meat and bone meal (MBM) is a high‐quality alternative protein source used to replace fishmeal (FM). However, the molecular mechanisms of over‐substituted FM by MBM resulted in growth reduction are still not clear. The objective of the study was to evaluate the effect of FM replacement by MBM on the concentration of postprandial free amino acid (FAA) and mRNA abundance of peptide and amino acid transporters in juvenile turbot (Scophthalmus maximus L.). Fish were fed with FM diet (60% FM), MBM diet (33% FM + 34.2% MBM) and MBM + AA diet (MBM diet with essential amino acid (EAA) added to match the AA profile of FM) for 30 days. Results showed that compared with the FM diet, MBM diet led to a reduction in FAA concentration peak values in plasma and muscle. MBM + AA diet significantly elevated the peak values of FAA concentrations to FM diet level in plasma, but not in muscle. Furthermore, compared with FM diet, MBM diet significantly increased gene expression of PepT1 and major amino acid transporters in intestine, whereas MBM diet greatly downregulated gene expression of T‐type amino acid transporter‐1, system ASC amino acid transporter‐2 and cationic amino acid transporter‐2 in muscle. Supplemented EAA did not ameliorate these different effects in intestine and muscle. Overall, this study provided a comprehensive explanation for the relationship between diet, FAA concentrations and AA transportations, which provides a molecular basis for further using MBM to replace FM in aquafeeds.     

Growth performance,digestive and absorptive capacity of on‐growing grass carp (Ctenopharyngodon idellus) fed with graded level of dietary fibre from soybean hulls

The present research evaluated the effects of graded levels of dietary fibre on growth, digestive and absorptive capacities, and the potential underlying mechanisms in on‐growing grass carp (Ctenopharyngodon idella). Grass carp (123.00 ± 0.70 g) were fed diets with graded levels of neutral detergent fibre (NDF: 97.8, 119.0, 140.2, 159.4, 181.2 and 201.6 g/kg diet) for 60 days. Besides, a 2‐week digestion experiment was conducted to explore the effect of dietary fibre on the apparent digestibility coefficients (ADC) of feed. The results showed that optimum dietary fibre level (140.2 or 159.4 g/kg diet) increased feed intake, per cent weight gain, specific growth rate and the ADC of dietary protein (p < .05); increased the activities of digestive and brush border enzyme; and up‐regulated intestinal amino acid transporter mRNA levels (SLC1A5 and SLC6A19b, p < .05) partially associated with activation of TOR signalling pathway. However, high dietary fibre levels (NDF levels ≥181.2 g/kg diet) were not conducive to the digestion and absorption of nutrients, resulting in the decline of growth performance. Thus, based on the quadratic regression analysis for per cent weight gain and feed efficiency, the optimum (143.9 and 134.5 g/kg diet) and maximum tolerance (189.8 and 171.2 g/kg diet) levels of dietary NDF were estimated for on‐growing grass carp.     

Fishmeal levels can be successfully reduced in white shrimp (Litopenaeus vannamei) if supplemented with DL‐Methionine (DL‐Met) or DL‐Methionyl‐DL‐Methionine (Met‐Met)

The main objective of this study was to evaluate the effect of methionine supplementation when reducing fishmeal levels in diets for white shrimp (Litopenaeus vannamei). Tested diets consisted of a positive control with 260 g/kg fishmeal (D1), two negative controls with 100 g/kg fishmeal and no amino acid (AA) supplementation (D2) or supplemented with lysine but not methionine (D3), and four additional diets with 100g/kg fishmeal supplemented with increasing levels of DL‐Met (1.0, 2.0 or 3.0 g/kg) (D4, D5, D6) or Met‐Met (1.0 g/kg) (D7). Each diet was fed to four groups of 30 shrimp for 8 weeks at a daily rate of 70 g/kg body weight. Reduction in fishmeal from 260 g/kg down to 100 g/kg did not significantly affect survival rate, feed conversion ratio (FCR), protein efficiency ratio (PER) or protein retention efficiency (PR%) of white shrimp. However, growth performance (final body weight, FBW; weight gain, WG; specific growth rate, SGR) was reduced when dietary fishmeal level was reduced from 260 g/kg (D1) to 100 g/kg without methionine supplementation (D2). The growth performance (FBW, WG and SGR) of shrimp was significantly increased by supplementation of the 100 g/kg fishmeal diet with increasing levels of DL‐Met (p < .05). Same performance as positive control (D1) was achieved with diets containing 100 g/kg fishmeal and supplemented with 3.0 g/kg DL‐Met or 1.0 g/kg Met‐Met. The highest values of growth performance (FBW, WG and SGR) were found in shrimp fed D6 and D7 diets, which were significantly higher than those of shrimp fed D2 and D3 diets (p < .05) but without statistical differences with shrimp fed D1, D4 and D5 diets (p > .05). The highest values of whole‐body and muscle protein contents were found in shrimp fed D1 diet, which were significantly higher than those of shrimp fed all other diets (p < .05). The highest value of intestinal tract proteolytic enzyme activity was found in shrimp fed Met‐Met‐supplemented diet (D7) and followed by the positive control diet (D1) and 3 g/kg DL‐Met‐supplemented diet (D6) (p < .05). The highest values of apparent digestibility coefficients (ADCs) of dry matter and crude protein were found in Met‐Met‐supplemented diet (D7) and followed by the positive control diet (D1) (p < .05). Shrimp fed the D1 diet showed the highest value of total essential amino acid (EAA) and was significantly higher than shrimp fed D2–D3 (p < .05) but without significant difference with shrimp fed D4–D7 (p > .05). In conclusion, results showed that same performance can be achieved with diets containing 260 or 100 g/kg fishmeal supplemented with 3.0 g/kg DL‐Met or 1.0 g/kg Met‐Met. Moreover, supplementation of limiting methionine in low‐fishmeal diets seems to improve the digestive proteolytic activity, improving digestibility of dry matter and protein, and eventually to promote growth of juvenile white shrimp in fishmeal reduction diets.     

The effects on some non‐enzymatic antioxidants and oxidative stress of Astacus leptodactylus (Esch., 1823) of starvation periods

This study investigated the effect of starvation (78 days) and refeeding (33 days) on the oxidative stress [malondialdehyde (MDA)] and the non‐enzymatic antioxidants [vitamin E (VE), vitamin C (VC), vitamin A (VA), beta carotene (βC) and reduced glutathione (GSH)] in the hepatopancreas, muscle and gill tissues of freshwater crayfish (Astacus leptodactylus). Crayfish were divided into three experimental groups: control (fed), starved (not fed) crayfish for 78 days and refeeding crayfish for 33 days after 78 days of starvation. The biochemical analysis of the tissues was conducted at 3, 18, 33, 48, 63 and 78 days of starvation and feeding and at 3, 18 and 33 days of refeeding. It was determined that crayfish can withstand starvation period of 78 days. In all of the periods, the MDA levels were significantly higher in the tissues of starved crayfish when compared with the control. The findings of this study demonstrate that starvation has a negative effect on the VE, VC, VA, βC and GSH levels in the crayfish. The measured parameters returned to control values after 33 days of the refeeding. Additionally, the starvation resulted in decreased levels of VE, VA and βC in the abdomen muscle of crayfish consumed by humans.