Anaesthetic efficacy and biochemical effects of 1,8‐cineole in rainbow trout (Oncorhynchus mykiss,Walbaum, 1792)

The aim of the present study was to investigate the anaesthetic efficacy and biochemical effects of 1,8‐cineole (cineole) in rainbow trout (Oncorhynchus mykiss). The fish were exposed to 200, 300, 400, 500, 600 and 800 μl/L cineole and time of induction of anaesthesia and recovery from anaesthesia were recorded. Thereafter, the fish haematological and biochemical responses to anaesthesia with different concentrations of cineole were studies. Moreover, the haematological and biochemical response of fish anaesthetized for 300 s with either cineole (283 μl/L) or eugenol (25 μl/L) were compared. Cineole at the concentrations of 200–800 μl/L induced stages 2, 3 and 4 anaesthesia within 109‐29.3, 226‐59 and 418‐117 s respectively. Increase in anaesthesia induction time led to higher stress responses and enzymes’ activity characterized by elevation in red blood cell (RBC), white blood cell (WBC), blood haematocrit and haemoglobin, and plasma cortisol, glucose and lactate levels, and aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase and creatine phosphokinase (CPK) activities. However, cineole concentrations had no significant effects on plasma alanine aminotransferase (ALT) activity and ions levels. Comparison of cineole with eugenol showed that cineole showed less change in blood RBC and plasma AST, ALP, CPK, cortisol and glucose levels compared to eugenol. In conclusion, cineole is efficient to anaesthetize trout at concentrations of 200–800 μl/L. Increase in cineole concentration shortens anaesthesia induction time, stress response and probably tissue damage. The concentrations of 600–800 μl/L cineol is recommended for rapid sampling as it causes the least stress and enzymatic responses. The present results suggest that cineole causes slightly lower side effects in trout compared to eugenol.     

Menthol and 1,8‐cineole as new anaesthetics in common carp,Cyprinus carpio (Linnaeus, 1758)

This study describes anaesthetic efficacy of menthol and 1,8‐cineole in common carp, Cyprinus carpio, in comparison with eugenol. Common carp fingerlings were exposed to eugenol: 5, 10, 15, 25, 35, 50, 75, 100, 150 and 200 ppm; menthol: 5, 10, 15, 25, 50, 100, 200, 300, 400, 500 and 600 ppm; 1,8‐cineole: 100, 150, 200, 300, 400, 500, 600, 700 and 800 ppm. Induction time and recovery time were recorded. Results showed that menthol and 1,8‐cineole anesthetized the fish at higher concentrations compared to eugenol. The fish exposed to menthol showed common fish behavioural responses to anaesthesia, similar to eugenol. But, 1,8‐cineole‐exposed fish showed tail‐up swimming, which was not observed before. Also, 1,8‐cineole failed to completely cease muscle tone. Exposure to 200 ppm eugenol and 600 ppm menthol resulted in 40% and 20% mortality, respectively. Induction time was exponentially dependent on anesthetic concentrations. Recovery time was linearly correlated to eugenol and menthol, but not 1,8‐cineole concentrations. Recovery time was exponentially dependent on induction time in the fish anesthetized with eugenol and menthol, but not 1,8‐cineole. Menthol and 1,8‐cineole are recommended for carp anaesthesia. Menthol is capable to anesthetize common carp within 1–3 min at 118–512 ppm. Common carp anesthetized with 108–133 ppm menthol recovers within 5 min. 1,8‐cineole failed to anesthetize common carp within less than 150 s at 300–800 ppm concentrations. However, it anesthetizes carp within 3 min at 595 ppm concentration. Also, 1,8‐cineole is not recommended for fish surgery. Both menthol and 1,8‐cineole were less efficacious than eugenol.     

Anaesthetic effect of eugenol at different concentrations and temperatures on black tiger shrimp (Penaeus monodon)

The anaesthetic effects of eugenol on Penaeus monodon were investigated at the different eugenol concentrations (60, 110, 160 and 210 mg/L), water temperature (21, 26 and 31°C), air exposure time (3, 6, 9 and 12 min) and body weight (2.62 ± 0.27, 6.34 ± 0.36 and 11.43 ± 0.33 g). The anaesthesia and recovery time were recorded. The results showed that the anaesthesia time of the shrimp decreased with the increase in the eugenol concentration and water temperature, and the recovery time increased with the increase of the eugenol concentration and the decrease of water temperature. Under the same eugenol concentrations, the recovery time increased with the increase of air exposure time and body weight. Under the eugenol concentration range of 60–210 mg/L, the recovered rate was 100%. The results indicated that eugenol is a safe and efficient anaesthetic for P. monodon.     

Alpinia galanga oil—A new natural source of fish anaesthetic

This study explores the anaesthetic activity of Alpinia galanga oil (AGO) in fish. Cyprinus carpio (koi carp) was used as a fish model. It was found that the induction time to stage 3 anaesthesia and the recovery time of the fish after exposure to AGO were exponentially and polynomially correlated to AGO concentrations. The viability of normal blood cells of koi carp anaesthetized with 500 mg/L AGO was found to be higher than 90% for normal red blood cells and white blood cells and 89% for peripheral blood nuclear cells indicating nontoxicity of AGO to the fish. A concentration of 300 mg/L of AGO was the most suitable for anaesthetizing koi carp due to the safety and effectiveness aspects as being ideally fitted to anaesthetic criteria. This concentration gave the induction time of 205.55 ± 5.07 s and the recovery time of 202.50 ± 9.30 s. Determination of stress biomarker such as blood cortisol and glucose as well as gene expression showed that the blood cortisol level of the fish anaesthetized with AGO was similar to normal levels. Moreover, blood glucose level was significantly less increased than those anaesthetized with tricaine methanesulfonate. Gene expressions of the fish cortisol receptor, cytochrome oxidase subunit1, heat shock protein 70 and Na⁺/K⁺‐ATPaseα₃ were significantly reduced after exposure to AGO indicating the advantages of AGO on fish stress reduction. Thus, AGO is a promising natural source for an alternative fish anaesthetics.     

Use of MS‐222 (tricaine methanesulfonate) and propofol (2,6‐diisopropylphenol) as anaesthetics for the tetra Astyanax altiparanae (Teleostei,Characidae)

The aim of this study was to evaluate the anaesthetic effect of MS‐222 and propofol and determine their optimal concentrations for safe handling of the tetra Astyanax altiparanae in the laboratory. The fish were separated by length into three classes: I (1.5–5.0 cm), II (5.1–8.0 cm) and III (greater than 8.1 cm). Pilot tests were performed to evaluate the appropriate anaesthetic concentrations for inducing the five possible anaesthetic stages: I – sedation; II – light anaesthesia; III – deep anaesthesia; IV – surgical anaesthesia; and V – spinal collapse. After defining the maximum and minimum concentrations required to induce stage IV anaesthesia, the animals were exposed to five intermediate concentrations (n = 10 fish/concentration) of each anaesthetic for 15 min. The animals were then transferred to clean water to evaluate the time required for recovery. In addition, blood glucose levels were measured for class II and class III fish subjected to the previously defined ideal concentrations for each of the tested anaesthetics (n = 10 fish/treatment). Both evaluated substances are suitable to anaesthetize A. altiparanae. The optimal MS‐222 concentration was 90 mg L^?1, and this result was similar for all three size classes. The optimal propofol concentrations for inducing surgical anaesthesia in the size classes I, II and III were 0.22, 0.23 and 0.27 respectively.     

Effects of flow velocity on growth,metabolism and nitrogen and phosphorus budget of Haliotis discus hannai Ino in a recirculating aquaculture system

To study the effects of different flow velocities on growth, metabolism and nitrogen (N) and phosphorus (P) budget of Haliotis discus hannai Ino in a recirculating aquaculture system, abalones (shell length: 45.71 ± 3.09 mm, body weight: 8.62 ± 0.75 g) were cultured at three different flow velocities with four replicates each: high (400 L/hr), medium (300 L/hr) and low (200 L/hr). At the end of the 70 days experiment, the survival rate, food intake and P utilization rate of abalones in the 200 L/hr group were significantly lower than those in any other group (p < .05), whereas the total ammonia nitrogen and nitrite nitrogen concentrations in the water were significantly higher than those in any other group (p < .05). The body weight specific growth rate, food conversion efficiency, abalone‐harvested N and P outputs and N utilization rate were ranked as follows: 300 > 400 > 200 L/hr (p < .05). In the 300 L/hr group, the activities of hexokinase, pyruvate kinase and lactate dehydrogenase were significantly lower than those in the 400 L/hr group, whereas the activity of succinate dehydrogenase was significantly higher than that in the other two groups (p < .05). Aerobic metabolism was the main source of energy acquisition for abalones in the 300 L/hr group. In the 400 L/hr group, the expression levels of manganese superoxide dismutase and thioredoxin peroxidase significantly increased from Day 35 to Day 70, but they still were significantly lower than the values in the 200 L/hr group (p < .05). In the 200 L/hr group, the expression levels of catalase and heat shock protein 70 were significantly higher than those in any other group, and they significantly increased from Day 35 to Day 70 (p < .05). These results indicate that maintaining flow velocity at 300 L/hr will minimize oxidative damage due to deterioration of water quality, reduce the energy consumption required for organisms to resist the impact of water flow, improve the N and P utilization rates of the recirculating aquaculture system, and reduce the cost of culturing abalones.     

Development of cryopreservation methods for silver barb (Barbodes gonionotus,Bleeker) spermatozoa by manipulation of cooling rates in dry shipper

The aim of this study was to develop a simple cryopreservation protocol for silver barb, Barbodes gonionotus, semen using a dry shipper. Freezing rates within the upper and lower chambers of dry shipper were recorded for 14 days post liquid nitrogen loading (dpl). To regulate freezing rates, straws (250 and 500 µl) wrapped with various insulators (polystyrene foam box, oxygen tube, silicone tube and electric wire) were frozen within the upper chamber. Straws containing semen diluted with Calcium‐free Hank's Balanced Salt Solution (Ca‐F HBSS) and 10% dimethyl sulphoxide were cryopreserved with or without insulators. Appropriate protocols were selected based on sperm quality during a 45‐day cryostorage. The upper chamber had potential as a freezing chamber within 9 dpl due to no significant (p > 0.05) change in freezing rates. High percentages of sperm motility and viability (p < 0.05) were observed when 250 µl straws with silicone tube (T4) frozen for 5 min, non‐insulated 500 µl straws (T9) and 500 µl straws with polystyrene foam box (T12) frozen for 1–5 min, having freezing rates of 43.1 ± 1.3, 71.3 ± 1.4 and 14.7 ± 0.4°C/min respectively. Dry shipper can be used as a freezing tool to cryopreserve silver barb semen.     

Nanoemulsion from essential oil of Pterodon emarginatus (Fabaceae) shows in vitro efficacy against monogeneans of Colossoma macropomum (Pisces: Serrasalmidae)

Outbreaks of diseases pose a major threat to sustainable aquaculture development worldwide. Application of herbal products to combat parasitic diseases provides an alternative approach for sustainable aquaculture. This study investigated the in vitro antiparasitic effects of an oil‐in‐water nanoemulsion prepared using the essential oil from Pterodon emarginatus, against monogeneans infesting Colossoma macropomum. Gill arches from C. macropomum (47.6 ± 14.5 g and 13.5 ± 1.4 cm) that were naturally parasitized by Anacanthorus spathulatus, Notozothecium janauachensis and Mymarothecium boegeri were immersed in different dispersions of the P. emarginatus nanoemulsions (0, 50, 100, 200, 400 and 600 mg/L). The major compounds presented in the essential oil of P. emarginatus were β‐elemene, β‐caryophyllene and α‐humulene. Characterization of these nanoemulsions showed that they have a small mean droplet size and low polydispersity index, which is concordant with stable systems. In this in vitro trial, the P. emarginatus nanoemulsion concentrations of 100, 200, 400 and 600 mg/L presented 100% helminthic efficacy against monogeneans of the gills of C. macropomum. The highest two concentrations used (400 and 600 mg/L) were seen to immobilize the parasites after only 15 min. Therefore, it would be worthwhile testing these concentrations in therapeutic baths against monogeneans of C. macropomum.     

Simultaneous effect of sex and dose on efficacy of clove oil,tricaine methanesulfonate, 2‐phenoxyethanol and propofol as anaesthetics in guppies,Poecilia reticulata (Peters)

We studied the simultaneous effect of sex and dose on anaesthesia efficacy to estimate, if possible, the lowest effective dose (LED) for clove oil, tricaine methanesulphonate (MS‐222), 2‐phenoxyethanol (2‐PE) and propofol in mature guppies. LED is the lowest dose needed to reach A5 stage in a mean time of 3 min, with mean recovery (R5) time of 5 min. We used four doses/anaesthetic: 25, 50, 75 and 100 mg/L for clove oil; 120,140,160 and 180 mg/L for MS‐222; 800, 1,000, 1,200 and 1,400 mg/L for 2‐PE, and 7.50, 8.25, 10.00 and 11.25 mg/L for propofol. Each dose was tested on 10 females and 10 males. Morbidity, mortality and behavioural changes were checked on two control groups (10 males and 10 females/group). Sedation (A3), A5 and R5 times were recorded. Significant interactive effect dose*sex on A5 time was found for each anaesthetic agent (p_dose*sex < .05). Except for MS‐222 (p_dose*sex = .284), significant interactive effect dose * sex on R5 time was found (p_dose*sex < .05). A5 time in females tended to be greater than in males, but, in general, R5 times were longer in males. Body size differences between males and females could explain these differences in MS‐222 on A5 time and for clove oil, 2‐PE and propofol on R5 time. No dose simultaneous meet LED′s conditions relating to both A5 and R5 times; therefore the lowest doses inducing A5 in a mean time of 3 min could be a safe guideline for anaesthetic procedure in both male and females.     

The use of clove oil, metomidate, tricaine methanesulphonate and 2-phenoxyethanol for inducing anaesthesia and their effect on the cortisol stress response in black sea bass (Centropristis striata L.)

Juvenile and adult black sea bass (Centropristis striata L.) were exposed to various concentrations of four anaesthetics to determine practical dosages for handling as well as for procedures such as bleeding, ovarian biopsy or tag implantation. In experiment 1, juveniles exposed to either 2.0 mg L^?1 metomidate, 15 mg L^?1 clove oil, 70 mg L^?1 tricaine methanesulphonate (TMS) or 200 mg L^?1 2‐phenoxyethanol (2‐PE) reached stage II of anaesthesia in 3–5 min and could be handled for weighing and measuring. All fish had completed recovery to stage III within 6 min. In experiment 2, the established concentrations of each anaesthetic were tested on juveniles to determine their ability to prevent a reflex to a subcutaneous needle puncture. All of the fish exposed to clove oil (20 mg L^?1) and 40% of the TMS‐treated (70 mg L^?1) fish reacted while none of the fish anaesthetized in metomidate (2.0 mg L^?1) or 2‐PE (200 mg L^?1) responded to the needle puncture. In experiment 3, metomidate (5.0 mg L^?1), clove oil (30 mg L^?1) TMS (125 mg L^?1) or 2‐PE (300 mg L^?1) were all effective for performing an ovarian biopsy or tag implantation on adults. In experiment 4, TMS (125 mg L^?1) exacerbated the cortisol response to a short handling stressor during a 30 min exposure. Fish anaesthetized in 2‐PE (300 mg L^?1), metomidate (5.0 mg L^?1) or clove oil (40 mg L^?1) had increased cortisol levels associated with the handling stressor but there were no further increases during the remainder of the experimental period. The results demonstrate that these anaesthetics are effective for sedation and anaesthesia of black sea bass and that the best choice is dependant upon the procedures to be performed.     

Effect of different water biofloc contents on the growth and immune response of gibel carp cultured in zero water exchange and no feed addition system

This study evaluated the growth and immune response of gibel carp (Carassius auratus gibelio) cultured under no feed addition biofloc technology (BFT) system at different total suspended solid (TSS) concentrations (10, 300, 600, 800 and 1,000 mg/L for group BF0‐NF, BF300‐NF, BF600‐NF, BF800‐NF and BF1000‐NF) for 30 days. The results demonstrated that bioflocs contained rich nutrients, and gibel carp eaten bioflocs showed higher weight gain, specific growth and survival. Digestive enzyme activities such as pepsin and amylase increased significantly in BF300/600/800/1000‐NF groups than those in BF0‐NF group. Antioxidant response including superoxide dismutase and total antioxidant capacity in serum and skin mucus was also enhanced significantly (p < .05). In addition, six immune‐related genes were examined by RT‐qPCR. Compared with BF0‐NF group, expression levels of immune genes intelectin (ITLN), dual specificity phosphatase 1 (DUSP 1), keratin 8 (KRT 8), myeloid‐specific‐peroxidase (MPO), c‐type lysozyme (c‐lys) and interleukin‐11 (IL‐11) were up‐regulated by 78.1‐, 23.9‐, 13.8‐, 138.8‐, 401.8‐ and 91.1‐fold, respectively. The highest expression values were observed at TSS of 600–800 mg/L. This study suggested that bioflocs can be uptaken by gibel carp as a food source, and have a potential to be used as a supplemental food for aquaculture.     

Effects of linalool on physiological responses of Cyprinus carpio (Linnaeus, 1758) and water physico‐chemical parameters during transportation

This study investigated the potential of linalool as an anaesthetic during transportation of common carp, Cyprinus carpio. The fish were transported at a loading density of ~103 g/L for 3 hr in 12 plastic bags (3 L water and 6 L pure oxygen) divided into four triplicated treatments: control (without linalool), L50 (50 mg linalool/L), L100 (100 mg linalool/L) and L200 (200 mg linalool/L). After 3‐hr transportation, serum physiological responses and water physico‐chemical parameters were compared among the treatments. Results showed that water total and un‐ionized ammonia increased and dissolved oxygen decreased in all treatments after transportation. Water total ammonia and dissolved oxygen levels in the linalool‐treated bags were significantly lower than the control. After transportation, the control fish showed significant elevation in serum cortisol and glucose levels; however, the levels were significantly lower than the linalool‐treated fish. All fish showed similar serum lactate levels, significantly lower than the value obtained before transportation. The control fish showed serum urea levels similar to the level obtained before transportation; however, the linalool‐treated fish had significantly higher serum urea levels compared to the control and before transportation levels. After transportation, all fish had decreased serum chloride and sodium levels compared to the before transport level. Overall, the present results showed that linalool is not beneficial for carp transportation in plastic bags, because it reacts with water oxygen, increases stress in fish, interferes with ammonia excretion and has no benefits in preventing ion loss.     

Integrated rearing system approach in the farming of mud crab,shrimp, fish,oyster and periphyton in bracksihwater pond

Diversification of production systems has been identified as one of the major strategies for the long‐term sustainability of brackishwater aquaculture. On the context of diversification, a 180‐day culture experiment was carried out where four farming systems were tested: monoculture of mud crab, Scylla serrata (T1), polyculture of mud crab, with mullet Mugil cephalus and shrimp Penaeus indicus (T2), polyculture of mud crab with mullet, shrimp and oyster Crassostrea cuttackensis (T3), polyculture of mud crab with mullet, shrimp, oyster along with periphyton net (T4) covering 10% of the water spread area. There was no significant difference in the growth performances of mud crab, shrimp and oyster among the treatment but a significantly higher (p < .05) final average body weight (ABW) of M.cephalus was recorded in T4 (121.19 ± 2.09 g). Production considering only the fed animals (excluding oyster) was significantly (p < .05) higher in T4 (2,115 kg/ha) compared to other treatments. The overall water quality parameters do not vary significantly (p > .05) among the treatment ponds. Although there was no significant difference in total ammonia nitrogen (TAN) and nitrite‐N, highest value was recorded in T2 (56.23 ± 39.47 µg/L and 185.53 ± 110.71 µg/L) and T1 (55.78 ± 32.26 µg/L and 180.83 ± 103.65 µg/L). The results from the current experiment based on water quality and growth of animals showed that there is no negative interaction between the mud crab and other animals stocked in the polyculture pond which indicated that polyculture provides an efficient and sustainable utilization of resources.     

Evaluating the use of garlic (Allium sativum) for the remedy of Cryptocaryon irritans in guppies (Poecilia reticulata)

Garlic (Allium sativum) has been shown to possess antimicrobial properties against a range of disease‐causing agents, including fish parasites. Our study aimed to investigate the potential use of garlic as a treatment against Cryptocaryon irritans infection, using guppies (Poecilia reticulata) as the fish model. Garlic was freeze‐dried, powdered, and used as it is or as an aqueous extract. The content of allicin, its main active ingredient, was 1.25 mg/g in garlic powder and 0.82 mg/ml in the aqueous extract, as determined by HPLC analysis. Aqueous garlic extract fully immobilized C. irritans theronts and protomonts within 20 and 40 min, respectively, following exposure to 5 µl/ml. Treatment trials were performed, in which infected fish were fed with garlic powder‐supplemented feeds (5%, 10%, or 20% supplementation), bathed in garlic aqueous extract (250 or 500 µl/L) and subjected to a combined treatment which included both feeding and bathing. Results revealed that the tested treatments failed to cure the infection, though reduction in infection intensity on the caudal fin, but not on the gills, was evident following dietary supplementation with 20% of powdered garlic in one of the trials.     

Determination of grapefruit (Citrus paradisi) peel extract bio‐active substances and its application in Caspian white fish (Rutilus frisii kutum) diet: Growth,haemato‐biochemical parameters and intestinal morphology

In this study, total phenolic and flavonoid contents of grapefruit peel extract (GPE) were equal to 117.3 ± 0.3 µg of gallic acid/mg and 39.30 ± 0.1 µg of quercetin/mg respectively. Caspian white fish (n = 180, 4 ± 0.9 g body weight) was fed with supplemented diets, including 0, 6.25, 12.5 and 25 mg of GPE/kg for 60 days at 25 ± 1°C. The growth performance was markedly improved in fish fed with 25 mg/kg of GPE compared to others (p < 0.05). Moreover, fish fed with 25 mg/kg of GPE showed a significant increase in red blood cell (2.65·10⁶ cell/mm³), white blood cell (17.75·10³ cell/mm³), packed cell volume (48%) and haemoglobin concentrations (8.75 g/dl) compared to the control (p < 0.05). However, the highest alanine aminotransferase (140 U/L), alanine transaminase (14.5 U/L), and alkaline phosphatase (18.5 U/L) were observed in control group. Morphological analysis of intestine revealed the highest amount of villus width (8.4 µM), height (32.86 µM) and surface area (342.7 µM²) in fish fed with 25 mg/kg of GPE (p < 0.05). In conclusion, supplementing feed with GPE at 25 g/kg can improve growth performance and haemato‐biochemical parameters of Caspian white fish fry.     

Autochthonous probiotic as growth promoter and immunomodulator for Astyanax bimaculatus cultured in water recirculation system

This work aimed to evaluate the probiotic Lactobacillus spp. in the microbiota of Astyanax bimaculatus reared in a water recirculation system. The experiment was randomized, using 800 postlarvae distributed into eight polyethylene containers, separated into two treatments, in quadruplicate: supplementation with Lactobacillus spp. (Probiotic) and no supplementation (Control). After 90 days of culture, 13 fish per container were anaesthetized. Three of these underwent microbiological evaluation, and haematological analyses were carried out on five. Immunological assessment was performed on another five fish. Zootechnical parameters were evaluated for all animals. In the microbiological evaluation, the probiotic group presented higher counts of lactic acid bacteria, but lower counts of Vibrio spp., Pseudomonas spp., and Staphylococcus spp., when compared with control. After haematological analysis, the supplemented group presented low haematimetric indices of Mean Corpuscular Haemoglobin Concentration and Mean Corpuscular Haemoglobin (MCHC and MCH, respectively) and higher amounts of total leucocytes, thrombocytes and circulating monocytes when compared with control. The immunological profile did not differ between treatments. In the zootechnical performance, probiotic group presented higher productivity, survival and apparent food efficiency when compared with control. Therefore, when Lactobacillus spp. was supplemented in the diet of A. bimaculatus, immunocompetence and the zootechnical performance of animals increased.     

Effects of dietary geniposidic acid on growth performance,flesh quality and collagen gene expression of grass carp,Ctenopharyngodon idella

This study was conducted to investigate the effects of dietary geniposidic acid (GA) on growth performance, flesh quality and collagen gene expression of grass carp (Ctenopharyngodon idella). The fish with an initial body weight of 47.1 ± 0.8 g were fed one of the seven diets, including control diet, Eucommia ulmoides (EU)‐supplemented diet (20 g/kg) and GA‐supplemented diets (200, 400, 600, 800 and 1,000 mg/kg GA) for 75 days. The growth performance and muscle proximate composition showed no difference among groups (p > .05). Dietary GA (200–1,000 mg/kg) increased the contents of total collagen and alkaline‐insoluble collagen in skin (p < .05), and high supplementation of GA (600–1,000 mg/kg GA) and EU increased the contents of total collagen, alkaline‐insoluble collagen and total amino acids (p < .05), but reduced the lipid level in muscle (p < .05). In collagen gene expression, EU and 200–1,000 mg/kg GA increased COL1A1 expression in muscle and skin (p < .05), but the expression of COL1A2 was increased only by high supplementation of GA (1,000 mg/kg, or 800–1,000 mg/kg) (p < .05). In conclusion, dietary GA improved the flesh quality of grass carp, and the supplementation level was estimated to be 600 mg/kg diet.     

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.     

The efficacy of Aqui‐S vet. (iso‐eugenol) and metomidate as anaesthetics in European eel (Anguilla anguilla L.), and their effects on animal welfare and primary and secondary stress responses

The effects of 40 mg/L metomidate and 300 mg/L iso‐eugenol (i.e. 600 mg/L Aqui‐S vet.) anaesthesia on the stress response in European silver eel during a process of complete anaesthesia, recovery and long‐term survival were studied. Metomidate is a hypnotic agent with no analgesia, whereas Aqui‐S vet. is a true anaesthetic. There was no difference between fish exposed to Aqui‐S vet. and metomidate, with respect to time to anaesthesia (mean 3.8 and 2.6 min respectively) and recovery (mean 7.6 and 6.5 min respectively). For the metomidate group, the plasma cortisol concentration increased to a peak during the recovery phase, and was significantly higher than for the Aqui‐S vet. group, which showed no such increase. Fish were kept in tanks with seawater for monitoring of long‐term survival (4 months) after anaesthesia treatment, and no mortality was observed in either group. The results indicate that metomidate is a potential stressor to European eels during exposure, and we do not recommend using metomidate in this species. Aqui‐S vet., however, seems promising as a stress‐reducing anaesthetic for European eel, and if used properly could improve animal welfare and survivability during and after common ecology‐related procedures, as capture, tagging and size measuring.     

Prevalence of stocked whitefish in River Kemijoki,Finland, inferred by micro X‐ray fluorescence analysis of otoliths

Micro X‐ray fluorescence (µ‐XRF) analysis of otoliths was evaluated as a method to estimate the proportion of stocked one‐summer‐old whitefish Coregonus lavaretus L. in catches of adult fish (n = 20) ascending the River Kemijoki to spawn. Laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS) analysis was applied as control. Polished otoliths were scanned with µ‐XRF to obtain strontium maps that were used to infer visually the provenance of the whitefish. Thirteen of the fish showed signs of being stocked as one‐summer‐old fingerlings. LA‐ICP‐MS was applied to determine the elemental composition in a spot outside the core of the otolith. The results were largely consistent with the visual inspection of the µ‐XRF mapped otoliths. In conclusion, µ‐XRF mapping successfully identified whitefish stocked as one‐summer‐old fingerlings. The vast majority of whitefish returning to the River Kemijoki to spawn were stocked fish.