Wheat grains fermented by fungal mycelia (<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> or <Emphasis Type="Italic">Lentinus edodes</Emphasis>) as alternative feed ingredients for juvenile rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

We investigated the effects of replacing non-fermented wheat grains with wheat grains fermented by fungal mycelia in the diet of juvenile rainbow trout (Oncorhynchus mykiss). We assessed growth performance, feeding parameters, and body composition in three experimental groups (0.33?±?0.01 g, in triplicates of 50 individuals each). The diets for all the groups contained ca. 43% protein and 19% lipids. Experimental diets were made by replacing the 100 g kg^?1 of wheat grains present in the basal diet (CTRL) with the same proportion of wheat grains fermented by Pleurotus ostreatus (PWD) or Lentinus edodes (LWD) mycelium. Fish were fed to apparent satiation twice a day for 56 days. Both, PWD and LWD, significantly increased fish body weight from day 28 onwards. Final body weight was 2.37?±?0.04 g (CTRL), 4.29?±?0.02 g (PWD), and 3.50?±?0.05 g (LWD), and feeding efficiency (%) was increased from 64.5?±?0.7 (CTRL) to 92.5?±?0.5 (PWD) and 84.8?±?1.5 (LWD). The experimental diets also improved nutrient retention efficiency (%): 30.0?±?0.5 (PWD), 27.7?±?1.1 (LWD), and 21.0?±?0.1 (CTRL), for crude protein; 40.3?±?0.6 (PWD), 31.0?±?1.8 (LWD), and 16.1?±?0.7 (CTRL), for ether extract; and 16.1?±?0.1 (PWD), 14.0?±?0.3 (LWD), and 11.6?±?0.6 (CTRL), for phosphorus. Body lipid content was highest for PWD followed by LWD and CTRL (81.4?±?1.4, 63.2?±?2.5, 42.3?±?2.6 g kg^?1, respectively), while viscerosomatic index was lowest for PWD (p?<?0.05). Liver glycogen in LWD and PWD fish (0.62?±?0.10 and 0.21?±?0.08% liver weight) was significantly higher than in CTRL fish (0.05?±?0.01% liver weight). Wheat-mycelium meals appear to be suitable dietary ingredients for improving juvenile rainbow trout growth and nutritional performance. These benefits vary according to the mushroom species used.     

Sperm vitrification of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>: effect of cryoprotectant solutions on sperm viability and spawning quality after artificial insemination

Vitrification is a fast freezing method with promising results for penaeids sperm cryopreservation. This study evaluated the efficiency of three cryoprotectant solutions for sperm vitrification and artificial insemination with cryopreserved spermatophores of Litopenaeus vannamei. The cryoprotectant solutions tested were 30% methanol, 2% soy lecithin, and 30% methanol?+?2% soy lecithin. Fully mature females were artificially inseminated with vitrified and fresh spermatophores as a control group. The vitrification method was efficient in maintaining high rates of sperm survival and membrane integrity. Although the egg fertilization was successfully attained by artificial insemination with cryopreserved spermatophores, low hatching rates suggested that possible DNA fragmentation of sperm cells should be further investigated. This is the first report of artificial insemination using vitrified sperm in penaeids.     

Protective role of antifreeze proteins on sterlet (<Emphasis Type="Italic">Acipenser ruthenus</Emphasis>) sperm during cryopreservation

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 μg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85?±?4% motility and 160?±?2 μm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44?±?9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 μg/mL of AFPIII (58?±?14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5?±?6% live cells, while the cryopreserved sperm only contained 26.6?±?14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 μg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 μg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.     

Effect of different permeable and non-permeable cryoprotectants on the hatching rate of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) embryos

The purpose of the present study was to investigate the toxicity of cryoprotectants on the hatching rate of rainbow trout (Oncorhynchus mykiss) embryos. Epiboly and first eye pigmentation stage embryos were immersed in six permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), propylene glycol (PG), ethylene glycol (EG), and acetamide (Ac), in concentrations of 1–5 M for 5 or 10 min and two non-permeable cryoprotectants, sucrose (Suc) (10%, 15%, 20%) and polyvinyl pyrrolidone (PVP) (5%, 10%, 15%) for 5 min. The embryos were then washed and incubated until hatching occurred. The toxicity of the cryoprotectant was assessed by the hatching rate. The results illustrated that permeable cryoprotectant toxicity for rainbow trout embryos increased in the order of PG < DMSO < MeOH < Gly < EG < Ac. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P < 0.05) with increased concentration and duration of exposure. There were no significant decreases in hatching rate of embryos treated with sucrose and PVP with the increase of concentration; sucrose had higher hatching rates than PVP. Rainbow trout embryos at first eye pigmentation stage exhibited greater tolerance to cryoprotectants than embryos at epiboly stage.     

Proteolytic digestive enzyme response in Japanese flounder larvae <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> fed two types of rotifers <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> species complex

Two common live feeds, the Brachionus plicatilis species complex SS-type and L-type were used to assess whether there were any differences in protein hydrolysis and digestive trypsin activity in first feeding Japanese flounder. There were no significant differences in hydrolysis activity at 2, 3 and 7 days after hatching (DAH). At 5 DAH, hydrolysis activity was significantly higher in larvae fed SS-type (p?<?0.05) at 50 kDa in 1.5- and 3-h incubation whereas L-type treatment had not completely hydrolyzed the proteins after 3 h at the same molecular weight. Larvae fed SS-type had significantly higher (p?<?0.05) trypsin activity at 3, 5, 6, 7 DAH. Contribution of live prey to trypsin fraction in larvae showed significantly higher (p?<?0.05) fraction for SS-type at 5 DAH (2.18?±?0.44%) and 6 DAH (2.04?±?0.29%) and the effect of exogenous trypsin from live prey was relatively low when compared to the total trypsin activity in larvae. This study discusses the differences in ability to digest proteins in Japanese flounder when fed different rotifer morphotypes and highlights the adaptability of this species to alternative rotifer morphotypes during its early developmental stages.     

Demographic and competition studies on <Emphasis Type="Italic">Brachionus ibericus</Emphasis> and <Emphasis Type="Italic">Proales similis</Emphasis> in relation to salinity and algal (<Emphasis Type="Italic">Nannochloropsis oculata</Emphasis>) density

We isolated the rotifers Brachionus ibericus and Proales similis from the sediment of shrimp tanks and studied their individual demographic characters and competition between them at two food levels (0.25?×?10⁶, 1.00?×?10⁶ cells ml^?1 of Nannochloropsis oculata at 25 °C) and salinities ranging from 10 to 30‰. Our hypothesis was that growth rates would be higher with increasing food levels and salinities. Observations were taken twice a day for life table studies and daily once for population growth experiments. Using survivorship and fecundity data, we derived various life history variables. Although the average life span (7.6?±?0.4 days) and gross reproductive rate (33.8?±?2.9 neonate female^?1 day^?1) of B. ibericus were higher than those of P. similis (average life span 5.4?±?0.6 days and gross reproductive rate 13.0?±?0.6 neonate female^?1 day^?1), the population growth experiments showed that P. similis had higher r values (0.32?±?0.005 day^?1) than B. ibericus (0.23?±?0.002 day^?1) at 1.0?×?10⁶ cells ml^?1 of N. oculata. The rotifer P. similis was more adversely affected due to the presence of B. ibericus than vice versa. The data are important for developing techniques for a large-scale culture of these rotifers as food in aquaculture.     

Sperm quality analysis of normal season (NG) and out-season by photoperiod manipulation (PG) of male rainbow trout broodstock (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Sperm quality parameters in rainbow trout (Oncorhynchus mykiss) were investigated during normal season spawning (November–January) and out-season spawning (July–August) treated with artificial photoperiod manipulation. Normal spawning males (n?=?15) were kept in an open concrete pond under natural condition. Out-season spawning males (n?=?15) were treated with artificial LED light (50 lm/m²) in a closed concrete pond. In these two experimental groups, five fish were used in each of three spawning periods. The mean weight and body length of males (2?+ years, n?=?30) were 1213.43?±?39.43 g and 45.08?±?0.62 cm, respectively. Sperms were collected from July to August 2016 in the out-season spawning or photoperiod-manipulated group (PG) (water temperature 14.21?±?0.31 °C) and from December 2016 to January 2017, in the normal season spawning group (NG) (water temperature 8.81?±?1.03 °C). Volume of sperm, osmolality of seminal plasma, density of sperm, percentage of motile spermatozoa (MOT), curvilinear velocity (VCL), and duration of motility were measured for each male. Seminal plasma osmolality, density of sperm, and the motility of duration were 358.47?±?37.24 and 308.87?±?44.09, 4.37?±?2.10 and 9.8?±?1.56, and 8.8?±?2.42 and 24.6?±?6.76 in PG and NG, respectively. Fertilization rate was 37.79?±?9.37% and 94.51?±?1.33% in PG and NG, respectively. Sperm quality parameters showed significant differences in most of the cases (p?<?0.05) and fertilization rate at eyed egg stage (150–160 degree-days) was significantly higher in normal season spawning group than the photoperiod-manipulated group (p?<?0.05). Though the rate of fertilization was low in out-season, it was able to get enough gametes in summer using only artificial light having no changes in other parameters.     

Cryopreservation of male and female gonial cells by vitrification in the critically endangered cyprinid honmoroko <Emphasis Type="Italic">Gnathopogon caerulescens</Emphasis>

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.     

Fish meal replacement with squilla (<Emphasis Type="Italic">Oratosquilla nepa</Emphasis>, Latreille) silage in a practical diet for the juvenile giant freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> de man, 1879

Replacement of fish meal with squilla silage in practical diet for the juvenile giant freshwater prawn, Macrobrachium rosenbergii, was evaluated. Five iso-nitrogenous and iso-caloric diets were prepared in which protein requirements were met with 100% fish meal (control, diet 1) and squilla silage replacing fish meal at 25, 50, 75 and 100% (diets 2, 3, 4 and 5, respectively). Water stability and water absorption of five feeds revealed significant differences (P?<?0.05). The weight gain varied between a maximum of 248.50?±?34.07% in diet 4 to lowest 132.45?±?14.34% in diet 1. The average daily gain in weight in grams varied from 0.091?±?0.01 (diet 5) to 0.154?±?0.04 (diet 3), whilst the specific growth rate and gross growth coefficient values were recorded highest in diet 4. Highest survival rates (86.66?±?5.77%) were recorded in both experimental diets 4 and 5. Low feed conversion ratio of 1.19?±?0.21 was recorded from diet 4, whilst the highest value of 2.16?±?0.09% was estimated from the diet 5. Proximate carcass composition showed highest crude protein content in post-larvae fed with diet 5 (47.24%) in contrast to lowest diet 1 (36.12%). Lipid values fluctuated significantly between 2.45 and 7.18% (P?<?0.05) between treatments. The ash content significantly increased from diet 1 (16.34%) to diet 5 (20.69%) (P?<?0.05). Highest feed efficiency (gain/feed) value was recorded in diet 4 (1.52?±?0.22), whilst the diet 5 showed lowest value (0.99?±?0.06). The results suggested that squilla silage can be utilised up to 75% of the protein in prawn diets for fish meal replacement.     

In vitro and in vivo evaluation of probiotic properties of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> in Kenyi cichlid, <Emphasis Type="Italic">Maylandia lombardoi</Emphasis>

In this study, Enterobacter cloacae bacterium was isolated from curd and its antibacterial potential against the pathogen Plesiomonas shigelloides was evaluated using the freshwater ornamental fish Kenyi cichlid (Maylandia lombardoi). Among the bacterial isolates, E. cloacae exhibited tolerance to acidic pH 2 and demonstrated the highest antibacterial activity against P. shigelloides in various in vitro assays. Dietary supplementation of E. cloacae with prebiotic supplement 2% mannan oligosaccharide improved the growth performance and reduced the toxic metabolites such as nitrite in culture tank water of Kenyi cichlid. In vivo application of E. cloacae with mannan oligosaccharide significantly elevated (p?<?0.05) white blood cell counts (88.47?±?2.15 10³ mm^?3) and respiratory burst activity (0.243?±?0.007) of Kenyi cichlid when challenged with P. shigelloides. Intestinal morphology of Kenyi cichlid fish treated with probiotic and prebiotic combinations showed improved intestinal architectures. The present findings confirm that the isolated bacterium E. cloacae is a potential probiotic and can be used effectively to prevent infection of P. shigelloides in freshwater ornamental fish culture.     

Growth and maturation in sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis> under high concentrations of dissolved oxygen

This study investigates the effect of high dissolved oxygen concentrations (DO) on sterlet Acipenser ruthenus maturation. Two groups, each comprising 246 individuals of 8-month-old juvenile sterlets, were reared for 37 months under high DO (group H, mean DO of 11.2 mg/L) or normal DO (group C, mean DO of 7.3 mg/L). Significant differences in body weight were observed between group H (638.7?±?191.9 g) and group C (572.2?±?151.9 g) in 12 months (P?<?0.05). Growth until 12 months occurred at a specific growth rate of 0.6%/day for group H and at 0.5%/day for group C. The food conversion rate of groups H and C was 5.2 and 5.8, respectively. The gonadosomatic index of group H was 3.1?±?1.1 for females and 3.1?±?9.7 for males, and showed no significant difference with group C (3.0?±?0.7 for females and 2.8?±?8.9 for males, P?>?0.05) in 24 months. Significant differences in follicle diameter in mature females were also observed. Group H included 68 mature females, with an average follicle diameter of 2318.0?±?175.3 µm, whereas group C included 56 mature females, with a smaller average follicle diameter of 2152.3?±?287.0 µm (P?<?0.001). However, the polarization index in group H (0.194?±?0.049) was higher than that in group C (0.178?±?0.042) (P?<?0.05). These results suggest that, for sterlet, rearing conditions with high DO can affect growth of both the body and follicle diameter of fish compared to normal rearing conditions.     

Growth,hematological and biochemical indices of common carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis> fed diets containing corn gluten meal

The effects of replacing fish meal (FM) with corn gluten meal (CGM) on growth and physiological performance were evaluated in common carp (Cyprinus carpio). Six experimental diets were formulated by substituting 0 (control), 20, 40, 60, 80, and 100% CGM protein for FM protein. The concentrations of dietary protein in the experimental diets were 27.8–29% and the P:E ratios were 14.7–15.46 mg/kJ. Eighteen fish with an initial weight of 13.5?±?0.1 g were allocated to each of 18 circular tanks (450 L) to give triplicate groups for each dietary treatment. The fish were fed to satiation for 8 weeks. At the end of the feeding trial, growth indices, body proximate composition, and hematological and biochemical parameters were measured. Blood samples were taken from six fish in each tank. Final weight and total length were significantly higher in fish fed 100% CGM (27.8?±?1.2 g and 11.9?±?0.3 cm) than for those fed the control (22.7?±?1.4 g and 10.9?±?0.5 cm) or 20% CGM (22.3?±?1.2 g and 11?±?0.4 cm) diets. No effect of FM replacement by CGM was observed for condition factor or hepatosomatic index (P?>?0.05). The highest value of protein productive value (14.31?±?0.65) was observed in fish fed 20% CGM (P?<?0.05). There were no significant differences in percentage body moisture and fat, but percentages of protein and ash were significantly different among experimental groups; the highest values of protein (15.6?±?0.24%) and ash (3.01?±?0.26%) were recorded in fish fed 40% CGM. For hematological parameters, the highest number of white blood cells (4.1?±?0.1?×?10³ mm^?3) was observed in fish fed 100% CGM (P?<?0.05). In addition, the highest hematocrit (42.1?±?0.7%) and triglyceride (294.11?±?23.82 mg dl^?1) were seen in fish fed the diet containing 40% CGM, while 80% CGM gave the highest cholesterol level (204.44?±?9.0 mg dl^?1; P?<?0.05). Replacement of FM with CGM had no negative effects on growth and physiological parameters of common carp fingerlings in this short (8 weeks) trial, suggesting that it may be feasible to replace FM with CGM in diets formulated for juvenile common carp.     

Correlation between C-reactive protein level,immunology, and hematology of a <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> infected with <Emphasis Type="Italic">Lactococcus garvieae</Emphasis>

The aim of this experiment was to evaluate the symptoms, variation, and correlation of hematological factors and some parameters of the innate immune response of the infected rainbow trout (Oncorhynchus mykiss) fish with Lactococcus garvieae bacteria. A total of 270 fish were divided into three groups including infected fish with a high and low concentration of L. garvieae and a control group without infection. The blood and tissue (brain, head, kidney, and spleen) samples were collected from each group (n?=?6 fish) at 0, 3, 14, and 21 days after treatment. The most observed symptoms during infection were lethargy, exophthalmia, and ascites. The mortality rates for the high and low dose-infected fish were defined as 60 and 25%, respectively. The infected groups had significantly (p?<?0.05) higher activity of serum lysozyme, myeloperoxidase, classical and alternative pathways of complement, serum bactericidal effects, and the specific antibody titer. Also, a significant (p?<?0.05) lower hematocrit and hemoglobin levels and higher white blood cell numbers were observed in the infected groups. A significant correlation was observed between the CRP levels and some of the hematological and immunological indices as bactericidal effects, classical complement pathways, lysozyme activity myeloperoxidase activity, white blood cell numbers, and hematocrit levels. The clinical symptoms, immune responses, and hematological indices variation among L. garvieae-infected fish are dependent on the duration and bacterial dose of the infection.     

Effects of dietary <Emphasis Type="Italic">myo</Emphasis>-inositol on growth,chemical composition and plasma chemistry of Amur sturgeon <Emphasis Type="Italic">Acipenser schrenckii</Emphasis>

The present study was conducted to demonstrate the dietary myo-inositol requirement and its effects on the growth, proximate composition and blood chemistry of Amur sturgeon (Acipenser schrenckii). Triplicate groups of 30 fish (initial weight 11.90?±?0.12 g) were fed different diets containing graded levels of myo-inositol (28.75, 127.83, 343.83, 565.81, 738.15 and 936.28 mg kg^?1) until satiation for 56 days. The fish were weighed after a 24-h fast, and six fish were used for whole body composition analysis. Further, the liver and muscle were sampled from another six fish for lipid analysis. The blood and liver were sampled from the remaining six fish for haematology and fatty acid analysis. The weight gain of fish increased with myo-inositol content, from the 28.75- to 343.83-mg kg^?1 myo-inositol treatment groups, and then stabilised. The liver lipid content and hepatosomatic index decreased significantly from 21.91 to 19.14% and from 3.20 to 2.76% with increased dietary myo-inositol supplementation, respectively. The whole body lipid content generally decreased from 6.33 to 5.55%. The content of liver-saturated fatty acids decreased significantly (28.13%) in the 936.28-mg kg^?1 treatment group. The content of plasma non-esterified fatty acids increased with the increase in dietary myo-inositol supplementation from 0.77 to 1.17 mmol L^?1, whereas the content of triglycerides significantly decreased from 4.62 to 3.28 mmol L^?1. In conclusion, the optimum myo-inositol requirement was found to be 336.1 mg kg^?1, based on weight gain in a two-slope quadratic broken-line model.     

The immune modulatory effect of oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> L.) essential oil on <Emphasis Type="Italic">Tilapia zillii</Emphasis> following intraperitoneal infection with <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

The current study aimed to evaluate the possible effect of Origanum essential oil on innate immune parameters as well as the hematological profiles of Tilapia zillii following challenge with Vibrio anguillarum. Fifty-four of Tilapia zillii weighing 180?±?10.2 g were randomly distributed into three identical closed recirculating seawater systems. The study included three groups (G1, G2, and G3) repeated in triplicates. Fish of the first two groups were fed on a basal diet without herbs, whereas fish of the last group were fed on a basal diet supplemented with Origanum essential oil at concentration 1 g kg^?1 for 15 days. Subsequently, fish of G2 and G3 subjected to a peritoneal inflammation by intraperitoneally injecting V. anguillarum (5.5?×?10⁵ CFU mL^?1), whereas fish of G1 injected with saline and served as control. Fish of all groups were then sampled at 4, 12, and 24 h post injection. No mortalities were observed in both basal and Origanum fed groups. However, some specimens of fish fed basal diet showed dorsal fin erosions, eroded mouth, and detached skin. Although the kinetics of RBCs, WBCs, Hb, and differential leukocyte values remained unchanged in fish fed different diets at the beginning of the trial, significant increases in those values were observed in fish fed Origanum essential oil at 12 and 24 h post injection. Similarly, an augmentation of plasma proteases, antiproteases, and lysozyme activities were recorded in fish fed Origanum essential oil at the same particular sampling points. A significant enhancement in plasma bactericidal capacity was only recorded in fish fed Origanum essential oil at 12 and 24 h post challenge compared to those fed basal diet. In conclusion, Origanum essential oil had a pronounced influence on the innate immunity and increased the fish resistance to V. anguillarum. These data gave insight into the potential use of Origanum in prophylactic strategies against threatening pathogens.     

Immunogene expression in head kidney and spleen of common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) following thermal stress and challenge with Gram-negative bacterium, <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

To evaluate the effect of thermal and microbial stress on the immune response of common carp (Cyprinus carpio L.), relative mRNA expression level of pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β] and other genes related to immune or stress response [inducible nitric oxide synthase (iNOS), heat shock protein 70 (Hsp70), superoxide dismutase one (SOD1), and glucocorticoid receptor (GR)] was measured by quantitative PCR (qPCR). In addition, total protein and total immunoglobulin level in blood plasma of experimental common carp was also assayed. All the above parameters were estimated 24 h post-challenge with Gram-negative bacterium, Aeromonas hydrophila. Common carp (54.89?±?6.90 g) were initially exposed to 20 °C (control group) and 30 °C (thermal stress group) water temperature for 30 days, followed by experimental challenge with 2.29?×?10⁸ colony forming unit/mL (CFU/mL; LD₅₀ dose) of A. hydrophila. Exposure of fish to thermal stress and subsequently challenge with A. hydrophila significantly (P?<?0.05) increases the IL-1β mRNA expression in head kidney and spleen of common carp by ~?39.94 and ~?4.11-fold, respectively. However, TNF-α mRNA expression in spleen decreased ~?5.63-fold in control fish challenged with A. hydrophila. Thermal stress and challenge with bacterium suppresses the iNOS and GR mRNA expression in spleen of common carp. Moreover, significant (P?<?0.05) increase in total protein content of blood plasma (~?43 mg/g) was evident in fish exposed to thermal stress and challenged with A. hydrophila. In conclusion, our study highlights the importance of elevated temperature stress and microbial infection in differential regulation of expression of several immunogenes in common carp.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Growth and fatty acid composition of <Emphasis Type="Italic">Octopus vulgaris</Emphasis> paralarvae fed with enriched <Emphasis Type="Italic">Artemia</Emphasis> or co-fed with an inert diet

The rearing of Octopus vulgaris paralarvae during its planktonic life stage is a major challenge, as mortality is currently very high and unpredictable. In this study, we examined the survival and growth rates, as well as the fatty acid composition, of O. vulgaris paralarvae fed on three different dietary treatments: group ArDHA was offered juvenile Artemia enriched with a lipid emulsion (Easy DHA-Selco^®); group ArMA was fed with juvenile Artemia enriched with a mixture of microalgae (Rhodomonas lens and Isochrysis galbana); and group ArMA+ID received the same Artemia as group ArMA complemented with an inert diet. Dietary treatments were tested in triplicate with homogenous groups of paralarvae (25 individuals l^?1) established in 50-l tanks, and the experiment was conducted for 15 days. The survival rate of 15-day post-hatch (-dph) paralarvae from groups ArMA (20 ± 8%) and ArMA+ID (17 ± 4%) tended to be higher than in group ArDHA (13 ± 5%), though these differences were not statistically different. The dry weight (DW) of 15-dph paralarvae increased by almost 60% in groups ArMA and ArMA+ID, and nearly 40% in group ArDHA, with respect to hatchlings. The fatty acid (FA) composition of paralarvae revealed a remarkable drop of docosahexaenoic acid (22:6n-3, DHA) from hatchlings to 15-dph paralarvae of all groups (P < 0.05). However, paralarvae from group ArDHA contained higher levels of DHA than those from ArMA and ArMA+ID (P < 0.05). Despite Artemia enriched with DHA-Selco^® contained three-times more DHA than Artemia enriched with microalgae, no beneficial effects of this dietary treatment were observed on the performance of paralarvae.     

<Emphasis Type="Italic">Curcuma longa</Emphasis> as additive in the diet for <Emphasis Type="Italic">Astyanax aff. bimaculatus</Emphasis>

The aim of the present study was to evaluate the effect of turmeric (Curcuma longa) as additive in the diet for Astyanax aff. bimaculatus. Fish (0.83 ± 0.04 g) were fed, for 60 days, with six diets containing 0.0, 20.0, 40.0, 60.0, 80.0, and 100.0 g turmeric kg^?1 feed. There was an increasing linear effect of turmeric on the thickness of the muscular layer, and height and width of the folds of the intestine. In the liver, a quadratic effect was observed of turmeric on the percentage of hepatocyte cytoplasm and a decreasing linear effect on the percentage of sinusoid capillaries. A quadratic effect was also observed of turmeric on the liver glycogen. There was no effect of turmeric on the antioxidant activity in the liver, carcass composition or productive performance of the fish. Thus, we concluded that Curcuma longa has trophic effects on the epithelium and the muscular layer of the intestine of A. aff. bimaculatus. Additionally, low levels of Curcuma longa cause increased deposition of liver glycogen and high levels cause reduction.     

Effect of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> K-C2 on the growth performance,amino acid content and gut microflora of amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis>

This study aimed to evaluate the effect of Lactococcus lactis K-C2 on the growth performance, microbial diversity, and release of free amino acids in the intestinal tract and the edible parts of young amberjack, Seriola dumerili. Fish were fed a diet with or without strain K-C2 (2?×?10¹⁰ cfu/g feed) for 25 days. The results indicated that the growth performance of fish in the treated group was significantly higher than those in the control group (p?<?0.05). The amount of five amino acids (aspartate, sarcosine, taurine, alanine, and arginine) in the gut content and 13 of 21 amino acids in the edible parts of fish in the treated group were significantly higher (p?<?0.05) than those in the control group. Sphingomonas, Propionibacterium, and Mycobacterium were observed in gut microflora of fish in both the control and treated groups. Staphylococcus and Kocuria were detected in one sample from the control and treated groups; Acinetobacter and Acidobacteria were found in one sample from the control group. L. lactis was only found in one sample in the treated group. In conclusion, the dietary administration of probiotic L. lactis stimulated growth, reduced feed consumption, and improved the nutritional value of cultured amberjack.