Effects of Dietary Lactic Acid Supplementation on the Activity of Digestive and Antioxidant Enzymes, Gene Expressions, and Bacterial Communities in the Intestine of Common Carp, Cyprinus carpio.

The present study investigated the effects of dietary lactic acid (LA) supplementation on the growth performance, intestinal digestive/antioxidant enzymes' activities, gene expression, and bacterial communities in common carp, Cyprinus carpio. Four diets were formulated to contain 0 g/kg LA (control), at 2.5 g/kg LA (2.5LAC), 5 g/kg LA (5LAC), and 10 g/kg LA (10LAC) and offered to the fish over a period of 56 days. The results showed that dietary 5 g/kg LA supplementation improved growth performance and feed efficiency in the fish. All LA treatments exhibited significant elevations in the intestinal trypsin and chymotrypsin activities, whereas the intestinal lipase, amylase, and alkaline phosphatase activities exhibited significant elevations in the 5LAC and 10LAC treatments. All LA treatments exhibited significant elevations in the intestinal heat shock protein 70, tumor necrosis factor-alpha, interleukin-1 beta, and defensin gene expressions, and the highest expression was observed in the 5LAC treatment. Additionally, dietary LA treatment significantly increased the lysozyme expression and Lactobacillus sp. population in the intestine of the fish, and the highest values were observed in the 5LAC and 10LAC treatments. Aeromonas sp. and Vibrio sp. populations decreased in the LA treatments, and the lowest Aeromonas sp. population was observed in the 10LAC treatment. The intestinal mucin2 and mucin5 expressions, and the hepatic reduced glutathione content, significantly increased, whereas hepatic glutathione peroxidase, glutathione reductase, and malondialdehyde significantly decreased in the 5LAC and 10LAC treatments. In conclusion, dietary 5 g/kg LA is recommended for common carp feeding to improve growth rate, antioxidant capacity, and intestinal health.

Parasitic fauna of farmed freshwater ornamental sutchi catfish (Pangasiandon hypophthalmus) and silver dollar (Metynnis hypsauchen) in Alborz province, Iran.

BACKGROUND: Parasitic infestations are one of the most significant problems in global aquaculture. In addition to direct economic losses due to significant mortalities, parasites may have a significant negative impact on the fish's behaviour, energetic demands, trophic position, interspecific competition, growth rate, and reproductive function. OBJECTIVES: We conducted the present study to evaluate the infection status of parasitic pathogens in freshwater sutchi catfish (Pangasianodon hypophthalmus) and silver dollar (Metynnis hypsauchen) farmed in Alborz province of Iran. METHODS: Between January and February 2021, 140 ornamental fish, including 70 sutchi catfish (P. hypophthalmus) and 70 silver dollar (M. hypsauchen), were delivered from various ornamental fish farms for parasitological analysis. Thorough macroscopic and microscopic examinations were performed to detect parasitic infections in delivered freshwater ornamental fish. RESULTS: A total of six parasite species, including five protozoan species (Nyctotherus piscicola, Trichodina heterodentata, Ichthyophthirius multifiliis, Protoopalina sp., and Hexamita sp.) and one monogenean species (Ancyrocephalus sp.), were identified in the examined fish. The overall prevalence of the recovered parasites was 46.43% (65/140) of the fish. CONCLUSIONS: In the current study, several parasites, including Hexamita sp., Ancyrocephalus sp., Protoopalina sp., and N. piscicola, were identified as the first records of the sutchi catfish (P. hypophthalmus) and/or silver dollar (M. hypsauchen), and these two fish species are known as new hosts for the isolated parasites in ornamental fish farms in Iran. Assessing the parasitic fauna of ornamental fish is therefore critical for preventing parasite introduction into adjacent provinces as well as neighbouring countries and promoting fish health.

Effects of Dietary Glycine Supplementation on Growth Performance, Immunological, and Erythrocyte Antioxidant Parameters in Common Carp, Cyprinus carpio.

The effects of dietary glycine supplementation, 0 (control), 5 (5 GL), and 10 (10 GL) g/kg, have been investigated on growth performance, hematological parameters, erythrocyte antioxidant capacity, humoral and mucosal immunity in common carp, Cyprinus carpio. After eight weeks feeding, the 5 GL treatment exhibited significant improvement in growth performance and feed efficacy, compared to the control treatment. Red blood cell (RBC) and white blood cell (WBC) counts, hemoglobin, hematocrit, neutrophil and monocyte counts/percentages, RBC reduced glutathione (GSH) content, and skin mucosal alkaline phosphatase, peroxidase, protease, and lysozyme activities were similar in the glycine-treated fish and significantly higher than the control treatment. Blood lymphocyte percentage decreased in the glycine-treated fish, but lymphocyte count increased, compared to the control fish. RBC glutathione reductase activities in the glycine-treated fish were similar and significantly lower than the control treatment. The highest plasma lysozyme and alternative complement activities were observed in GL treatment. The glycine-treated fish, particularly 5 GL, exhibited significant improvement in RBC osmotic fragility resistance. Dietary glycine had no significant effects on RBC glutathione peroxidase activity, plasma immunoglobulin, eosinophil percentage/count, and hematological indices. In conclusion, most of the benefits of dietary glycine supplementation may be mediated by increased glutathione synthesis and antioxidant power.

Bioaccumulation of Some Hazardous Heavy Metals in Three Edible Fishes Caught from Gorgan Bay, Caspian Sea.

A survey was conducted to evaluate heavy metal accumulation in edible fishes caught from Gorgan Bay, as a part of the Caspian Sea ecosystem. Fish samples including mullet, roach, and common carp were just randomly prepared from fishing locations in three different areas of Gorgan Bay including estuary, channel, and Miankale wetland twice the fishing seasons. A total of 135 samples were measured using an atomic absorption spectrometer to 4 heavy metals of arsenic (As), lead (Pb), cadmium (Cd), and copper (Cu) (a total of 540 metal samples). The highest accumulation of As was related to the mullet of estuary, and the lowest contamination was related to the mouth of the channel. In relation to Cd, the highest pollution in all three species related to the fishes caught from the estuary and the lowest accumulation related to the Miankaleh wetland. In the case of metal Pb, results showed the most contamination related to the Mullet caught from Miankaleh wetland and the lowest accumulation related to the roach of the mouth of the channel. In all three fish species, the highest contamination of Cu was in the estuary and the lowest was in the fish caught from Miankaleh wetland. In general, the highest accumulation belonged to all three species located in estuaries. The probable reason can be pouring river pollution to the bay more than wetlands and channel. Due to the enclosure of the Caspian Sea and consequently the Gulf of Gorgan, limiting the fishing times and points to the periods of least incoming pollutants is very helpful for public health, especially food hygiene and fishing from this ecologically sensitive and vulnerable basin.

Anesthesia of rainbow trout with citronellal: Efficacy and biochemical effects.

The aim of this study was to assess the anesthetic efficacy and biochemical effects of citronellal on rainbow trout, Oncorhynchus mykiss. The fish were exposed to 200-800 µl/L citronellal to find the anesthesia induction and recovery times. The deep anesthesia stage was reached within 117-613 s, using 800-200 µl/L citronellal, respectively, and all fish recovered within 240-420 s. To assess biochemical responses, the fish were exposed to 200, 400, 600, and 800 µl/L of citronellal and blood-sampled after deep anesthesia. The results showed that anesthesia led to significant elevations in blood erythrocytes, hematocrit, hemoglobin, mean corpuscular volume, plasma lactate, and lactate dehydrogenase; these parameters exhibited increasing trends as citronellal concentration increased. These results along with significantly higher plasma lysozyme activity, cortisol, and glucose levels in 200 µl/L treatment suggest that an increase in citronellal concentration significantly mitigates hypoxia/stress responses in the fish. Significant elevations in plasma alkaline phosphatase activity and malondialdehyde level were observed in the 200 µl/L treatment, which suggests induction of hemolysis and oxidative stress in this treatment. There was a tendency to higher ammonia levels along with the increase in citronellal concentration, which might be due to early ammonia accumulation because of lower opercular movement in the fish. Except for blood hematocrit and mean corpuscular volume, there were no significant differences between the 600 and 800 µl/L citronellal treatments. In conclusion, high concentrations of citronellal are more suitable for trout anesthesia and blood sampling than low concentrations. Based on the present study, 600-800 µl/L citronellal is recommended for trout anesthesia.

Food sources for camptandriid crabs in an arid mangrove ecosystem of the Persian Gulf: a stable isotope approach.

Crabs of the family Camptandriidae are the most dominant burrowing crabs inhabiting arid mangrove forests of the Persian Gulf. They play important roles in the structuring and functioning of mangrove ecosystems by modulating biogeochemical processes and cycling of nutrients, serving as important ecosystem engineers. We analysed stable carbon (13C) and nitrogen (15N) isotope values of three camptandriid crabs (Opusia indica, Nasima dotilliformis, and Manningis arabicum) and their potential food sources in the Hara Biosphere Reserve, northern Persian Gulf. A Bayesian mixing model was used to estimate the contribution of potential food sources for consumers. The results showed that to some degree, all the four sources selected contributed to the camptandriid diets, but microphytobenthos made the most important contributions to the diet of the consumers. Mangroves do not appear to be a significant source of carbon in the diet of camptandriid crabs in the arid mangrove system of the Persian Gulf. Rather, they offer favourable growing conditions, thus boosting microphytobenthos production and availability for consumers which prefer a high nutritional and palatable source.

Effects of dietary thyme (Zataria multiflora) extract on antioxidant and immunological responses and immune-related gene expression of rainbow trout (Oncorhynchus mykiss) juveniles.

Effects of dietary hydroalcoholic extract of Zataria multiflora (ZE) on growth performance, plasma and hepatic antioxidant capacities, and humoral and skin mucus immune parameters were evaluated in rainbow trout (Oncorhynchus mykiss) juveniles. in vitro tests showed that ZE had antioxidant property comparable to butylated hydroxytoluene (BHT) at 100-200 µg/mL concentrations, although its antioxidant property was lower than BHT at concentration below 100 µg/mL. Moreover, ZE had anti-bacterial activity against Aeromonas hydrophila, which was 30-50% lower than that of tetracycline. After feeding the fish with diets supplemented with 0 (CT, 1 (ZE1), 2 (ZE2), and 3 (ZE3) g/kg ZE for eight weeks, there were no significant differences in growth performance and feed efficiency among the treatments; however, the fish in ZE2 and ZE3 treatments showed significantly higher survival than the fish in CT treatment. Blood leukocyte counts, plasma globulin, total immunoglobulin, lysozyme and bactericidal activity against A. hydrophila in ZE2 and ZE3 groups were significantly higher than that of CT group. All the ZE-treated groups had higher plasma complement activity compared to the CT group. Mucosal lysozyme and bactericidal activities of the ZE2 fish were significantly higher than the other treatments. Expression of tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, and lysozyme genes increased in head kidney of the fish treated with ZE; the highest increases were related to the ZE2 treatment. Plasma total antioxidant (TA) activities of ZE2 and ZE3 treatments were significantly higher than that of the CT treatment. Plasma and hepatic superoxide dismutase (SOD) and catalase (CAT) activities of ZE2 group were significantly higher than the other treatments. Plasma malondialdehyde (MDA) levels were significantly lower in ZE2 treatment, compared to the other treatments. However, hepatic MDA level of ZE2 treatment was significantly lower than those of the ZE1 and CT treatments. In conclusion, dietary ZE supplementation level of 2 g/kg is suggested for rainbow trout feed supplementation to augment fish survival, antioxidant and immune strength.

Oral immunization of trout fry with recombinant Lactococcus lactis NZ3900 expressing G gene of viral hemorrhagic septicaemia virus (VHSV).

This study has investigated the ability of Lactococcus lactic (NZ3900) carried G gene of viral haemorrhagic septicaemia virus (VHSV) under nisin-controlled gene expression (NICE) system in rainbow trout (O.Mykiss). Two groups of trout fry (7 ± 0.65 g) were immunized with 1 × 1010 cfu/g and 1 × 108 cfu/g recombinant L. lactis NZ3900, two groups of fish were fed 1 × 1010 cfu/g and 1 × 108 cfu/g L. lactis vector free, and one group was fed by the basal diet as a control. Oral immunization was done on days 1-7 and boosting was performed on days 15-21. The relative expression of IFN-1 and MX-1 genes significantly increased in head kidney of vaccinated fish depend on vaccine dosage compared to the control group. Fish in vaccinated group also showed elevated VHSV-specific antibody levels compared to the control groups. Relative percent survival (RPS), under virulent isolate VHSV challenge were estimated 62%, 78% for 108 cfu/g 1010 cfu/g feed vaccinated groups 21 days post-vaccination, while groups fed similar doses of L. lactis vector free illustrated 22% and 27% RPSs, respectively. The significant reduction of viral loads (transcript levels of N gene) were detected in the immunized groups. Increased weight gain and decreased feed consumption in vaccinated group attributed to the probiotic effect were also observed. In conclusion, our results demonstrate the ability of recombinant L. lactis as oral vaccine against VHS in rainbow trout, which can be considered as effective method against different fish pathogens.

Cyprinid herpesvirus 3 (CyHV-3) transmission and outbreaks in Iran: Detection and characterization in farmed common carp.

This study shows the presence of Cyprinid Herpesvirus-3 (CyHV-3) in common carp (Cyprinus carpio) from Iranian carp farms with cumulative mortality up to 80% during 2015-2016. Pathological signs of disease such as gill necrosis, sunken eyes, and increased slime secretion on the skin and fins were observed in affected fish. The extensive fusion of secondary lamellae with necrotic cells, margination of chromatin, and formation of intranuclear inclusion bodies in gill tissues were also observed by histopathological examination. Most tubular epithelial cells and some hematopoietic cells showed intranuclear inclusion bodies in the kidney. The Iranian CyHV-3 isolates showed identity with Asian strains, and displayed the I++ II+ allele of the Asian lineage, as revealed by sequence analysis of the TK gene, Marker I, and Marker II. The detected isolates were also similar to those detected from koi in the same region of Iran, suggesting the probable transmission of CyHV-3 from ornamental to farmed cyprinids. This represents the first report of CyHV-3 from Iranian farmed common carp to the best of our knowledge.

Humoral immune responses and gill antioxidant-related gene expression of common carp (Cyprinus carpio) exposed to lufenuron and flonicamide.

The aim of this study was to investigate plasma proteins, total immunoglobulin (Ig), lysozyme and complement (ACH50) levels, and gill superoxide dismutase (SOD) and catalase (CAT) gene expression in common carp (Cyprinus carpio), following exposure to lufenuron (LUF) and flonicamide (FL). Fish were distributed in 12 tanks as three quadrupled treatments: control (fish were kept in pesticide-free water), LUF [fish exposed to 10% of LUF LC50 (4.3 mg/L)], and FL [fish exposed to 10% of FL LC50 (0.1 mg/L)]. The plasma parameters were assessed after 7 and 21 days exposure to pesticides, whereas the gene expressions were assessed after 21 days. The results showed that LUF exposure significantly decreased plasma total protein and globulin levels compared to the control group. Both pesticide significantly decreased plasma total Ig levels compared to the control group; however, LUF exhibited a greater effect. There were no significant effects of pesticides or sampling time on plasma ACH50 activity. Pesticides and sampling time interacted to affect plasma lysozyme activity. Seven days after exposure, both pesticides significantly increased lysozyme activity, and the effect of FL was greater than LUF. Nevertheless, there was no significant difference in plasma lysozyme activities among the pesticides, 21 days after the exposure. Both pesticides significantly decreased SOD and CAT gene expression, nevertheless, FL exhibited greater effects than LUF. In conclusion, both pesticides induced immunosuppression in the fish, though such effects were more severe in LUF group, compared to the FL. These pesticides negatively affect expression of gill antioxidant genes, and the FL effects were greater than the LUF.

Expression of immune, antioxidant and stress related genes in different organs of common carp exposed to indoxacarb.

The aim of the present study was to investigate the effects of chronic exposure of common carp (Cyprinus carpio) to indoxacarb on immune, antioxidant and stress gene expression. After 21 days exposure to 0, 0.75, 1.5 and 3 ppm indoxacarb, expression of IL-1ß, IL-8, IL-10, TNF-α, IFN-γ, SOD, CAT, HSP70, IGF-I and IGF-II were assessed in liver, kidney and gills. In general, exposure to low concentration of indoxacarb increased inflammatory cytokine gene expression (IL-1ß, IL-8, IL-10, TNF-α and IFN-γ) and inhibits inflammatory cytokines' expression at higher concentrations. The assessment of antioxidant gene expression (SOD and CAT) in different organs indicate that they were increased by low concentrations of indoxacarb to deal with primary oxidative situation. However, higher concentrations of indoxacarb caused reduction in oxidative gene expression. IGF genes expression in liver significantly increased at a concentration of 0.75 ppm treatment, then it decreased at 1.5 ppm indoxacarb and increased again by increasing in the indoxacarb concentration to 3 ppm. The expression of HSP70 in kidney showed a significant elevation in 0.75 and 1.5 ppm treatments compared with 3 ppm treatment and the control group. The expression of this gene in liver was significantly increased in 1.5 and 3 ppm treatments. The same pattern of expression was also observed in gill. Overall, indoxacarb exposure affects common carp health at transcription levels. Changes in the genes expression generally suggest that indoxacarb exposure led to interference in inflammation, oxidative stress and tissue damage.

Dietary Application of the Probiotic Lactobacillus plantarum 426951 Enhances Immune Status and Growth of Rainbow Trout (Oncorhynchus mykiss) Vaccinated Against Yersinia ruckeri.

This study was aimed to assess the effect of oral application of Lactobacillus plantarum (2 × 107 CFU g-1 feed) as a probiotic on growth performance and immune status of vaccinated rainbow trout (29.5 ± 2 g) to yersiniosis at 16 ± 2 °C for 72 days. Fish were randomly allocated into 12 fiber glass tanks (4100 L) at a density of 80 fish per tank (240 fish per treatment). The results revealed that the activity of lysozyme and alkaline phosphatase was significantly higher in immunized fish fed with diet supplemented with probiotic (vaccine +probiotic) than that in the immunized group fed with basal diet (vaccine group) while no significant differences in levels of hematological parameters, complements, total IgM, proteins, and the intestine lactic acid bacteria (LAB) were detected. Also, significantly a better growth performance in terms of feed conversion ratio, weight gain, and thermal growth coefficient was seen in the vaccine + probiotic group than that in the vaccine group. These results indicate that feeding probiotic after vaccination can enhance the efficacy of immersion vaccination to Yersinia ruckeri.

Effects of dietary 1,8-cineole supplementation on physiological, immunological and antioxidant responses to crowding stress in rainbow trout (Oncorhynchus mykiss).

The aim of the present study was to investigate beneficial effects of dietary 1,8-cineole (cineole) supplementation on physiological, immunological and antioxidant responses of rainbow trout (Oncorhynchus mykiss) to crowding stress. The fish were fed for 50 days with diets containing 0 (control), 0.05, 0.1, 0.25, 0.5 and 1% cineole prior to exposure to a 14-day crowding stress. Serum stress markers (cortisol, glucose, lactate, T4 and T3), antioxidant responses [catalase (CAT) and superoxide dismutase (SOD) activities and total antioxidant capacity (TAC) and malondyaldehyde (MDA) levels] and immune responses [lysozyme and alternative complement (ACH50) activity, and total immunoglobulin (Ig) levels], and blood leukocyte (WBC) and differential counts were measured before and after crowding stress. Results showed that 1% cineole was capable to reduce the basal and stress-induced cortisol elevation and increase the serum T3 levels after stress. Increase in dietary cineole levels significantly decreased serum cortisol, glucose and lactate levels. Increase in dietary cineole levels significantly increased serum CAT, SOD, lysozyme and ACH50 activities, and TAC and eosinophil levels, and decreased MDA and monocyte levels. After the stress, there was no significant difference in the control group CAT and SOD activities compared to the basal values; however, CAT activities decreased and SOD activities increased in the cineole-treated groups compared to the basal values. Nevertheless, the control group had significantly lower CAT and SOD activities compared to the fish treated with 0.1-1% cineole. Cineole significantly increased blood WBC and serum lysozyme, ACH50 and total Ig. Moreover, cineole administration significantly mitigated the stress-induced decrease in total Ig levels as well as increase in leukocyte count. The cineole-treated fish had higher survival and growth performance compared to the control group. Although all levels of cineole (0.05-1%) showed beneficial effects on different tested factors, 0.5 and 1% levels had beneficial effects on most of the tested factors; thus, are recommended for dietary inclusion to suppress adverse effects of stress in trout.

Modification of saltwater stress response in Cyprinus carpio (Linnaeus, 1758) pre-exposed to pesticide indoxacarb.

To evaluate the effects of indoxacarb on saltwater stress response in Cyprinus carpio, the fish were pre-exposed to indoxacarb (0, 0.75, 1.5 and 3mg/L denoted as CP, 0.75IT, 1.5IT and 3IT, respectively) for 21 days and then released to saltwater. A negative control (CN) group was included (the fish were held in indoxacarb-free water for the entire experiment). The fish were sampled immediately (0h) and 24, 48 and 72h after the salinity exposure for the analysis of plasma cortisol, glucose and sodium, chloride, potassium and calcium levels. All fish pre-exposed to 3mg/L indoxacarb, died after the first day of salinity challenge. CP showed typical cortisol response after the salinity challenge, but, cortisol response of the fish pre-exposed to indoxacarb (0.75IT and 1.5IT) was blocked. Plasma glucose increased significantly in all groups compared to the CN; however, this elevation had no consistent trend in 0.75IT and 1.5IT which indicated interference in glucose response due to indoxacarb exposure. Plasma sodium increased (compared to CN) in all groups after the salinity challenge. However, elevation in plasma chloride and potassium was significantly different among the groups and the indoxacarb-treated fish showed slightly sooner ionic disturbance. The results clearly indicate that indoxacarb impairs stress response of C. carpio and the fish may not be able to respond normally to additional stressors, which threatens their survival.

Toxic effects of copper sulfate and copper nanoparticles on minerals, enzymes, thyroid hormones and protein fractions of plasma and histopathology in common carp Cyprinus carpio.

Differences in toxicological effects of dissolved copper and copper nanoparticles were studied in common carp, Cyprinus carpio. The fish were exposed to 0.25mgL-1 copper as copper sulfate (0.25Cu), 0.25mgL-1 copper as copper oxide nanoparticles (0.25NCu) and 25mgL-1 copper as copper oxide nanoparticles (25NCu) over 14days. Plasma biochemical, enzymatic and hormonal characteristics, and liver and kidney histopathology were examined at the end of the experiment. The results showed that both forms of copper had no significant effects on plasma calcium levels, however, significantly increased plasma phosphorous levels, compared to control group (no added copper). Plasma alanine transaminase (ALT) activity increased in 0.25Cu and 25NCu treatments compared to the control and 0.25NCu treatments. Nanoparticle copper exposure significantly decreased plasma alkaline phosphatase (ALP) activity compared to the control and 0.25Cu treatments. Only copper sulfate exposure caused plasma aspartate transaminase (AST) elevation. Both copper forms increased plasma T4 and free T4 (FT4); however, copper sulfate effect was higher than nanoparticle copper. Copper sulfate exposure increased plasma albumin fraction, whereas, 25mgL-1 copper nanoparticle exposure increased plasma α2-globulin fraction compared to the control. Both copper forms damaged the fish liver and kidney, however, copper sulfate caused more severe damages compared to nanoparticle copper. Overall, except for plasma ALP and α2-globulin fraction, dissolved copper seems to be more toxic than nanoparticle copper in common carp.