Dietary Olive Leaf Extract Differentially Modulates Antioxidant Defense of Normal and Aeromonas hydrophila-Infected Common Carp (Cyprinus carpio) via Keap1/Nrf2 Pathway Signaling: A Phytochemical and Biological Link.

Olive leaves are an immense source of antioxidant and antimicrobial bioactive constituents. This study investigated the effects of dietary incorporation of olive leaf extract (OLE) on the growth performance, hematobiochemical parameters, immune response, antioxidant defense, histopathological changes, and some growth- and immune-related genes in the common carp (Cyprinus carpio). A total of 180 fish were allocated into four groups with triplicate each. The control group received the basal diet without OLE, while the other three groups were fed a basal diet with the OLE at 0.1, 0.2, and 0.3%, respectively. The feeding study lasted for 8 weeks, then fish were challenged with Aeromonas hydrophila. The results revealed that the group supplied with the 0.1% OLE significantly exhibited a higher final body weight (FBW), weight gain (WG%), and specific growth rate (SGR) with a decreased feed conversion ratio (FCR) compared to the other groups (p < 0.05). An increase in immune response was also observed in the fish from this group, with higher lysosome activity, immunoglobulin (IgM), and respiratory burst than nonsupplemented fish, both before and after the A. hydrophila challenge (p < 0.05). Similarly, the supplementation of the 0.1% OLE also promoted the C. carpio's digestive capacity pre- and post-challenge, presenting the highest activity of protease and alkaline phosphatase (p < 0.05). In addition, this dose of the OLE enhanced fish antioxidant capacity through an increase in the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and decreased hepatic lipid peroxidation end products (malondialdehyde-MDA), when compared to the control group, both pre- and post-infection (p < 0.05). Concomitantly with the superior immune response and antioxidant capacity, the fish fed the 0.1% OLE revealed the highest survival rate after the challenge with A. hydrophila (p < 0.05). A significant remarkable upregulation of the hepatic sod, nrf2, and protein kinase C transcription levels was detected as a vital approach for the prevention of both oxidative stress and inflammation compared to the infected unsupplied control group (p < 0.05). Interestingly, HPLC and UPLC-ESI-MS/MS analyses recognized that oleuropein is the main constituent (20.4%) with other 45 compounds in addition to tentative identification of two new compounds, namely oleuroside-10-carboxylic acid (I) and demethyl oleuroside-10-carboxylic acid (II). These constituents may be responsible for the OLE exerted potential effects. To conclude, the OLE at a dose range of 0.66-0.83 g/kg w/w can be included in the C. carpio diet to improve the growth, antioxidant capacity, and immune response under normal health conditions along with regulating the infection-associated pro-inflammatory gene expressions, thus enhancing resistance against A. hydrophila.

Assessment and Bioaccumulation of Heavy Metals in Fish Feeds, Water, and Some Tissues of Cyprinus carpio Cultured in Different Environments (Biofloc Technology and Earthen Pond System).

Fish feed quality is the main determinant of fish flesh quality, so it is important for successful aquaculture. The current study determines the concentration of heavy metals in fish feeds (A and B), water, and their bioaccumulation in gills, liver, and muscle of C. carpio cultured in different environments (biofloc technology and earthen pond systems). In addition, the correlation between heavy metals in fish feeds with bioaccumulated metals in fish tissues was also determined. Results revealed that most heavy metal concentration was significantly greater (P < 0.05) in feed B than in feed A but in permissible range, while all the heavy metal concentration was notably higher in earthen ponds than in biofloc technology. Result from the bioaccumulation factor and concentration of the metals showed that heavy metals were highly accumulated in the fish liver followed by gills. The metal concentration in fish feeds and fish edible parts (muscle) was lower than the WHO standard level; however, the amount of Pb was higher in the fish muscle, liver, and gills, which is harmful for human consumption and also for fish health. Though the correlation test revealed that all of the metals from the feeds were positively correlated to the metals in fish tissues, but most of the estimated correlation was significant and linearly correlated. It can be concluded that producers must measure feed quality correctly to avoid heavy metal contamination because it may assimilate and accumulate in the food chain.

Influence of biosynthesized nanoparticles exposure on mortality, residual deposition, and intestinal bacterial dysbiosis in Cyprinus carpio.

Nanotechnology has revealed profound possibilities for the applications in applied sciences. The nanotechnology works based on nanoparticles. Among nanoparticles, silver nanoparticles largely introduced into aquatic environments during fabrication. Which cause severe contamination in the environment specially in freshwater fish. Therefore, the current study was a pioneer attempt to use the animal blood to fabricate AgNPs and investigate their toxicity in Cyprinus carpio (C. carpio) by recording mortality, tissue bioaccumulation, and influence on intestinal bacterial diversity. For this purpose, fish groups were exposed to different concentrations of B-AgNPs including 0.03, 0.06, and 0.09 mg/L beside the control group for 1, 10, and 20 days. Initially, the highest concentration caused mortality. The results revealed that B-AgNPs were significantly (p < 0.005) accumulated in the liver followed by intestines, gills, and muscles. In addition, the accumulation of B-AgNPs in the intestine led to bacterial dysbiosis in Cyprinus carpio. At the phylum level, Tenericutes, Bacteroidetes, and Planctomycetes were gradually decreased at the highest concentration of B-AgNPs (0.09 mg/L) on days 1, 10, and 20 days. The genera Cetobacterium and Luteolibactor were increased at the highest concentration on day 20. Moreover, the principal coordinate analysis (PCoA) based on Bray-Curtis showed that the B-AgNPs had led to a variation in the intestinal bacterial community. Based on findings, the B-AgNPs induced mortality, and residual deposition in different tissues, and had a stress influence on intestinal homeostasis by affecting the intestinal bacterial community in C. carpio which could have a significant effect on fish growth.

Ultrastructural studies of photoreceptor cell degeneration with organophosphate and its regeneration in Cyprinus carpio communis L.

Organophosphates are highly neurotoxic to aquatic fauna if they enter the water bodies as runoff, thus affecting the nervous system of the fishes. The present study was undertaken to investigate the vision changes, especially on the photoreceptor layer of the retina, of Cyprinus carpio communis L. when exposed to monocrotophos, an organophosphate. Fish were exposed to three sub-lethal concentrations of LC50, i.e. 0.038 ppm (1/10 LC50), 0.062 ppm (1/6 LC50), and 0.126 ppm (1/3 LC50), to observe the changes in the photoreceptor cells at the behavioral, histopathological and ultrastructural levels. Further, acetylcholinesterase activity was also evaluated. Behavioral changes, such as long resting period, inactivity, increase in air gulps and decrease in opercular and fin movements, were observed. A semi-quantitative analysis of the histological sections showed shrinkage in retinal layers at 0.038 ppm concentration of monocrotophos. At 0.062 ppm, the disappearance of the outer nuclear layer was observed and at the highest concentration of 0.126 ppm, damage in all retinal layers involving necrosis of the outer segment of the photoreceptor cells was observed. Further, at the ultrastructural level, detachment of photoreceptor cells and damage in the inner and outer segments of the photoreceptors were observed in an increasing dose-dependent manner. A reduction in the acetylcholinesterase level was observed in the treated groups. The treated fish were then transferred to toxicant-free water for 60 days to study self-regeneration, but no regeneration was observed in photoreceptor cells of the fish retina. This study shows that exposure to of monocrotophos effectively damages and disturbs the functioning of photoreceptor cells of retina of C. carpio communis L., thus affecting its vision.

Evaluation of acute and sublethal effects of chloroquine (C18H26CIN3) on certain enzymological and histopathological biomarker responses of a freshwater fish Cyprinus carpio.

In this study the toxicity of antimalarial drug chloroquine (CQ) on certain enzymological (GOT, GPT and LDH) and histopathological alterations (Gill, liver and kidney) of a freshwater fish Cyprinus carpio was studied after acute (96 h) and sublethal (35 days) exposure. The median lethal concentration (96 h) of CQ was 31.62 mg/ml. During acute treatment (CQ at 31.62 mg/ml) the treated fish groups showed a significant increase in GOT and GPT activities in blood plasma; whereas LDH activity was decreased when compare to control groups. To analyse the effects of drug at the lowest concentration, the fish were exposed to 3.16 mg/ml (1/10th of 96 h LC50 value) for 96 h. In sublethal treatment (3.16 mg/ml) GOT activity increased up to 14th day and decreased during the rest of the exposure period (21, 28 and 35th day). A biphasic response in GPT activity was observed. LDH activity was found to be increased throughout the study period (35 days) compare to control groups. The alterations in enzyme activities in blood plasma were found to be significant at p < 0.05 (DMRT). Many histopathological changes in vital organs such as gill, liver and kidney of fish were observed in CQ treated group (acute and sub-lethal) compare to normal group. The alterations in the enzymological and histopathological study in the present investigation indicate that the drug CQ has toxic effects on non-target organisms. We conclude that the alterations in enzymological parameters and histopathological changes can be used as biomarker to assess the health of the aquatic organism/environment. Further data on molecular studies are needed to define the mode of action and toxicity of these emerging pollutants.

Effect of butylated hydroxytoluene (BHT) on the cryopreservation of common carp (Cyprinus carpio) spermatozoa.

The aim of the present study was to test the effects of butylated hydroxytoluene (BHT) on the cryopreservation of common carp spermatozoa. BHT is widely used in the cryopreservation of the spermatozoa of different animal species and successfully sustains the characteristics of spermatozoa during freezing and thawing, but it has not previously been used with fish. After sampling, common carp spermatozoa were diluted with an extender composed of modified Kurokura's extender, 10% DMSO, and 10% egg yolk containing 0.0001, 0.001, 0.01, 0.1, 1, 2.5, 5, or 10mM BHT and subsequently frozen in liquid nitrogen. The post-thaw spermatozoa characteristics (i.e., progressive motility percentage (%), duration of progressive motility (s), fertilization rate (%), and eyed-eggs rate (%)) were evaluated and compared with those of the control group. There were significant increases in the percentage of progressive motility and the duration of progressive motility at the concentrations of 0.1 and 0.001mM BHT (P<0.05). The duration of post-thawed spermatozoa progressive motility at 0.001mM BHT was significantly greater than that of the other groups (39.6±0.4s, P<0.05), and the fertilization rates and eyed-eggs rates were also higher following the 0.1 and 1mM BHT treatments. BHT at concentrations of more than 1mM caused sperm immobility during the preparatory stages of the sperm freezing. We concluded that 0.001-0.1mM BHT can be beneficial for the cryopreservation of common spermatozoa.