Carp Blood Research Articles

Rapid conversion and reversible conjugation of glutathione detoxification of microcystins in bighead carp (Aristichthys nobilis)

The glutathione and cysteine conjugates of microcystin (MC-GSH and MC-Cys, respectively) are two important metabolites in the detoxification of microcystins (MCs). Although studies have quantitated both conjugates, the reason why the amounts of MC-GSH are much lower than those of MC-Cys in various animal organs remains unknown. In this study, MC-RR-GSH and MC-RR-Cys were respectively i.p. injected into the cyanobacteria-eating bighead carp (Aristichthys nobilis), to explore the biotransformation and detoxification mechanisms of the two conjugates. The contents of MC-RR, MC-RR-GSH, MC-RR-Cys and MC-RR-N-acetyl-cysteine (MC-RR-Nac, the acetylation product of MC-RR-Cys) in the liver, kidney, intestine and blood of bighead carp in both groups were quantified via liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). In the MC-RR-GSH-treated group, the MC-RR-Cys content in the kidney increased 96.7-fold from 0.25 to 0.5h post-injection, demonstrating that MC-RR-GSH acts as a highly reactive intermediate and is rapidly converted to MC-RR-Cys. The presence of MC-RR in both MC-RR-GSH- and MC-RR-Cys-treated groups indicates, for the first time, that MC conjugation with the thiol of GSH/Cys is a reversible process in vivo. Total MC-RR concentrations dissociated from MC-RR-Cys were lower than those from MC-RR-GSH, suggesting that MC-RR-Cys is more capable of detoxifying MC-RR. MC-RR-Cys was the most effectively excreted form in both the kidney and intestine, as the ratios of MC-RR-Cys to MC-RR reached as high as 15.2, 2.9 in the MC-RR-GSH-treated group and 63.4, 19.1 in the MC-RR-Cys-treated group. Whereas MC-RR-Nac could not be found in all of the samples of the present study. Our results indicate that MC-RR-GSH was rapidly converted to MC-RR-Cys and then excreted, and that both glutathione and cysteine conjugates could release MC-RR. This study quantitatively proves the importance of the GSH detoxification pathway and furthers our understanding of the biochemical mechanism by which bighead carp are resistant to toxic cyanobacteria.

Comparative Study of the Effect of Different Anticoagulants on Blood Cell Morphology in Common Carp ( C. carpio ) and Rainbow Trout ( O. mykiss )

Abstract. Hematological assessment is an important tool in evaluating the fish health status. This tool corroborated with biochemical, parasitological and bacterial examinations aids the researchers in monitoring fish welfare status. For relevant blood samples using the right harvesting methodology with the right anticoagulant is imperative. This study presents a comparison between two different anticoagulants like Li-Heparin and ethylenediaminetetraacetic acid (EDTA) in common carp ( Cyprinus carpio ) and rainbow trout ( Oncorhynchus mykiss ). Two common carps and two rainbow trouts were submitted for blood harvesting using Li-Heparin and EDTA containers. Blood smears were prepared using a Romanowsky staining procedure and examined via light microscopy. The results of the study revealed significant in vitro hemolysis and red blood cell morphologic alterations in the common carp blood samples when harvested on EDTA in contrast with the Li-Heparin anticoagulant. The white blood cells showed no significant changes induced by neither of the two anticoagulants. No significant anticoagulant induced cell morphological alterations were noticed in the rainbow trout. It is concluded that EDTA is not the appropriate anticoagulant for hematologic evaluation in common carp.

Dietary modulation of immune response and related gene expression profiles in mirror carp (Cyprinus carpio) using selected exotic feed ingredients

An investigation was conducted to evaluate the effect of feeding selected exotic ingredients on immune responses and expression of immune related genes in mirror carp (Cyprinus carpio). Fish were fed diets for a total of 56days. Fishmeal served as the main protein source in the control diet and two experimental diets consisted of fishmeal fixed at 34% provision of protein and the remaining 66% protein was provided either by earthworm meal (EW diet) or a combination of whey protein concentrate (8%) and casein (58%) (WPC diet). At the start of the trial fish were injected intraperitoneally with Aeromonas hydrophila bacterin and samples of carp blood were taken for gene expression, haematological and serological analysis. Compared to fish fed fishmeal, a significant increase in mRNA expression of the pro-inflammatory cytokines IL-1β (24h post injection) and TNFα (at 12h and 48h post injection) was observed in fish fed EW. Moreover a similar trend was observed for complement 3 (C3) gene, where fish fed EW showed significant elevations in mRNA expression values at both 12 and 48h post injection compared to control fed fish. In contrast, fish fed WPC showed a significant decrease in C3 and TNF-α mRNA expression compared to fish fed fishmeal (48 h post injection). Fish fed EW and WPC showed a significant increase in peripheral leukocyte levels compared to fish fed fishmeal 14d post injection. Fish fed fishmeal presented significantly higher circulatory IgM levels at 7d post injection compared to fish fed EW and WPC. In contrast, fish fed EW and WPC showed a significant increase in IgM levels at 28d post injection. The present study shows an up-regulation of all immune related genes in fish fed EW, compared to fish fed fish meal, which is indicative of an acute inflammatory response. In contrast it appears feeding WPC has an immunosuppressive effect towards TNFα and C3 expression in carp blood. This may be offset by the fact that feeding WPC to carp appears to aid in the increased production of circulatory IgM and leukocyte levels compared to fishmeal fed fish.

Diclofenac-induced oxidative stress in brain, liver, gill and blood of common carp (Cyprinus carpio)

Due to its analgesic properties, diclofenac (DCF) is one of the most commonly used non-steroidal anti-inflammatory drugs (NSAIDs). While residue from this pharmaceutical agent has been found in diverse water bodies in various countries, there is not enough information of its potential toxicity on aquatic organisms, particularly in species which are economically valuable due to their high consumption by humans, such as the common carp Cyprinus carpio. This study aimed to evaluate potential DCF-induced oxidative stress in brain, liver, gill and blood of C. carpio. The median lethal concentration of DCF at 96h (96-h LC50) was determined and used to establish the concentration equivalent to the lowest observed adverse effect level (LOAEL). Carp specimens were exposed to this concentration for different exposure times (12, 24, 48, 72 and 96h) and the following biomarkers were evaluated: lipid peroxidation (LPX) and the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Also, the DCF and 4-hydroxy DCF was determined by LC-MS/MS. Results show a statistically significant LPX increase (P<0.05) in liver and gill mainly as well as significant changes in the activity of the antioxidant enzymes evaluated in these organs, with respect to controls (P<0.05). The DCF concentrations decreased in water system and increased in the carp. The DCF biotransformation to 4-hydroxy DCF was observed to 12h. The pharmaceutical agent DCF is concluded to induce oxidative stress on the common carp C. carpio, with the highest incidence of oxidative damage occurring in liver and gill. Furthermore, the biomarkers employed in this study are useful in the assessment of the environmental impact of this agent on aquatic species.

Fusion of Tat-PTD to the C-terminus of catfish growth hormone enhances its cell uptakes and growth-promoting effects

Fish growth hormone has been long regarded as a promising feed additive in aquaculture for its significant growth promotion effect. To improve its efficiency in oral applications, we generated a Silurus asotus growth hormone-Tat (saGH-Tat) fusion protein and investigated its effect on biological activities relevant to transduction and growth promotion. In this study, saGH, saGH-Tat, Green fluorescent protein and saGH fused protein (GFP-saGH) and GFP-saGH-Tat were all highly expressed in Escherichia coli strain BL21 (DE3) and confirmed by Western blot. The transduction effect of Tat protein transduction domain (Tat-PTD) was verified by observing increased GFP fluorescence in separated intestine cells or frozen sections in GFP-saGH-Tat treated fish. The recombinant saGH and saGH-Tat were tested for biological activity by specific binding with crude liver membrane extracts as well as in a growth promotion assay via oral administration. saGH-Tat treatment demonstrated strengthened receptor binding and accelerated growth-promoting effects. Under optimal concentrations of hormone (1μg/g of fish body weight), saGH-Tat treated fish exhibited an 87.25% growth increase compared to 61.3% in saGH-treated fish. The response of downstream signal pathway factors were analyzed by measuring insulin like growth factor-I (IGF-I), growth hormone receptor (GHR) mRNA expression in the liver, and growth related digestive enzymes (trypsin and chymotrypsin), and all indicate significant up-regulation. Further confirmation of increased GH level in carp blood by Tat fusion was shown by flow cytometry. These results suggested that Tat-PTD fused to the C-terminus saGH greatly enhances its biological activities, making it a promising agent for increasing productivity in aquaculture.

Comparative proteomics analysis of carp seminal and blood plasma

Comparative proteomics analysis of carp seminal and blood plasma

Microcystin‐LR induces cytotoxicity and affects carp immune cells by impairment of their phagocytosis and the organization of the cytoskeleton

Microcystin-LR (MC-LR) is the main isoform of hepatotoxin produced by cyanobacteria, which occur worldwide in the aquatic environment. The present study investigated the in vitro toxic MC-LR effects on immune cells isolated from the blood of carp. Cells were exposed to different MC-LR concentrations ranging from 0.01 to 1 µg ml(-1) for 2, 6 and 24 h. In addition, the effect of the toxin on the phagocytic activity of leukocytes and on actin and tubulin re-organization in phagocytic cells was studied. We observed that MC-LR induces apoptosis in lymphocytes 2 h after incubation, whereas high toxin concentrations induced necrosis in lymphocytes in a time- and concentration-dependent manner. Incubation of the cells for 2 h with 0.1 and 1 µg ml(-1) MC-LR inhibited phagocytosis without affecting apoptosis or glutathione (GSH) levels. Moreover, at this time point and with these concentrations, the toxin also induced a significant re-organization of the actin cytoskeleton in phagocytes, which subsequently collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. These results suggest that both phagocytes and lymphocytes are targets for MC-LR and the disturbances of phagocytosis may impair the balance of the immune system.

Pro- and anti-inflammatory cytokine expression in carp blood and head kidney leukocytes exposed to cyanotoxin stress – An in vitro study

Cyanotoxins are toxic, secondary metabolites produced by different species of cyanobacteria that are present all over the world in aquatic environments. No data are available about the molecular mechanisms underlying the stress associated with exposure of fish immune cells to low concentrations of cyanotoxins. The purpose of this study was to determine whether the expression of cytokines that underlie immune regulation are changed after incubation of fish leukocytes with pure cyanotoxins: microcystin- LR (MC-LR), anatoxin-a (Antx-a), or an extract containing Antx-a. The study investigated the relative gene expression of four important cytokines, IL-1β, TNF-α, IL-10, and TGF-β, in carp head kidney and blood leukocytes exposed to toxins at concentrations of 0.01 or 0.1 μg/ml for 4 h. The data showed that pure toxins could induce dysregulation of pro-/anti-inflammatory cytokine expression. Expression of cytokine IL-1 β was highly upregulated following Antx-a exposure, whereas MC-LR induced merely moderate reactions. The expression of TNF-α mRNA was significantly suppressed in blood and head kidney cells incubated with toxins at the higher concentration. These results showed that pure toxins dysregulated the expression of pro-inflammatory cytokines IL-1β and TNF-α more promptly than the anti-inflammatory cytokines TGF-β and IL-10. In contrast, the studies demonstrated a clearly downward trend of pro-inflammatory cytokines and an upward trend of anti-inflammatory cytokines in leukocytes exposed to an extract containing defined concentrations of Antx-a. This study suggests that cyanotoxins present in aquatic environments may exert immunotoxic effects by altering the transcription of important mediators of the fish immune system.

Protective role of propolis in chlorpyrifos-induced changes in the haematological parameters and the oxidative/antioxidative status of Cyprinus carpio carpio

The protective effect of propolis on haematological parameters and antioxidant status were evaluated in the blood and various tissues of carp exposed to chlorpyrifos (CPF). The fish were exposed to sublethal concentrations of CPF (0.040 and 0.080mg/L) for 10days, and propolis (10mg per kg of fish weight) was simultaneously administered. Samples of the blood and tissue (liver, kidney, and gill) were collected at the end of the experiment and analysed for their oxidant–antioxidant status, including the malondialdehyde (MDA) levels, and the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity. The samples were also measured for changes in the haematological parameters, such as the red blood cell (RBC) and white blood cell (WBC) counts, the haemoglobin concentration (Hb), the haematocrit (Ht) level, and the erythrocyte indices: the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC). The findings of this study demonstrated that CPF had a negative effect on the haematological parameters and the antioxidant enzyme activities of the fish; this toxic effect was neutralised by the administration of propolis. The present results suggest that propolis can be effective in the protection of CPF-induced toxicity in fish.

Expression analysis of heat shock genes in the skin, spleen and blood of common carp(Cyprinus carpio)after cadmium exposure and hypothermia

Heat shock proteins are chaperones that play a pivotal role in controling multiple regulatory pathways such as stress defense, hormone signaling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, the expression patterns of four well-known heat shock genes (hsp70, hsc70-1, hsc70-2 and hsp90α) were characterized in the skin, spleen and blood cells of the common carp, under unstressed conditions and after Cd2+ treatment or hypothermia. The examined genes were expressed in a tissue-specific manner: hsc70-2 was expressed constitutively, and was at best only slightly inducible; hsp90α exhibited a high basic expression in all three tissues, whereas hsc70-1 did so only in the blood cells, the expression of hsp70 proved to be below the level of detection in unstressed fish. Cold shock induced the expression of hsp genes in the spleen (hsp90α) and blood cells (hsp70, hsc70-1 and hsp90α), while Cd2+ treatment has no effect on the expression pattern. The highest inducibilities were detected in the skin: for hsp70 an induction of at least 20-fold after cadmium exposure, for hsc70-1 of at least 30-fold and for hsp90α of 3-fold after hypothermia.

Response of juvenile crucian carp (Carassius auratus) to long-term ammonia exposure: feeding, growth, and antioxidant defenses

We investigated the effect of long-term ammonia exposure on feeding, growth, and some antioxidant parameters of liver, gill, and blood of juvenile crucian carp (Carassius auratus). In chronic exposure experiments, juvenile crucian carp were exposed to different concentrations of NH3–N (0.107, 0.214, 0.321, 0.428 mg L−1) for 45 days. Ammonia in the external environment significantly impaired the feeding and growth of juvenile crucian carp; additionally, enzymatic (superoxide dismutase (SOD) and catalase (CAT)) and non-enzymatic (glutathione (GSH) and malondialdehyde (MDA)) antioxidants in fish liver showed their protective functions significantly when oxidative stress occurred, but specific results were variable depending on time, ammonia concentration, and the tissue.

Direct-binding quartz crystal microbalance immunosensor to detect carp metallothionein

Metallothionein (MT) is a valuable biomarker against xenobiotic heavy metal contamination in organs and blood of fish. In this study, MT was induced in carp blood by cadmium injection and was purified to homogeneity by DEAE-cellulose ion exchange column chromatography to be used dually as a standard and immunogen. A sensitive detection on carp MT was then followed using a batch-type direct-binding quartz crystal microbalance immunosensor system installed with a well cell. Among antibody immobilization methods tested, two methods that were carried out via a heterobifunctional thiolation cross-linker, and via the cross-linker and protein G showed their respective advantages in simplicity and sensitivity. When analyzed using the immunosensor for carp MT in the concentration range of 50–4000 ng/mL, the limit of detection around 250 ng/mL was obtained. Based on its reasonable sensitivity and real-time presentation for the sensor signal, the immunosensor of this study was presumed as a screening tool to monitor possible heavy metal contamination in a fish farm and imported fish.

Bioconcentration Factor of Perfluorinated Compounds in Blood of Common Carp, Cyprinus Carpio

PP-30-070 Background/Aims: The effects of Perfluorinated compounds (PFCs) on common carp (Cyprinus carpio) were investigated using blood samples. Methods: Target chemicals comprise 9 species of PFCs including 3 sulfonates (PFBS(C4), PFHxS(C6), PFOS(C8)) and 6 carboxylates (PFOA(C8), PFNA(C9), PFDA(C10), PFUnA(C11), PFDoA(C12), PFTrA(C13)). Carps were exposed to PFCs at the same concentration ranges (negative control, 5 and 50 ng/mL) and exposure period. Experiments were performed in totally 49 days 21 days of uptake stage followed by 28 days of depuration. Results: The results showed that PFOS was detected at as high as 5411.1 and 39944.2 ng/mL at low (5 ng/mL) and high (50 ng/mL) concentrations, respectively. At high concentration exposure, the concentration of PFCs was 5–8 times higher than that of low concentration exposure. Conclusion: Bioconcentration factor (BCF) was calculated in which BCF values can be sorted as PFOS > PFH × S > PFBS. After the depuration process, PFOS in carps exposed to high concentration was detected at highest level which is 5–7 times higher than that of carps exposed to low concentration.

Disodium EDTA used as anticoagulant causes hemolysis in common carp blood

Disodium EDTA used as anticoagulant for common carp blood caused a significant increase and high variability in hematocrit readings comparing to the heparinized samples. Na2EDTA induced erythrocyte swelling, causing cell membrane disruption (hemolysis). The nuclei released from destroyed cells changed shape from oval to round, and underwent gradual swelling and vacuolation, followed by karyolysis. The degree of observed changes was similar at all Na2EDTA concentrations (0.1, 0.5, and 1.0 mg/mL); on the other hand, percentage of abnormal and destroyed cells showed considerable differences among blood sampled from various carp individuals. The obtained results revealed that Na2EDTA caused gradual damage to erythrocytes, and thus should not be applied as anticoagulant for blood analysis of common carp.

양식 잉어 및 뱀장어에 경구투 fluoroquinolone계 항생제의 체내 잔류량의 변화

The distribution of fluoroquinolones was investigated in the carp (Cyprinus carpio) and eel (Anguilla japonica) after an oral dose of 20 mg/kg body weight/day for 35 days. Blood samples were collected at 5 and 10 hours and 1, 2, 3, 5, 7, 9, 13, 20, 30, and 35 days after treatment. The fluoroquinolone concentrations were determined high- performance liquid chromatography with an ultraviolet detector. The recovery rates of fluoroquinolones in the fish samples ranged from 91.4-96.6, 91.2-96.5, and 90.4-98.6% for concentrations of 0.1, 0.5, and <TEX>$1.0\;{\mu}g/g$</TEX>, respectively. In the blood of carp, ciprofloxacin, enrofloxacin, norfloxacin, and sarafloxacin reached maximums level of 11.03, 9.37, 9.10, and <TEX>$9.81\;{\mu}g/g$</TEX> 10 hours, 1 day, 10 hours, and 10 hours after treatment, respectively. In the eel blood, these reached maximum levels of 12.65, 11.18, 11.91, and <TEX>$8.74\;{\mu}g/g$</TEX> all at 10 hours. Carp sample concentrations of ciprofloxacin, enrofloxacin, norfloxacin, and sarafloxacin were not measurable 20, 30, 20, and 20 days after treatment, respectively, in all experiments (<LOQ; limit of quantitation). Eel sample concentrations of ciprofloxacin, enrofloxacin, norfloxacin, and sarafloxacin were not measurable 20, 35, 30, and 20 days after treatment, respectively, in all experiments (<LOQ).

Detoxifying effect of Nelumbo nucifera and Aegle marmelos on hematological parameters of Common Carp (Cyprinus carpio L.).

The objective of this study was to investigate the efficacy of Nelumbo nucifera and Aegle marmelos on common carp exposed to sub-lethal concentrations of combined heavy metals (5 ppm) under laboratory conditions. The fish were treated with Nelumbo nucifera (500 mg/kg bwt) and Aegle marmelos (500 mg/kgbwt) for 30 days as a dietary supplement. The blood biochemical parameters of the fish were evaluated by analyzing the level of red blood cells (RBC), packed cell volume (PCV), hemoglobin concentration, glucose, cholesterol, iron and copper. The findings of the present investigation showed significant increase in hemoglobin (p<0.001), RBC (p<0.01) and PCV (p<0.01) of herbal drug-treated groups compared with metal-exposed fish. Conversely, glucose and cholesterol level in blood of common carp showed significant reduction compared with heavy-metal-exposed groups. All the values measured in Nelumbo nucifera and Aegle marmelos treated fish were restored comparably to control fish. Our results confirmed that Nelumbo nucifera and Aegle marmelos provide a detoxification mechanism for heavy metals in common carp.

Enumeration of Carp Blood Leukocytes with Flow Cytometry

Enumeration of Carp Blood Leukocytes with Flow Cytometry

Extraction of genomic DNA using a new amino silica monolithic column

A new amino silica monolithic column was developed for DNA extraction in a miniaturized format. The monolithic column was prepared in situ by polymerization of tetraethoxysilane (TEOS) and N-(beta-aminoethyl)-gamma-aminopropylmethyldimethoxysilane (AEAPMDMS). DNA was loaded in 50 mM tris(hydroxylmethyl)aminomethane-EDTA buffer at pH 7.0 and eluted with 300 mM potassium phosphate solution at pH 10.0. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 56 ng DNA with an extraction efficiency of 71 +/- 5.2% (X +/- RSD). When the amino silica monolithic column was applied to extract genomic DNA from the whole blood of crucian carp, an extraction efficiency of 52 +/- 5.6% (X +/- RSD) was obtained by three extractions. Since the chaotropic-based sample loading and organic solvent wash steps were avoided in this procedure, the purified DNA was suitable for downstream processes such as PCR. This amino silica monolithic column was demonstrated to allow rapid and efficient DNA purification in microscale.

Comparative study of erythrocytes of polyploid hybrids from various fish subfamily crossings

We have undertaken comparative studies of the number and phenotypes of erythrocytes in the peripheral blood of red crucian carp (RCC), blunt snout bream (BSB), and their hybrids, including triploids, tetraploids, and pentaploids. The results indicate that the mean nuclear volume of erythrocytes in peripheral blood increases regularly with increasing ploidy. Furthermore, many more mature erythrocytes have a dumbbell nucleus in the peripheral blood of polyploid hybrids compared with their diploid parents. With the increase in ploidy level, the percentage of such erythrocytes increases. The polyploid hybrids also have a large number of erythrocytes with abnormal shapes. For example, round and tear-shaped erythrocytes have been observed in the peripheral blood of polyploid hybrids. Since the erythrocytes in polyploid hybrids with their larger volume and lower specific surface area are unfavorable for the conveyance of oxygen, morphological variations of erythrocytes might improve defective blood circulation.

Effect of a subchronic exposure to Simazine on energetic metabolism of common carp (Cyprinus carpio)

The energetic parameters, such as glycogen, glucose, proteins, lactate and adenosine triphosphate (ATP) content and lactate deshydrogenase (LDH) activity in tissues and blood of carps from simazine (2-chloro-4,6-bis(ethylamino)-s-triazine) contaminated waters were investigated. In order to confirm the field results, a laboratory experiment was designed in which carps were exposed to simazine at the concentration level of 45 μ g. L− 1 (10-fold of the amount found in natural waters) for 90 days. Fish from a contaminated reservoir showed low glycogen concentrations in hepatopancreas and muscle, while fish in another contaminated reservoir showed high LDH activity together with an increase in lactate content in muscle. Laboratory findings did not confirm field results, and fish exposed to simazine did not show alterations in the parameters studied. The results suggest that carps were not stressed by the presence of the simazine at the concentration levels found in both studies and the mechanisms of defense covered the energetic demand.