Seasonal short-term effects of naltrexone on LH secretion in male carp (Cyprinus carpio L.)

In order to evaluate the influence of the season (the stage of gonad maturity) on the modulatory role of endogenous opioid peptides in LH secretion in fish, sexually mature male carp (Cyprinus carpio L.) were intravenously injected with naltrexone-opioid receptor antagonist (5 or 50 microg kg(-1)) in the period of natural spawning (June) or gonad recrudescence (December). Moreover, the possible involvement of the dopaminergic system was studied in fish pre-treated with pimozide (dopamine receptor antagonist) and in intact fish. Blood samples were taken every minute, up to 10 min after naltrexone injection. In June, naltrexone significantly lowered LH levels in comparison to saline injected males. In December, there were no differences between saline and naltrexone-injected carps. In fish pre-treated with pimozide, neither in June nor in December were any significant differences in LH levels between control group and the groups injected with naltrexone found. The results showed that, in male carp, LH secretion under the influence of naltrexone depends on the stage of gonad maturity what suggests that the feedback of gonadal steroids on LH release could be mediated by the endogenous opioids. The role of dopamine in these processes is also discussed.     

Microsatellite markers in common carp (Cyprinus carpio L.)

Microsatellite markers of the poly (CA) type in common carp ( Cyprinus carpio L.) are described. Clones containing a (CA) repeat were isolated from a common carp genomic library and sequenced. The number of repeats found was high compared to mammals but comparable with other teleost fishes. Classification of the repeats (perfect, imperfect and compound) are compared with the Atlantic cod ( Gadus morhua L.), rainbow trout ( Oncorhynchus mykiss ), and Atlantic salmon ( Salmo salar L.). A total of 41 primer sets were designed and tested for polymorphism on a test panel of eight animals (derived from outbred lines, inbred lines and gynogenetic clones). Thirty-two markers were found to be polymorphic. The heterozygosity in the outbred animals was 60·4%, 51·1% in the inbred animals and 0% in the gynogenetic clones. The average number of alleles among the eight animals was 4·7 per marker. Six markers (18·8%) gave an additional polymorphic amplification product besides the polymorphic amplification product in the expected size range. The possibility that these loci are tetraploid is discussed. The polymorphic loci described for common carp will be valuable as genetic markers for use in population, breeding, and evolutionary studies.     

Genetic differences in natural antibody levels in common carp (Cyprinus carpio L.)

In mammals, natural antibodies (Nabs) are mostly of the IgM isotype and can bind to a particular antigen or pathogen even if the host has never been exposed. Despite their early detection and abundance, the exact role and genetic control of Nabs remain unclear. We have used an indirect ELISA with three different antigens (keyhole limpet haemocyanin, chicken ovalbumin and bovine serum albumin) to demonstrate the ubiquitous presence of Nabs in common carp. Serum levels of Nabs increased with age, i.e. 10-month-old fish showed higher levels than 4-month-old fish. Also, fish grown in earth ponds showed higher levels of Nabs than fish grown in a clean environment of UV-treated water. Furthermore, we show that Nabs are present in different levels in the serum of carp lines with a different genetic background, suggestive of a genetic control. These genetic differences were independent of antigen, age and environment. Genetic differences in levels of Nabs could not unequivocally be related to differences in survival under farmed conditions. The possibilities for using levels of Nabs as marker criterion for selection for genetic disease resistance are discussed.     

Soybean meal induces intestinal inflammation in common carp (Cyprinus carpio L.)

The development of soybean meal (SBM) induced enteritis in the hindgut of the omnivorous common carp (Cyprinus carpio L.). The developed condition was assessed when carp, continuously fed on animal protein, were transferred to a diet in which 20% of the protein was replaced by SBM. After week 1, most of the inflammation parameters were already present, but at week 3, a strong aggravation of the condition was observed which included a shortening of the mucosal folds, the disappearance of the supranuclear vacuoles, an increased number of goblet cells, a thickened lamina propria and sub-epithelial mucosa with increased numbers of basophilic granulocytes as well as a decreased uptake capacity of enterocytes (impaired endocytosis and microvilli). Contrary to previous observations made with respect to Atlantic salmon, common carp start to recover from the fourth to the fifth week after switching to SBM feeding. At this stage, the supranuclear vacuoles refill and most of the parameters revert to basal levels. During the enteritis process, a real-time quantitative PCR analysis was conducted to measure the expression of inflammatory and anti-inflammatory cytokine genes in the isolated intraepithelial lymphocytes (IEL). The pro-inflammatory interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha1 (TNF-alpha1) genes were up-regulated during the inflammation process while the anti-inflammatory interleukin 10 (IL-10) was down-regulated after an initial up-regulation at week 1. Transforming growth factor beta (TGF-beta) expression showed an up-regulation from week 3 onwards despite the high Ct value and the low primer efficiency shown. This study confirms the contribution of IEL (mainly T-like cells) and basophils in the enteritis process. In addition, the results show a clear involvement of up- and down-regulated cytokine genes in both the onset and recovery of the SBM-induced enteritis in the hindgut of carp.     

Effect of different levels of phytase on growth and mineral deposition in common carp (Cyprinus carpio,L.)

The effect of phytase (Ronozyme P) on growth and mineral deposition in common carp (Cyprinus carpio L) was evaluated. Five diets (treatments T1‐T5) were prepared to contain about 36.6% DM proteins and 0.55% DM phosphorus. Diet 1 had no supplement; diet 2 was supplemented with 2.80 g inorganic phosphorus (P) (Na₂HPO₄) kg^?1. Diets 3, 4 and 5 were supplemented with 500, 750 and 1000 U phytase kg^?1. After 126‐day feeding trials, fish grew from 39.9 g to 158 g, 262 g, 155 g, 166 g and 172 g in diets T1‐T5, respectively. There were no differences (P > 0.05) in mean weight gain, SGR, feed intake or feed conversion ratio of fish in T1, T3, T4 and T5. T2 fish had the best (P < 0.05) growth performance. Apparent P digestibility was significantly higher in T2, while faecal P discharges were significantly reduced in T3, T4 and T5. Fish in T2, T3 and T4 had higher (P < 0.05) bone P than that in T1. Vertebral Mg (mg g^?1 DM) of fish in T3, T4 and T5 were higher (P < 0.05) than that of fish in T1. Bone Mn showed the same trend. Data indicated that the enzyme exhibited no clear improvement for a higher P availability.     

Response of common carp (Cyprinus carpio L.) leucocytes to hormone-induced stress

The influence of cortisone on leucocytes composition in the blood of common carp (Cyprinus carpio L.) is studied. Following the hormone injection, the relative number of leucocytes decreased and the number of neutrophils and blast-form cells increased in the leucocyte spectrum of experimental fish.     

Monoclonal antibodies against spermatozoa of the common carp (Cyprinus carpio L.)

Summary Eleven monoclonal antibodies that recognize membrane determinants on spermatozoa of the carp Cyprinus carpio L. have been produced. Indirect immunofluorescence revealed that these determinants are uniformly distributed on the surface of head and midpiece. Most of them are also present on the outer membrane of precursor sperm cells. Although none of the monoclonal antibodies reacted with carp somatic tissue, five monoclonal antibodies were positive for surface membrane determinants of oogonia and early prophase oocytes in carp ovary. Preliminary analysis of the testis and ovary of three other species of fish showed that some carp determinants are shared with germ cells from Barbus conchonius, Clarias lazera, or Salmo gairdneri.Abbreviation WCS Wageningen Carp Sperm antibody     

The effects of anticoagulants on hematological indices and blood cell morphology of common carp (Cyprinus carpio L.)

Hematological parameters (Ht, Hb, RBC, WBC, PLT), erythrocyte size, and osmotic fragility, differential leukocyte count, ROS production in common carp blood collected on three anticoagulants: heparin (10 IU/mL, Na2EDTA (0.1, 0.5, and 1 mg/mL), and sodium citrate (0.3 mg/mL) were compared. Na2EDTA caused partial blood hemolysis in Ht tubes which made Ht measurement impossible, and resulted in high variability of the results. Both, citrate and Na2EDTA increased sensitivity of red blood cells to hemolysis. Na2EDTA also induced erythrocyte anisocytosis and anisonucleosis. Na2EDTA significantly increased ROS production but no effect of anticoagulants on WBC, PLT or differential leukocyte count was observed. The obtained results show that Na2EDTA should not be used for evaluation of red blood cell parameters and erythrocyte morphology, and for ROS production measurement in common carp. Heparin proved to be the most appropriate anticoagulant to use for this species, although Na2EDTA and sodium citrate may be used for WBC and leukocyte differential count evaluations.     

Physiological compartmentation in gonadal tissue of the common carp (Cyprinus carpio L.)

Summary Physiological compartmentation in carp (Cyprinus carpio L.) gonads was investigated after intracardial injection of horseradish peroxidase (HRP) and two mouse anti-carp-sperm monoclonal antibodies.Immunohistochemistry revealed that a physiological barrier exists in carp testis for HRP and mouse IgG monoclonal antibody around the central lumina of the tubules in which the spermatozoa are located, but not around the cysts containing the precursor germ cells. The results with HRP were confirmed by electron microscopy. Mouse IgM monoclonal antibody did not penetrate the spermatogenic cysts. Probably because of its large size, it was almost exclusively located inside blood capillaries and only sparsely in the interstitial tissue.In the ovary, HRP was regularly distributed in the gonadal tissue, whereas the IgG antibody was predominantly localised on oogonia and early prophase oocytes. The results indicate that in contrast with the testis, no barrier around germ cells exists in the carp ovary.     

鲤鱼微卫星突变速率和模式(英文)

利用150个微卫星分子标记在F1代家系的基因型分析过程中,共有27600个等位基因从亲本向子代传递,其中在5个微卫星座位上检测到6个突变的等位基因。对突变的等位基因数目进行统计分析后得出:鲤鱼平均每个世代每个微卫星座位的突变速率为2.53×10-4。在发现突变的5个位点中,经测序发现,突变序列中插入1个以上的重复单元就导致了突变的发生。这些突变表明,鲤鱼的微卫星突变没有遵循严格的渐变突变模型(stepwise mutation model,SMM)。该文关于鲤鱼微卫星突变速率和模式的研究将会对统计鲤鱼有效群体的统计提供有效参数。     

Structure,organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.     

Differential macrophage polarisation during parasitic infections in common carp (Cyprinus carpio L.)

In many parasitic infections both classically activated macrophages (caMF) and alternatively activated macrophages (aaMF) play a pivotal role. To investigate if both types of macrophages also play an important role during parasitic infections in fish, we infected carp with either Trypanoplasma borreli or Trypanosoma carassii and determined the activation state of the head kidney leukocytes (HKL). Nitrite production was used as read-out for caMF and arginase activity as read-out for aaMF. Basal nitrite production and arginase activity of HKL were moderately different between the two infections. Differences were observed, however, after ex vivo re-stimulation of HKL. Re-stimulation with LPS and T. borreli lysates increased nitrite production by HKL of T. borreli-infected fish. Re-stimulation with cAMP increased arginase activity in HKL of T. carassii-infected fish. Our results indicate that T. borreli-infected carp are more prone to increase nitrite production by caMF while T. carassii-infected fish are more prone to increase arginase activity by aaMF.     

The effects of starvation and refeeding on plasma amino acid levels in carp, Cyprinus carpio L., 1758

Plasma amino acid (AA) levels of carp, Cyprinus carpio L., 1758, were analysed after various periods of starvation as well as after 12 days of refeeding. The levels were compared to control groups, which had been previously fasted for 24 h. A positive correlation between dietary and plasma essential amino acid (EAA) concentrations was observed in all of the control groups.
The effect of starvation on the dynamics of AA concentration was different according to the period of starvation. Fasting already produced a decrease of total α-AA levels at 2 days, and these low levels were maintained until 5 and 8 days. These short periods of fasting affected the levels of EAA (especially branched-chain AA) more than those of non-essential amino acids (NEAA). The only AA that increased was Ala, which rose at 5 days of starvation, surpassing the levels of the control group. These high levels were maintained until 19 days of starvation.
A different situation was observed at 19 days, when an important increase of total α-AA levels was produced, the branched-chain AA being the most notable among EAA and Glu/Gln among NEAA. Later, at 50 days of starvation, total α-AA, EAA (except Leu and Ile) and NEAA decreased.
After 50 days of starvation, 12 days of refeeding did not modify the levels of EAA and NEAA, and their concentrations were lower than those of the control group, which presented an increase of total AA at this time. The differences observed on the changes of individual AA levels are discussed.     

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5'-terminal untranslated region (UTR) of 285 bp, a 3'-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.     

Genetic control of glucosephosphate isomerase (GPI) in common carp (Cyprinus carpio L.)

In common carp, a freshwater fish species of tetraploid origin, GPI enzymes are present in two variants: GPI-A and GPI-B. GPI-A is coded by two loci segregating for two (GPI-A 1*) and six (GPI-A2*) alleles. Experimental crosses of the ornamental (Koi) variety of common carp revealed that GPI-B is coded by only one locus (GPI-B*). Another GPI-B* locus must have been silenced in the process of functional diploidization. It was also shown that the GPI-A2* locus segregated independently from the GPI-B* locus, demonstrating that the loci are located on different chromosomes.     

The effects of 3-methylcholanthrene on macrophage respiratory burst and biotransformation activities in the common carp (Cyprinus carpio L.)

The sensitivity of phagocytic cell function as a bioindicator of pollution stress by polycyclic aromatic hydrocarbons was evaluated in the common carp (Cyprinus carpio L). The time course response of the head-kidney macrophage respiratory burst was measured 1, 2, 3, 5 and 7 days after intraperitoneal injection of a prototypical Cyp 1A inducer (3-methylcholanthrene). This immune activity was compared to the rate of induction of total cytochrome P450, ethoxyresorufin O-deethylase activity (EROD) and glutathione S-transferase activity (GST) in the liver and head-kidney. 3-methylcholanthrene (40 mg kg(-1)) caused a rapid increase in the macrophage respiratory burst. This response was maximal at day 3 post exposure and coincided with maximum induction of cytochrome P450 and EROD activity in liver and head-kidney. Moreover, alpha-naphtoflavone, which functions as both an Ah receptor antagonist and an inhibitor of cytochrome P450 1A activity, reversed the 3-methylcholanthrene induction of immune and enzymatic parameters measured, suggesting metabolic processes. Taken together these results suggest that the induction of macrophage oxidative function may be an equally sensitive marker of exposure to polycyclic aromatic hydrocarbon as the induction of biotransformation activities and confirm that responses mediated by the Ah receptor are similar, if not identical, to those of mammals.     

Divergent selection for antibody production in common carp (Cyprinus carpio L.) using gynogenesis

A base population (n = 101) of carp, consisting of a single hybrid cross, was immunized with the hapten-carrier complex DNP-KLH. to perform a divergent selection for antibody response. Measurement of the DNP-specific antibody response at 12 and 21 days postimmunization, allowed the classification of a low number of individual carp as early/high (10%) or late/low (13%) responders. Three individuals defined as early/high and three defined as late/low responding, were gynogenetically reproduced to obtain corresponding homozygous progenies within one generation only. Upon immunization with DNP-KLH, the antibody response was found to be significantly higher in the early/high responder homozygous offspring. Although the homozygosity of the offspring apparently caused a (s)lower antibody response (compared with the base population), the differences between the high and low responder offspring do indicate a genetic influence on the antibody response. The realized heritability (h2) for antibody production was estimated at 0.37 ± 0.36. The present study provides the basis for a divergent selection of homozygous inbred carp lines with a genetically controlled difference in antibody response. These inbred lines will allow us to investigate relationship(s) between immune responsiveness and resistance to infectious diseases in fish.     

Cortisol affects testicular development in male common carp, Cyprinus carpio L., but not via an effect on LH secretion

Previous work showed that prolonged elevated cortisol levels, implicated in the stress adaptation, inhibits testicular pubertal development in male common carp, as well as an impairment of the synthesis of the 11-oxygenated androgens. This may be a direct effect of cortisol on the testis or via the gonadotropin secretion by the pituitary. The aim of the present study was to investigate whether cortisol has an effect on pituitary LH secretion. Juvenile common carp were fed with cortisol containing food pellets. Elevated cortisol levels blocked the increase in testosterone levels and pituitary LH content, but induced higher plasma LH levels at the end of puberty. The in vitro LH release capacity was correlated to the pituitary LH content. At the final stage of pubertal development, when a significant difference in pituitary LH content was observed, sGnRHa-induced LH release was also decreased. Testosterone has been shown to induce development of pituitary gonadotrophs, leading to an increase in LH content and GnRH-inducible LH release, but a decrease in plasma LH levels. We observed decreased plasma testosterone levels as a consequence of prolonged cortisol treatment. It is hypothesised that cortisol inhibits the testicular testosterone secretion and thereby, prevents LH storage. In vitro, this leads to a reduced GnRH-inducible LH release, but in vivo to increased LH plasma levels. It is very unlikely that the impaired testicular development is due to an effect of cortisol on LH secretion.