Preliminary Results Concerning the Influence of Human Kisspeptin on LH Secretion in Prussian Carp (Carassius gibelio) Females at the Stage of Ovarian Recrudescence and Spawning Season.

The aim of the experiments was to evaluate the influence of human kisspeptin on LH secretion of Prussian carp (Carassius gibelio) females during ovarian recrudescence and spawning season. For the experiments, human kisspeptin KISS1 (0.1 mg kg(-1) of body weight--b.w.), GnRH analogue (Des Gly(10), D-Ala(6)) GnRH-A (20 µg kg(-1) b.w.) and dopamine antagonist (pimozide) (5 mg kg(-1) b.w.) were used alone or in combinations. At 3, 6, 12, 24 hours after injection(s) blood samples were collected from all fish. LH levels were measured in plasma with the use of the ELISA method. KISS1 did not show any significant effects on spontaneous LH secretion in both tested seasons. At 12 hours sampling time (both stages of gonad maturity) a combination of tested compounds (GnRH-A+KISS1) significantly increased LH release in comparison with the control. In the stage of gonad recrudescence KISS1 significantly increased LH secretion evoked by pimozide at 24 hours. A combination of three components: KISS1, GnRH-A, and pimozide significantly decreased LH secretion in comparison to LH secretion evoked by GnRH-A and pimozide during stage of gonad recrudescence. These results suggest that kisspeptin is involved in seasonal control of reproduction in Prussian carp. The possible interaction of kisspeptin and the dopaminergic system is also discussed.

Interaction between type I interferon and Cyprinid herpesvirus 3 in two genetic lines of common carp Cyprinus carpio.

Cyprinid herpesvirus 3 (CyHV-3) infection in common carp Cyprinus carpio L. and its ornamental koi varieties can induce the severe systemic disease known as koi herpesvirus disease. This disease is characterised by a rapid replication and spreading of the virus through multiple organs and results in a fast onset of mortality (starting on Day 6 post infection) in up to 100% of infected fish. During the first phase of viral infections, type I interferons (IFNs) have generally been proven to be essential in inducing an innate immune response; however, very little is known about the type I IFN response to herpesviruses in fish. The aim of this work was to study the type I IFN responses during CyHV-3 infection in 2 genetically divergent lines of common carp which presented differing survival rates. Our results show that CyHV-3 induced a systemic type I IFN response in carp, and the magnitude of type I IFN expression is correlated with the virus load found in skin and head kidney. In this in vivo experimental setup, the level of type I IFN response cannot be linked with higher survival of carp during CyHV-3 infection.

The Effect of [(D-Ala6, Pro9NEt)mGnRH-a + Metoclopramide] (Ovopel) on Propagation Effectiveness of Two Breeding Lines of Common Carp (Cyprinus carpio L.) and on Luteinizing Hormone and 17α,20ß-Dihydroxyprogesterone Levels in Females during Ovulation Induction.

The study evaluates the impact of Ovopel on the reproductive effectiveness of carp from Polish line 6 and Lithuanian line B and the release of luteinizing hormone (LH) and 17α,20ß-dihydroxyprogesterone (17α,20ß-DHP) in females from these lines during ovulation induction. The levels of both hormones were determined in blood plasma samples taken just before the priming injection of Ovopel (0 h), at the time of administering the resolving dose of Ovopel (12 h), and after the next 12 h (24 h). Following Ovopel treatment, the mean egg weight obtained for line 6 was higher, but not statistically different, than that observed for line B. Egg quality, on the other hand, was significantly higher in line B. Female provenance did not significantly affect the number of eggs and living embryos after 70 h incubation. However, the total egg number for line 6 was higher. The mean number of living embryos (70 h) was similar for both lines. LH concentrations at 0, 12, and 24 h were not statistically different between the lines. A comparison of LH concentrations between ovulated and non-ovulated females at different sampling times revealed no significant differences either within or between the lines. Statistically significant differences in LH levels were found for both ovulated and non-ovulated females from a given line between the sampling times. The results for 17α,20ß-DHP were similar, with only one difference: 24 h after the priming dose of Ovopel, 17α,20ß-DHP levels in ovulated fish were significantly higher compared with non-ovulated females, but only in line 6.

Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections.

Interferons (IFNs) are secreted mediators that play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for two highly contagious viruses: spring viraemia of carp virus (Rhabdovirus carpio, SVCV) and the Cyprinid herpesvirus 3 (CyHV-3), which belong to Rhabdoviridae and Alloherpesviridae families, respectively. Both viruses are responsible for significant losses in carp aquaculture. In this paper we studied the mRNA expression profiles of genes encoding for proteins promoting various functions during the interferon pathway, from pattern recognition receptors to antiviral genes, during in vitro viral infection. Furthermore, we investigated the impact of the interferon pathway (stimulated with poly I:C) on CyHV-3 replication and the speed of virus spreading in cell culture. The results showed that two carp viruses, CyHV-3 and SVCV induced fundamentally different type I IFN responses in CCB cells. SVCV induced a high response in all studied genes, whereas CyHV-3 seems to induce no response in CCB cells, but it induces a response in head kidney leukocytes. The lack of an IFN type I response to CyHV-3 could be an indicator of anti-IFN actions of the virus, however the nature of this mechanism has to be evaluated in future studies. Our results also suggest that an activation of type I IFN in CyHV-3 infected cells can limit the spread of the virus in cell culture. This would open the opportunity to treat the disease associated with CyHV-3 by an application of poly I:C in certain cases.

Roundup in the Reproduction of Crucian Carp (Carassius carassius): An In Vitro Effect on the Pituitary Gland and Ovary.

Roundup, the most popular herbicide in global agriculture, is regarded as an endocrine disruptor causing alterations of important hormones at the hypothalamic-pituitary-gonadal axis as well as impairment of gametogenesis. The whole pituitary glands of crucian carp (Carassius carassius) were incubated for 3 h in the medium containing Roundup (0-control, 1 and 10 ng/mL). The level of luteinizing hormone (LH), and mRNA transcript abundance of kisspeptin (kiss-1) and its receptor (gpr54), were determined. The isolated ovarian fragments were incubated for 24 h in the presence of Roundup and the following effects on reproductive parameters were determined: the final oocyte maturation and ovulation, structural changes in follicles, secretion of 17,20ß-progesterone (17,20ß-P) as well as mRNA transcript abundance of the luteinizing hormone receptor (lhr), estrogen receptors (erα, erß1, erß2), and zona radiata (chorion) proteins (zp2 and zp3). Roundup inhibited final oocyte maturation and decreased the percentage of ovulated eggs, and furthermore, caused structural changes in the ovarian follicular components. There were no significant changes in the measured hormone levels and analyzed genes mRNA transcript abundance. Summing up, obtained results indicate that Roundup may adversely affect oocyte maturation and the quality of eggs, suggesting that exposure to this herbicide can lead to reproductive disorders in fish.

Protective effect of melatonin on cadmium-induced changes in some maturation and reproductive parameters of female Prussian carp (Carassius gibelio B.).

The aim of this study was to determine whether melatonin (Mel), which is a known antioxidant and free radical scavenger, could perform the role of a preventive agent against the toxic effects of cadmium (Cd2+) on mortality, fish growth, gonadosomatic index (GSI), luteinizing hormone (LH) secretion, the response to hormonal stimulation of spawning, and also tissue accumulation of Cd in Prussian carp females. These females received melatonin implants and were exposed to 0.4 or 4.0 mg/L of Cd (as CdCl2·2.5H2O) over either a 5- or 3-month period, followed by further 2 months of purification in clear water. Negative changes caused by exposure to cadmium in the water were as follows: higher fish mortality, lower body weight, increased accumulation of cadmium in the brain and ovary, lowered GSI, impaired spontaneous LH secretion during exposure, and impaired LH secretion during stimulation of spawning. All of these effects were observed in the group of fish exposed to 0.4 and/or 4.0 mg Cd/L but did not occur or were less pronounced in the groups exposed to cadmium in the presence of melatonin released from the implants. During depuration, in the group of fish which had been exposed to the highest Cd concentration, we observed a significant improvement in fish survival rate, body growth, inhibition of further cadmium accumulation in tissues, and gradual return of spontaneous LH secretion as well as normalization of the GSI value to the control group levels. In conclusion, these findings indicate that melatonin can be a preventive agent for some toxic effects on fish reproduction induced by environmental cadmium contamination.

The effect of aromatase inhibitor, fadrozole, on sGnRHa stimulated LH secretion in goldfish (Carassius auratus) and common carp (Cyprinus carpio).

The aromatase inhibitor, fadrozole, was applied to common carp and goldfish in order to examine its ability to increase the spontaneous and sGnRHa stimulated LH secretion. First, trials in goldfish in 2003 showed fadrozole's moderate ability to potentiate sGnRHa-stimulated LH secretion. However, this ability was much weaker than that obtained with dopamine antagonist, pimozide. There was no ovulation in fadrozole-treated fish. Several experiments on goldfish and carp during the two consecutive years with the different treatment regimes and doses of fadrozole did not confirm the optimistic results obtained in 2003. This shows that fadrozole is unable to replace the antidopaminergic drugs being used in fisheries practice.

Differential effects of morphine and naltrexone on the in vitro LH secretion from male and female carp pituitary gland.

The in vitro effects of morphine (10(-10), 10(-8), 10(-6) or 10(-5) M) or/and naltrexone (10(-6) or 10(-8) M) on LH release from male and female carp (Cyprinus carpio L.) dispersed pituitary cells (obtained from fish at the time of late gonad recrudescence) were investigated. Morphine alone at the lowest tested concentration (10(-10) M) increased LH secretion from the cells of males. On the contrary, in female cell incubations the highest concentrations of morphine (10(-6) or 10(-5) M) significantly lowered LH levels. Naltrexone alone (at both tested concentrations) had no influence on LH secretion, neither in males nor in females. However in the incubations of female cells it antagonised the influence of morphine at 10(-10) or 10(-8) M. In male cell incubations naltrexone abolished the stimulatory action of morphine at 10(-10) M. The results suggest that in the in vitro culture of carp pituitary cells LH secretion is modulated by the opioids which affect the release of this gonadotropin through the typical opioid receptors and that the mu type of these receptors is involved in this process. The effects of opioid agonist and antagonist depend on the stage of gonadal maturity and the sex of fish i.e. the actual level of sex steroids.

The effects of naltrexone, an opioid receptor antagonist, on plasma LH levels in common carp (Cyprinus carpio L.).

Naltrexone-an opioid receptor antagonist, was administered intraperitoneally to sexually mature male and female common carp in the prespawning period, in order to investigate its effects on spontaneous or sGnRH-A-stimulated LH secretion. Naltrexone and sGnRH-A were injected at the same time. The possible involvement of a dopaminergic system in this process was studied in males pre-treated with pimozide (a dopamine receptor antagonist) 12 h before naltrexone and/or sGnRH-A administration. Blood samples for the analysis of carp LH concentrations were taken just before the injections and then after the injections, serial sampling during 24 h was performed. In male carp, naltrexone (500 or 5000 microg kg(-1)) decreased spontaneous LH release, but there were no effects of naltrexone on sGnRH-A-stimulated LH secretion. In males pre-treated with pimozide, a similar response to naltrexone injection (500 microg kg(-1)) as in pirnozide non-treated fish, was observed. The highest dose of naltrexone, 5000 microg kg(-1), significantly stimulated LH release, in response to sGnRH-A administration in pimozide pre-treated males. In female carp, contrary to males, naltrexone at a dose of 500 microg kg(-1), caused significant stimulation of spontaneous LH release. These data indicate that endogenous opioid peptides modify LH secretion in sexually mature carp. In males, they stimulate LH secretion, acting rather on the hypothalamic GnRH system and in females, opioids inhibit LH release by the influence on the dopaminergic system.

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.