Long-term cortisol treatment inhibits pubertal development in male common carp, Cyprinus carpio L

The onset and regulation of puberty is determined by functional development of the brain-pituitary-gonad (BPG) axis. Stress has been shown to interfere with reproduction and the functioning of the BPG axis. The response to chronic and severe stress may require much energy and force the organism to make adaptive choices. Energy that is normally available for processes like growth, immune response, or reproduction will be channeled into restoration of the disturbed homeostasis. Cortisol plays a key role in the homeostatic adaptation during or after stress. In the present study, immature common carp were fed with cortisol-containing food pellets covering the pubertal period. We showed that cortisol caused an inhibition of pubertal development, by affecting directly or indirectly all components of the BPG axis. The salmon GnRH content of the brain was decreased. Luteinizing hormone- and FSH-encoding mRNA levels in the pituitary and LH plasma levels were diminished by long-term cortisol treatment, as was the testicular androgen secretion. Testicular development, reflected by gonadosomatic index and the first wave of spermatogenesis, was retarded.     

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.     

Corticosteroids affect the testicular androgen production in male common carp (Cyprinus carpio L.).

Our previous experiments to study the effect of stress adaptation on pubertal development in carp showed that repeated temperature stress and prolonged feeding with cortisol-containing food pellets, which mimics the endocrine stress effects, retarded the first waves of spermatogenesis and decreased 11-ketotestosterone (11KT) plasma levels. The objective of the present study was to investigate whether the decrease in plasma 11KT is caused by a direct effect of cortisol on the steroid-producing capacity of the testis or by an indirect effect, such as a decrease in plasma LH. Pubertal and adolescent isogenic male common carp (Cyprinus carpio L.) were fed with either cortisol-containing food pellets or control food pellets over a prolonged period. Our results indicate that cortisol has a direct inhibitory effect on the testicular androgen secretion independent of the LH secretion. Furthermore, the pubertal period is critical to the influence of cortisol regarding testicular androgen secretion, because the effect is no longer observed at adolescence.     

Larval development of carp, Cyprinus carpio L., in acidic water

Laboratory experiments measuring survival and growth at various pH levels and two temperatures indicate that the threshold of pH of water below which larval development of carp is stopped is 5.0–5.2, the development of some individuals being stopped within the pH range 5.0–5.5. Inhibition of development is due to the inability of a larva to fill its swimbladder with air, this preventing swimming and leading to death from starvation. In water of pH 5.5–6.0 the development may be undisturbed, though slower. The effect of water acidification is reflected in slower growth rate and prolongation of the larval stages of development.     

The effect of digested sludge on the common carp, Cyprinus carpio L.

The effects of sewage sludge on the survival of the common carp, Cyprinus carpio, were investigated. It was noted that, when the concentrations of the sludge used for rearing carp exceeded 0.6%, the fish died within 42 days, whereas those reared in 0.2% sludge remained alive and healthy during the trial period. The body and the gills of the dead fish were covered by a veil-like film which looked like coagulated mucus and was formed by the heavy metal ions from the sludge reacting with some constituents of the mucus secreted by the gill. The histopathological symptoms of the various organs further demonstrated that internal injury in liver and kidney was also an important feature of the intoxicants as well as external damage, especially the particles observed around the gills. It was concluded that the optimum concentration of sludge to any species of fish should be determined by testing a series of concentrations before using it as a supplementary fish feed or fish pond fertilizer.     

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.     

Multiple acute temperature stress affects leucocyte populations and antibody responses in common carp, Cyprinus carpio L

Stress is a potential factor causing increased susceptibility of fish to pathogens. In this study, stress-induced immunological changes that may contribute to a decreased immune status were investigated. A 3 h drop in ambient water temperature of 9 degrees C was used as a relative mild and acute stress model for carp. Effects of this stressor on the dynamics of leucocyte populations were determined with specific monoclonal antibodies. The relative number of circulating B-lymphocytes in the total leucocyte population decreased significantly within 4 h after the onset of single or multiple cold shocks. This decrease was reversible, as B-lymphocyte numbers were restored within 24 h. Most probably, a redistribution of B-lymphocytes contributed to this phenomenon. In head kidney, an increase was measured in the relative number of B-lymphocytes. Granulocyte numbers showed opposite reactions: the percentage of granulocytes in the total leucocyte population nearly doubled in circulation and decreased significantly in the head kidney. This demonstrates that in vivo, a mild stressor differentially alters the distribution of leucocytes. In stressed carp, the percentage of apoptotic lymphocytes in blood is significantly higher compared with the unstressed animals. B-lymphocytes as well as Ig- lymphoid cells contributed to this increased apoptosis. Labelling of blood lymphocytes with a polyclonal antiserum against the glucocorticoid receptor also showed, besides B-lymphocytes, part of the Ig- lymphoid cell population to be glucocorticoid receptor positive. As the distribution of B-lymphocytes was substantially affected, the effect of temperature stress on T-lymphocyte-independent (trinitrophenyl-lipopolysaccharide) and T-lymphocyte-dependent (dinitrophenyl-keyhole limpet hemocyanin) humoral antibody responses was determined. Kinetics of the primary antibody response to the T-lymphocyte-independent antigen showed lower antibody titres in stressed carp during the onset of the immune response, implying a slower development of the antibody response against the T-lymphocyte-independent antigen.     

Sex steroids and their involvement in the cortisol-induced inhibition of pubertal development in male common carp,Cyprinus carpio L

The onset and regulation of puberty is determined by functional development of the brain-pituitary-gonad (BPG) axis. Sex steroids produced in the gonads play an important role in the onset of puberty. Stress interferes with reproduction and the functioning of the BPG axis, and cortisol has frequently been indicated as a major factor mediating the suppressive effect of stress on reproduction. Prolonged elevated cortisol levels, implicated in stress adaptation, inhibited pubertal development in male common carp (Cyprinus carpio). Cortisol treatment caused a retardation of pubertal testis development and reduced the LH pituitary content and the salmon GnRHa-stimulated LH secretion in vitro. A reduced synthesis of androgens also was observed. These findings suggest that the cortisol-induced inhibition of testicular development and the maturation of pituitary gonadotrophs are mediated by an effect on testicular androgen secretion. In this study, we combined cortisol treatment with a replacement of the testicular steroid hormones (testosterone and 11-oxygenated androgens) to investigate the role of these steroids in the cortisol-induced suppression of pubertal development. The effect of cortisol on spermatogenesis was independent of 11-ketotestosterone, whereas the effect on the pituitary was an indirect one, involving the testicular secretion of testosterone.     

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.     

The effect of temperature on survival of carp fry, Cyprinus carpio L., in acidic water

Studies of carp fry, Cyprinus carpio , showed that temperature elevation within the range of 18–26° C increases the mortality caused by lowering the pH of water. It was found that 26-h acclimation in acidic water (mean pH 4.22) prolongs the survival of fish in water at pH 4.02–4.20. Carp fry obtained less oxygen in water at pH 4–0, as compared to pH 7–0. The differences–though insignificant–pointed to an adverse effect of acidification on the respiratory processes.     

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.     

Investigation on carp, Cyprinus carpio L. gynogenesis

A reliable method for gynogenetic mass-production has been developed in carp ( Cyprinus carpio L.). The effect of different cold shock treatments applied after fertilization were examined. The yield of viable diploid gynogenetic animals could be significantly increased when the cold shock was applied 5 min or 15 min after fertilization. A negative correlation has been found between the effectiveness of these two treatments, so both were used on separate batches of eggs. By means of gynogenesis, the crossover frequency and map distance of the transferrin locus have been determined. Successful pilot experiments were made on grass carp ( Ctenopharyngodon idella Val.) and sheatfish ( Silurus glanis L.) with respect to gynogenetic mass production.     

Induced triploidy in carp, Cyprinus carpio L.

A method for mass production of triploid carp was developed by the cold treatment of eggs after fertilization. Triploidy was demonstrated by cytophotometry and chromosome preparation techniques. Of the haploid embryos, 100% showed morphological abnormalities and died soon after hatching. Haploid embryos of maternal and paternal origin occurring in the course of cold treatment were distinguished by using a genetic marker. Among morphologically normal larvae, the frequency of triploidy was 100%, and the viability of the triploid embryos, larvae and the juvenile fish did not differ from the diploid control. Sex differentiation started in the triploid fish, but was greatly retarded. Gonad size and the small number of growing oocytes suggest that most of the triploid individuals will be sterile. Triploid carp grew at the same rate as diploid controls in the laboratory test.     

Determination of the surface area of common carp,Cyprinus carpio L.

A wrap method adaptation combined with AutoCAD2005 and Scion Image for Windows were used to determine the surface area of a fish. Compared with the corresponding r² and F of many models, the most accurate formula: S = 752.15W^0.675 (r² = 0.999, F = 18362.94, P < 0.0001) for estimating the surface area of common carp was obtained. Similarly, the fin formula: S = 1834.12W^0.708 (r² = 0.992, F = 2690.47, P < 0.0001) was also obtained for the same purpose. It was proven that these two formulae gave good estimates of surface and fin areas of four strains of common carp: Yellow‐river carp, fancy carp, mirror carp and Xingguo red carp.     

A quantitative study of testicular germ cell populations in masculinized neomale common carp (Cyprinus carpio L.)

The main objective of the present study was to investigate the effects of 17 alpha-methyltestosterone treatment upon the testicular germ cells of gynogenetic masculinized neomale common carp (Cyprinus carpio L.) in comparison with diploid carp. Gynogenetic common carp progeny (mean body weight, BW, 2.6+/-0.3g; mean total length, 10.4+/-0.5 cm) were treated for a period of 40 days with 17 alpha-methyltestosterone (MT) at a dose of 100mg kg(-1). The oral administration of MT resulted in 61.5-100% of fish exhibiting male gonads. The masculinized neomales exhibited reduced (P<0.05) body weight (BW=22.9+/-0.8) but significantly increased (P<0.05) mean testis weight (2.1+/-0.3) and mean gonadosomatic index (GSI=9.5+/-0.2%) in comparison with fish not treated with MT (BW 54.8+/-1.3; GSI=0.61%). Furthermore, treatment with MT also resulted in an increased number of fish exhibiting abnormal gonads. However, neomales did not exhibit abnormalities in the development of sperm ducts. MT treatment significantly increased germ cell volume, nuclear diameter, nuclear volume and cyst volume (P<0.01 in all cases) in MT-treated fish compared to untreated fish. The area occupied by seminiferous tubules, the number of Sertoli cells and germ cells per cyst, and the number of Leydig cells were significantly (P<0.05) greater in fish treated with MT. The carp neomales exhibited approximately 20-60% more Sertoli cells per cyst (P<0.05). Leydig cell nuclear volume and Leydig cell individual volume were significantly reduced in MT-treated groups (P<0.05) compared with untreated groups. In conclusion, our study strongly suggests that the abnormal gonadal structure evident in masculinized neomales could be explained by a combination of MT-induced genetic (homozygosity) and anabolic effects (upon germ and somatic cells).     

Specific dynamic action and feeding metabolism in common carp,Cyprinus carpio L.

• 1.1. Oxygen uptake attributable to Specific Dynamic Action (SDA) was measured in common carp, Cyprinus carpio L. (63.6–84.0 g) fed on 20, 35 and 50% dietary protein at 0.40 to 1.00% ration levels at 28°C.
• 2.2. After feeding both SDA magnitude and mean peak oxygen consumption increased directly with dietary protein and ration levels. SDA duration was not significantly related to dietary protein but significantly increased with ration levels.
• 3.3. SDA coefficients were 8.99, 13.51 and 15.94% with 20, 35 and 50% dietary protein showing a direction relationship to the protein content. The SDA coefficient did not change with ration size.
• 4.4. SDA models resulting from this work are of great interest for the aquaculturist, as post-feeding oxygen requirements in an intensive fish culture can be predicted where dietary protein and ration levels are known.

     

The eye lens weight and age in the common carp, Cyprinus carpio L.

The validity of the use of the weight of dried eye lenses as an indicator of age in the common carp, Cyprinus carpio L., is tested. There is a significant difference between males and females for fish of two and three years old. Otherwise no significant difference is noted between sexes. The linear relationship between the dry weight of eye lenses and age + 1 are: In Y + 1= 0.5202 + 16.4928 X ( r = 0.93) for female and ln Y + 1=0.4276 + 16.7364 X ( r = 0.94) for male.
The method for determining age is valuable for fish until three years old. However for older fish, in spite of a correlation coefficient between lens weight and age and even with a significant difference between annual groups, the age determination may be misleading due to the overlap in lens weights. In conclusion therefore this method may not be useful for back calculation of growth, for production estimates or studies in which accurate estimates of age are needed.