Induced diploid gynogenesis and polyploidy in the ornamental (koi) carp Cyprinus carpio L.

The results of a series of experiments conducted in our laboratory on the ornamental common carp (koi), aimed at optimizing heat-shock chromosome-set manipulation procedures, are described. The timing of heat-shock initiation was expressed in the relative unit of embryological age (₀) in order to standardize this parameter, the absolute time for heat-shock initiation being calculated from duration of one ₀ at two different pre-treatment water temperatures. Heat shocks were applied within the periods of 0.05–0.60 ₀ and 1.20–2.20 ₀ which, respectively, cover the successive phases of the 2nd meiotic division and the 1st cleavage. The highest production of diploid gynogenetic offspring was observed when heat shocks were initiated at 0.15–0.25 ₀ and at 1.5 ₀, after insemination, corresponding to anaphase of meiosis-II, and metaphase of the 1st cleavage, respectively. Similar results were obtained irrespective of the different pre-treatment water temperatures, thus confirming the possibility of standardizing heat-shock timing by ₀.     

Spontaneous diploidization of maternal chromosome set in ornamental (koi) carp, Cyprinus carpio L.

An unusually high frequency of spontaneous diploidization in a maternal chromosome set (SDM) was discovered in one ornamental carp (koi) female in an experiment on these fish for induced gynogenesis. Spontaneous appearance of diploid embryos in the gynogenetic offspring (intact eggs × irradiated sperm) and appearance of triploids among control fish (intact eggs × intact sperm) obtained from koi female and males of edible carp indicated spontaneous diploidization of maternal chromosomes (SDM). Possible cytological processes causing this phenomenon are discussed.     

Vascularization and related distribution of leucocytes in carp, Cyprinus carpio L., head kidney

The distribution of lymphoid cells in the carp head kidney was investigated in relation to the vascular system. Blood vessels in the head kidney were histologically identified into arteries, sinusoids and two types of veins: renal veins and portal, which were distinguished by India ink injection into the caudal vein. By histological and histoplanimetrical observations it was found that the head kidney contained a number of lymphoid cells, which mainly aggregate around the connections between the portal veins and sinusoids, and that the cellular density of the aggregations was higher than in the thymus.
Pigment-containing cell clusters were also observed around these connections. This arrangement of the blood vessels suggests that it is one of the structures able to trap foreign materials, and the occurrence of the lymphoid clusters around the portal veins is a phylogenetic sign of the morphological division between granulopoietic and lymphatic tissues in the carp head kidney.     

Sperm production and steroidogenesis in testes of the common carp, Cyprinus carpio L., at different stages of maturation

Testes from carp, Cyprinus carpio L., at five different maturational stages from immature through to spermiation and regression were incubated with or without addition of carp hypophysial homogenate (chh) for 8 or 20 h. Concentrations of steroids and spermatozoa were measured in the medium and the residual tissue examined histologically. There was an increase in the area of the germinal cysts containing spermatozoa, the percentage of the testis which they occupied and in the production of spermatozoa as the gonadosomatic index (GSI) increased, but this was unaffected either by incubation or by pretreatment with chh. The major steroid in plasma and in in vitro testicular cultures from all of the maturing fish captured in winter was 1 I-ketotestosterone. The production rate of this steroid in virro was unaffected by GSI, while plasma levels tended to increase with GSI. 17.20β-Dihydroxy-4-pregnen-3-one was detectable in significant amounts in only a few spermiating fish in summer, but was stimulated more in incubations with chh in maturing winter than in summer spermiating or post-spawning fish. 17,20a-Dihydroxy-4-pregnen-3-one was not detectable in incubations, but plasma concentrations tended to increase towards spermiation and were positively correlated with the size of the cyst. After spawning, fish had low plasma steroid levels and failed to respond in vitro to pituitary extract, indicating a testicular post-spawning refractoriness.     

Temperature induced metabolic costs in carp, Cyprinus carpio L., during warm and cold acclimatization

The oxygen consumption of 6 carps was measured in a flow-through respirometer when water temperature was reduced from 23 to 7°C and increased from 23 to 33°C as well as from 11 to 32°C. The metabolic reaction of Cyprinus carpio L. was assessed at 3 levels: standard, routine and spontaneous. The Standard Metabolic Rate increased and the Q₁₀ decreased with rising temperatures. A quadratic relation was established between metabolic rates and temperature level. The enery exenditure above Standard Metabolic Rate induced by temperature changes was 6.65 ± 1.97 kJ kg^-0.8 d^-1 (x ± S.E., N = 6).
Costs for temperature acclimatization amounted to 29 %, 24 % and 9 % of the energy required for standard, routine and spontaneous action metabolism, respectively.

Zusammenfassung

Temperaturinduzierte Stoffwechselerhöhungen bei Karpfen , Cyprinus carpio L., während Warm- und Kaltakklimatisierung
Wir haben untersucht, wie der Stoffwechsel von Cyprinus carpio im Standard-, Routine- und Spontanniveau während einer Kalt- und Warmakklimatisierung im Bereich von 7–33 °C reagiert. Dieses Verhalten wurde mit Hilfe eines Durchfluß-Respirometers ermittelt. Die "Standard Metabolic Rate" nahm zu und der Q₁₀ verringerte sich mit steigender Temeratur.
Eine quadratische Beziehung besteht zwischen den Stoffwechselraten und dem Temperaturniveau. Die durch Temperaturänderun induzierte Stoffwechselerhöhung über die Standardrate wurde für den Meßbereich mit 6.65 ± 1.97 kJ kg^-0.8 d^-1 (x ± S.E., N = 6) ermittelt.
Der Anteil temperaturinduzierter Aufwendungen entsprach 29%, 24% und 9% des Standard-, Routine- und Spontanverbrauches an Energie.     

A comparison of the pond culture of carp, Cyprinus carpio L., in Britain and mainland Europe

In recent years the requirement for carp in the U.K. has escalated, mainly due to the restocking market. This demand cannot be met from mainland Europe because of import restrictions and home production has increased. Freshwater coarse fish farming has been historically unimportant in Britain and few scientific data exist for pond culture under British conditions. In general, continental guidelines are followed but the growing-on ponds are smaller. The climate and the nature of the sites have often resulted in unreliable spawning. First year survival and growth tends to be poorer but growth rates in the second year are comparable. Carp for restocking ( c. 300 g) can be reared in 2 years and carp for the table ( c. 1000 g) can sometimes be produced in 3 years. Levels of production, however, compare favourably with more traditional carp culture, up to 480 kg ha⁻¹ per year using extensive cultivation and up to 2200 kg ha⁻¹ per year with intensive rearing and pelleted foods. The sites in Britain for which data exist have, however, been eutrophic in contrast to the more mesotrophic sites on the mainland. This apparent difference in base, line productivity may explain the relatively good production performance so far in Britain.     

Effects of temperature, oxygen and carbon dioxide on osmotic fragility of carp, Cyprinus carpio L., erythrocytes

The in vitro effects of gases and temperature on the osmotic fragility of carp erythrocytes were studied. At the three different temperatures analyzed (5, 11 and 20°C) there was no noticeable modification in erythrocyte membrane osmotic resistance. Osmotic fragility of red blood cells was altered by CO₂ and air treatment, as compared to the standard procedure. This suggests the need to take into account a possible moderate hypoxia that develops in the routine procedure of nucleated erythrocyte osmotic fragility tests.     

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.     

Respiration of the carp, Cyprinus carpio L., at 10 and 20° C and the effects of hypoxia

Oxygen consumption of carp acclimated at 10 and 20° C has been measured under routine conditions. Some complications and precautions necessary in continuous flow respirometry are discussed. Routine V o₂ at different levels of hypoxia have been determined. Individual variation leads to scatter in the data and different methods of plotting the relationship between V o₂ and P o₂ are proposed; attention is drawn to differences between inlet (or ambient) P o₂ and inspired P o₂. Using certain criteria a 'critical' oxygen tension of about 95 mm Hg was found at 20° C; Q ₁₀ values are about 2 at normoxia and some suggestions of an increase near to the critical oxygen tension were found. Blood samples from the dorsal aorta showed rising Pa,o₂ of 16 mm Hg which increased to 70–80 mm Hg when P _insp was 90 and they then fall as the inspired oxygen is lowered. During periods of deep hypoxia (25 mm Hg) blood lactate concentration increases steadily and indicates an increasing dependence on anaerobic mechanisms.     

An in situ estimate of daily food consumption and alimentary canal evacuation rates of common carp, Cyprinus carpio L.

Daily food consumption and alimentary canal evacuation rates for common carp, Cyprinus carpio L., were estimated at 4-week intervals from April to October 1979. Both the rate of food evacuation and its relationship to temperature were described by an exponential function. Gut evacuation rates ranged from 6.0% of gut contents per hour at 9°C to 32.7% h⁻¹ at 26.5°C. Temperature accounted for 72–91% of the variation in gut evacuation rate among months. Daily food consumption varied from a low of 3.87 mg g⁻¹ of fish wet wt per day in April at 14°C (0.39% of wet body wt) to a high of 40.75 mg g⁻¹ (4.08%) in August at 26.5°C. Although a greater quantity of food appeared to be consumed during the morning in spring and early summer and a greater amount in the evening after July, there was not a statistically significant time of day for peak feeding activity. The field method described here seemed to provide reasonable estimates of food consumption compared with other field and laboratory studies.     

Cellular effects of adrenosterone feeding to juvenile carp, Cyprinus carpio L., effect on liver, kidney, brain and muscle protein and nucleic acids

Different groups of juvenile common carp (3–4 g) were fed diets supplemented with adrenosterone at doses of 1.0, 2.5 and 10.0 mg kg^-1 for 60 days and the effect on food conversion efficiency and cellular growth responses of liver, kidney, brain and muscle were investigated. At the end of the hormone feeding phase an increase of 40–75% was observed in food conversion efficiency. No change in hepato-somatic and viscero-somatic index was observed, but brain and kidney weights in relation to body weight was decreased compared with the controls. Variable changes in protein, RNA/DNA, protein/RNA and protein/DNA were seen in liver, kidney, brain and muscle. Feeding of the steroid for 60 days increased the water content of the muscle but ash contents of the muscle were decreased. No change in the total muscle lipids and total nitrogen content were observed. After the removal of the drug from the feed, some of the changes reported were ameliorated.     

The effect of feeding 11-ketotestosterone on the food conversion efficiency and tissue protein and nucleic acid contents of juvenile carp, Cyprinus carpio L.

Juvenile common carp were fed 11-ketotestosterone for 60 days with the diet and the effect on food conversion efficiency, organ weights and protein and nucleic acids (RNA, DNA) content of the liver, kidney, brain and muscle were observed. Feeding of the steroid increased the food conversion efficiency of all the experimental groups studied as compared with controls. A decrease in weight of brain, liver and kidney in relation to body weight was noticed after 60 days of the hormone feeding. No change in the visceral weight was observed. These changes in relative weights of the organ were ameliorated 30 days after the withdrawal of the steroid from the food. The weight of the viscera decreased compared with the control weight during this time. Feeding of the hormone brought variable changes in the total proteins, RNA/DNA, protein/RNA and protein/DNA in all the organs studied. In addition to these findings, changes in the moisture, total lipid and ash contents of the muscle were also observed. The results are discussed in the light of existing knowledge of the effect of anabolic-androgenic steroids on the growth processes of different organisms.     

Analysis of expressed sequence tags (EST) obtained from common carp,Cyprinus carpio L., head kidney cells after stimulation by two mitogens,lipopolysaccharide and concanavalin-A

A representative cDNA library from mRNA obtained from lipopolysaccharide and concanavalin-A-induced head kidney cells of carp, Cyprinus carpio, was constructed. Two hundred single pass and partially sequenced clones (AU183343 to AU183542) were generated from expressed sequence tags (ESTs) and these were searched for homology in the DDBJ/GENBANK with blastN and blastX programs. Clones matching known genes were classified according to their function and distribution. One hundred and twenty-nine genes showed homology with known genes in databases, whereas 71 (35.5%) clones did not show any significant homology to sequences in the public database. Known genes also showed homology to fish genes deposited in the database. Twenty-two clones (11%), encoding 16 different sequences, were identified as putative biodefense and oncogenes, associated with an immune response. High expression of lysozyme (3%) was detected. Putatively identified biodefense-related sequences such as Lectin type 2, MHC class II invariant chain, mcl-1a and lysozyme were aligned with known homologues from the database and the percentage identity determined. A time course evaluation of gene expression due to mitogen stimulation by RT-PCR revealed the above mentioned gene homologues were switched on early during the cell proliferation.     

Comparative analysis of variability of three mitochondrial genes of cytochrome oxidase complex (cox1, cox2, and cox3) in wild and domestic carp (Cyprinus carpio L.)

For the first time, we studied the polymorphism of three mitochondrial genes of the cytochrome oxidase complex (cox1, cox2, and cox3) in natural populations of wild carp living in the Volga, Amur, and Don River Basins, as well as in European Hungarian carp and two pedigree lines of Ropsha carp of domestic breeding. The highest level of nucleotide and haplotype diversity in the studied samples was detected for the cox1 gene (?? = 0.61, h = 100%). Two lines of the Ropsha carp (?? = 0.61, h = 100%) and the Far East population of Amur wild carp from Shershikh strait (Am: ?? = 0.20, h = 70%) were the most polymorphic for three genes. The second sample of Amur wild carp from the Amur River (Ac), as well as the samples of Volga and Don wild carp and Hungarian carp had lower values of variability. The presence of two main genealogical lines of the wild carp and carp was demonstrated based on the total sequence of three genes, as well as the corresponding amino acid sequences in the studied area. One of these lines (line I) is typical of the sample of Amur wild carp (Am) and three members of the Ropsha carp. Line II is developed by sequences of Volga, Don, and Amur wild carp (Ac), as well as European Hungarian carp and seven other members of the Ropsha carp. Three to four sublines, which differ in nucleotide and amino acid substitutions, were found within the lines. Possible reasons for the origin of genomic variability in wild carp, as well as in European and Russian breeds of carp, are discussed.     

Investigation of long-term stress induced by several stressors by determination of the concentration of different blood plasma components in a model of Prussian carp (Carassius auratus gibelio BLOCH, 1783) and Common carp (Cyprinus carpio L., 1758)

Several compounds (carbohydrates, proteins, hormones, etc.) were used in fish to quantify the level of stress. Our investigations focused on two parameters of the blood plasma: plasma glucose and serum/plasma fructosamine (SeFa) that has not been tested on fish as yet. Experiments were conducted on two fish species. The concentrations of these components were investigated on Common carp (Cyprinus carpio L., 1758) and on Prussian carp (Carassius auratus gibelio BLOCH, 1783) from the G?d?ll?-Isaszeg pond system by creating conditions different from ideal. Stress effects caused a fluctuating tendency in blood plasma glucose levels each week for both Common carp and Prussian carp, thus, there was no steady growth. However, SeFa concentrations exactly followed stress effects, moreover, it tolerated short-term negative effects (handling of fish, blood sampling) and did not cause alterations at individuals blood samplings. This experimental method can offer assistance to farmers in the daily routine (e.g. in fish transport) and in the technology of propagation.