Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.     

Cryopreservation of sperm in common carp Cyprinus carpio: sperm motility and hatching success of embryos

In this study, fish sperm cryopreservation methods were elaborated upon for ex situ conservation of nine strains of Bohemian common carp. Common carp sperm were diluted in Kurokura medium and chilled to 4 degrees C and dimethyl sulfoxide was added. Cryotubes of sperm with media were then cooled from +4 to -9 degrees C at a rate of 4 degrees C min(-1) and then from -9 to -80 degrees C at a rate of 11 degrees C min(-1), held for 6 min at -80 degrees C, and finally transferred into liquid N(2). The spermatozoa were thawed in a water bath at 35 degrees C for 110 s and checked for fertilization yield, hatching yield of embryos, and larval malformations. Fresh and frozen/thawed sperm were evaluated for the percentage and for the velocity of motile sperm from video frames using image analysis. The percentage and velocity of sperm motility at 15 s after activation of frozen/thawed sperm was significantly lower than that of fresh sperm (nine males). ANOVA showed a significant influence of fresh vs frozen/thawed sperm on fertilization rate (P < 0.0001), but differences in hatching rate and in larval malformation (0-6.8%) were not significant, and different males had a significant influence on fertilization and hatching rate (P < 0.003 and P < 0.007, respectively). Multiple range analysis (LSD) showed significant differences between fresh and frozen/thawed sperm regarding fertilization rate (68 +/- 11 and 56 +/- 10%, respectively) and insignificant differences between fresh and frozen/thawed sperm on the hatching rate (50 +/- 18 and 52 +/- 9%, respectively). The percentage and velocity of fresh sperm motility were correlated, respectively, with the fertilization yield of frozen/thawed sperm at the levels r = 0.51 and r = 0.54.     

The effect of tannin concentration and egg unsticking time on the hatching success of tench Tinca tinca (L.) larvae

The influence of dose and time of eggs exposure in tannin solution on tench embryonic development, survival of embryos and percentage of larvae hatched were studied. The eggs obtained under controlled conditions from 11 tench females were fertilized with semen from 7 males and then rinsed for 1 h in Woynarovich solution (40 g urea, 30 g NaCl per 10 dm³ of water). Random samples (50 cm³) of the fertilised eggs were placed into three tannin solutions 0.05, 0.1 or 0.15% for 30, 60 or 90 s, then they were transferred to Weiss jars where incubation at 25°C took place. Tannin solution of 0.05% for 30–90 s or 0.1% for 30 s allowed effective unsticking of eggs and resulted in a very high per cent of hatched larvae. Extending the eggs rinsing time to 60 s in 0.10 and 0.15% solutions resulted in a high mortality of embryos during hatching because of hardened egg capsule.     

Egg incubation time and hatching success in tench Tinca tinca (L.) related to the procedure of egg stickiness elimination

The experiment showed different results after a short (2 min) enzyme alcalase Merck EC 3.4.21.14 (5.0 ml L^?1 concentration) treatment of tench eggs in contrast to the traditional methods of eliminating egg stickiness involving milk solution (50 g L^?1) treatment for 70 min followed by the addition of a talc suspension (33 g L^?1) for 10 min or treatment by fine clay suspension (20 g L^?1) for 60 min or talc suspension (33 g L^?1) for 80 min. The alcalase enzyme treatment resulted in decreased egg stickiness compared with the conventional milk/clay/talc treatments, indicated by lower duration of egg incubation and higher hatching rates (anova for hatching rate, P < 0.0084). The highest hatching rate (93.2%) was achieved using the enzyme; the lowest (31.3%) was using a talc suspension (control hatching rate was 86.2%). Duration of egg incubation at degree‐days (D°) after enzyme treatment (58.6 D°) was about 4–5 h shorter than the classical method using milk solution and talc suspension (63–65 D°). Prolongation in the latter classical method may also be explained by a hardening of the egg envelopes.     

Urinary bladder, ionic composition of seminal fluid and urine with characterization of sperm motility in tench (Tinca tinca L.)

A small urinary bladder attached to the seminal duct in caudal part of the abdominal cavity was registered for the first time in dissected males of tench. The urinary bladder wall was of whitish color and the bladder contained 0.5–2 ml of urine. When collected in the experiment, the tench sperm was white‐colored. Spermatozoa density is highly variable due to contamination by urine, and the latter additionally activates spontaneous motility of the spermatozoa. Seminal fluid contains ions such as Na⁺ (18.4 ± 1.3 mm ), K⁺ (1.9 ± 0.6 mm ), Ca²⁺ (0.6 ± 0.2 mm ) and Mg²⁺ (0.5 ± 0.1 mm ), leading to osmolality of 230 ± 82 mOsmol kg^?1 depending on the dilution by urine. Urea was detected in urine samples uncontaminated by sperm with an osmolality of 85 ± 58 mOsmol kg^?1. Urine also contained high concentrations of ions such as Na⁺ (30.9 ± 8.9 mm ), K⁺ (4.3 ± 2.9 mm ), Ca²⁺ (0.9 ± 0.5 mm ) and Mg²⁺ (0.6 ± 0.2 mm ). The spontaneous sperm activation by urine was up to 100%, but could be prevented by collection in an immobilizing solution. Motility was observed for 90–100% spermatozoa just after their transfer to distilled water or in a swimming medium (SM, 30–45 mm KCl) with a velocity of 120–140 μm s^?1. A flagellar beat frequency of 60–70 Hz and forward motility lasted up to 80 s in distilled water, and up to 180 s in SM at room temperature.     

The haematology of gynogenic tench, Tinca tinca L., and of recessively homozygous colour tench strains

Two wild‐coloured strains of tench (the first meiotic gynogenic generation MeiG₁, and their control diploid half siblings) and three recessively homozygous colour strains (golden, blue and alampic) were examined for the determination of basic haematological indices. The MeiG₁ strain had higher erythrocyte counts than diploid controls or the blue and alampic strains (P < 0.001), and had a higher blood haemoglobin content than all three colour strains (P < 0.001). No differences were detected among strains for haematocrit, mean corpuscular haemoglobin, or mean corpuscular volume. Both the lowest leucocyte count (P < 0.001) and leucocrit value (P < 0.001) were found in the alampic tench, and may result from a negative pleiotropic effect of this recessive homozygous genotype (bbgg). In agreement with previous findings in tench, the differential leucocyte count revealed lymphocytes to be the dominating white blood cells; their rate was about 90% in both the wild‐coloured and blue strains, and less in the other two strains (83–84%). Neutrophil granulocytes were most abundant in the MeiG₁ strain. Eosinophil granulocytes were detected only in the golden strain, and were not common (0.2%).     

A three-state MWC analysis of oxygenation in tench (Tinca tinca) hemoglobin

Summary Oxygen equilibria in tench hemoglobin were analysed according to a three-state MWC model. In addition to theT andR states of the traditionally used two-state model, the three-state model introduces an additional state, theS state, when organic phosphates bind to theT-structure hemoglobin. Under conditions covering natural red cell pH values and nucleoside triphosphate-hemoglobin ratios, it was possible to closely fit experimental data to the three-state equation with constant values of the association constantsK _R ,K _T , andK _S , and with only the allosteric constantsL andM varying with effector conditions. Thus, in contrast to a twostate analysis of oxygen equilibria, the three-state analysis was consistent with the basic assumption of the MWC model, that heterotropic ligands only affect allosteric constants and not association constants. The temperature-dependence of the three-state parameter values showed that in the presence of nucleoside triphosphate the dominance of theS state over theT state was most pronounced at low temperatures. Furthermore, the numerical values of the enthalpy and entropy change of oxygenation were lower in theS state than in theT andR states, and the enthalpy and entropy change for the allostericSR transition were much larger than for theTR transition.Abbreviations Hb hemoglobin - Y fractional O₂ saturation - ATP adenosine triphosphate     

Entrainment to light of circadian activity rhythms in tench (Tinca tinca)

The present article analyzes locomotor activity rhythms in Tinca tinca. To that end, three different experiments were conducted on 24 animals (20 g body weight) kept in pairs in 60-liter aquaria fitted with infrared sensors connected to a computer to continuously record fish movements. The first experiment was designed to study the endogenous circadian clock under free-running conditions [ultradian 40:40 min LD pulses and constant dark (DD)] and after shifting the LD cycle. Our results demonstrate that tench has a strictly nocturnal activity pattern, an endogenous rhythm being evident in 45.8% of the fish analyzed. The second experiment was conducted to test the influence of different photoperiods (LD 6:18, 12:12, 18:6, and 22:2) on locomotor activity, the results showing that even under an extremely long photoperiod, tench activity is restricted to dark hours. The third experiment examined the effect of light intensity on locomotor activity rhythms. When fish were exposed to decreasing light intensities (from 300:0 lux to 30:0, 3:0, and 0.3:0 lux) while maintaining a constant photoperiod (LD 12:12), the highest percentage of locomotor activity was in all cases associated with the hours of complete darkness (0 lux). In short, our results clearly show that (a) tench is a species with a strictly nocturnal behavior, and (b) daily activity rhythms gradually entrain after shifting the LD cycle and persist under free-running conditions, pointing to their circadian nature. However, light strongly influences activity rhythms, since (c) the length of the active phase is directly controlled by the photophase, and (d) strictly nocturnal behavior persists even under very dim light conditions (0.3 lux). The above findings deepen our knowledge of tench behavior, which may help to optimize the aquacultural management of this species, for example, by adjusting feeding strategies to their nocturnal behavior.     

Response of the tench (Tinca tinca L.) to potassium nitrate enriched water

When the tench ( Tinca tinca L.) was exposed to a slight increase, 8.5 mg/l, in the potassium content of the water, metabolic and hormonal changes occurred which lasted more than four weeks. An initial phase of lipolysis was followed by a partial consumption of glycogen reserves, which in turn was followed by a phase of gluconeogenesis. Hyperglycaemia persisted throughout the experiment. The ion distribution in erythrocytes and liver changed relatively early; there was no observable change in water content. These changes are similar to those observed during the osmoregulation of fish.     

Response of juvenile tench Tinca tinca (L.) to the anaesthetic 2-phenoxyethanol

The response of tench Tinca tinca aged 40–171 days post‐hatch (22–49 mm TL) to the anaesthetic 2‐phenoxyethanol was studied at 25°C. The lowest effective concentration of 2‐phenoxyethanol increased with age, while the highest safe concentration decreased. The fish aged 40 days post‐hatch required a significantly (P ≤ 0.05) shorter time to become anaesthetized than did older fish. The recovery time after 15 min of exposure to 2‐phenoxyethanol at 0.45 g dm^?3 was significantly shorter in the 40‐day‐old fish than in older fish. In juveniles of the same age, induction time or recovery time did not depend on their size or condition (Fulton's coefficient). At 25°C, 2‐phenoxyethanol at 0.5 g dm^?3 may be used to efficiently and safely anaesthetize T. tinca juveniles.     

Determination of substrate preferences of tench, Tinca tinca (L.), under controlled experimental conditions

The substrate preference of tench Tinca tinca (L.), 33 2‐year‐old individuals, mean size 10 ± 0.36 cm total length, was determined using four different substrates (concrete, artificial vegetation, sand and mud) in a 26 m long maquette. The species showed a high preference (100%) for the mud. Six more trials were performed combining the substrates by pairs (concrete–vegetation, concrete–mud, concrete–sand, mud–vegetation, mud–sand, sand–vegetation). Selection order was: mud (100% of the individuals when compared with concrete and sand and 98.26 ± 2.68% when compared with artificial vegetation), artificial vegetation (100% of the individuals when compared with concrete and 99.81 ± 0.76% when compared with sand), sand (98.29 ± 3.66% when compared with concrete) and concrete.     

Ultrastructure of spermatozoa of tench Tinca tinca observed by means of scanning and transmission electron microscopy

Structure of tench (Tinca tinca L.) spermatozoa was investigated by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spermatozoa of 26.1+/-3.8 microm total length possessed typical primitive simple structure, called "aqua sperm", without acrosomal head structures. It was probably the smallest spermatozoon described among cyprinid fishes. Heads were mostly composed of dense and slightly granular material, which appeared to be fairly homogeneous except for the occasional appearance of vacuoles. The midpiece remained separated from the flagellum by the cytoplasmic channel; it was cylindric/cone-shaped, 0.86+/-0.27 microm in length and 1.17+/-0.24 microm in width at proximal part. The proximal centriole was located in the "implantation fossa". The distal centriole appeared almost tangential to the nucleus and it functioned as a basal body for the flagellum. It had an orientation of 140 degrees with respect to the distal centriole. The sperm flagellum with 25.45+/-2.47 microm of total length had no any fin. The diameter of the flagellum perpendicular to the plane of the doublet of central microtubules was 173.67+/-20.45 nm and horizontal plane of the central microtubules was 200.71+/-20.45 nm. Peripheral doublets and the central doublet of microtubules measured 23.39+/-3.18 and 35.88+/-4.44 nm in width, respectively. The diameter of a microtubule was only 9.14+/-2.97 nm. A vesicle was attached to the most basal region of the flagellum and located just under plasma membrane of the flagellum.     

Influence of dietary composition on growth and energy reserves in tench (Tinca tinca)

The effects of different protein, lipid and carbohydrate diets on growth and energy storage in tench, Tinca tinca L., were studied. Over a 2-month period fish were fed four different diets: control, protein-enriched, carbohydrate-enriched and lipid-enriched. The best growth rates were obtained with the control and protein-enriched diets; the carbohydrate diet produced the worst results (lowest specific growth rate, weight gain, nutritional index and hepatosomatic index). These results suggest that it is not advisable to reduce dietary fish protein below 35%, and that it is not possible to obtain a protein-sparing effect of either lipids or carbohydrates, at least in our experimental conditions. The high-protein diet resulted in the storage of energy excess as muscle proteins and hepatic glycogen. Tench fed the high-carbohydrate diet stored carbohydrates as muscle glycogen and reduced plasma triglycerides. Finally, both liver and muscle lipid content were in positive correlation to dietary lipid.     

Successful gonadal development and maturation of tench (Tinca tinca L.) in small concrete ponds

The experiments were performed in a tench farm from autumn until the spawning season (June–July). Tench broodstocks from natural habitats were maintained in 25 × 6 × 1 m concrete ponds and fed on commercial trout pellets. Females and males were separated and maintained under natural photoperiod and temperature conditions at densities around 2 kg m^?2. Water flow throughout was supplied at the rate of 15 L s^?1. When females showed external signs of advanced gonadal development, induction of spawning was made by luteinizing hormone releasing factor (LH‐RH) synthetic analogue administration at three different periods of the reproductive season (June–July). A single intramuscular injection (20 μg kg^?1 body weight) was administered to 110 mature females selected from a total of 150. The females were stripped 42 h (22°C) after hormone administration. The mean rate of stripped females to the number injected was 77%. Mean relative egg weight in relation to the weight of the stripped females was 5.61%. More than 90% of the males provided semen without hormonal induction. Differences in egg production and external egg quality were observed at different times of the spawning period. It was proven that tench maintained in small concrete tanks and fed on artificial diets were able to reach gonadal maturation.     

PCR-RFLP assays to distinguish the Western and Eastern phylogroups in wild and cultured tench Tinca tinca

The tench Tinca tinca is a valued table fish native to Europe and Asia, but which is now widely distributed in many temperate freshwater regions of the world as the result of human-mediated translocations. Fish are currently being transplanted between watersheds without concern for genetic similarity to wild populations or local adaptation, and efficient phylogeographic markers are therefore urgently needed to rapidly distinguish genetically distinct geographical populations and to assess their contribution to the hatchery breeds and to the stocked wild populations. Here, we present a new method to distinguish recently discovered and morphologically undistinguishable Western and Eastern phylogroups of the tench. The method relies on PCR-RFLP assays of two independent nuclear-encoded exon-primed intron-crossing (EPIC) markers and of one mitochondrial DNA (mDNA) marker and allows the rapid identification of the Western and Eastern phylogroup and also of three geographical mtDNA clades within the Eastern phylogroup. Our method will enable researchers and fishery practitioners to rapidly distinguish genetically divergent geographical populations of the tench and will be useful for monitoring the introduction and human-mediated spread of the phylogroups in wild populations, for characterization of cultured strains and in breeding experiments.     

Age,growth and sex ratio of tench Tinca tinca (L., 1758) in Seyhan Dam Lake,Turkey

The study investigated the age, growth and sex ratio of Tinca tinca L., 1758 in Seyhan Dam Lake, Turkey and determined its first population structure data in the lake. Fishermen had introduced tinca into the lake between 2001 and 2002. A total of 1284 T. tinca (778 males and 506 females) were captured between January and December 2007 by gill nets of various mesh sizes. The sex ratio was 1 : 1.53 females to males. Total lengths and weights ranged from 12 to 29 cm and 27 to 403.3 g. Ages in both sexes ranged from 1 to 5 years. For all individuals the length‐weight relation and von Bertalanffy growth parameters were W = 0.063 × L^2.51 (r = 0.981), and L = 50.86 cm, k = 0.112 year^?1, t_o = ?1.551 year.     

Topographical relation between the olfactory bulb and the olfactory tract in tench (Tinca tinca L)

Electrical activity was recorded from single cells in the olfactorybulb when electrically stimulating the medial and lateral olfactorytract and when stimulating the olfactory epithelium with aminoacids. Bulbar units excited by stimulation of the medial olfactorytract were found in the medial and middle parts of the bulb.Neurones in the dorso-lateral part of the bulb were excitedby stimulation of lateral tract. Units inhibited by stimulationof the lateral or medial olfactory tracts had a reversed distributionwith the majority found in the medial or lateral parts of thebulb respectively. The chemicals tested induced changes in thedischarge of units mainly situated in the lateral part of thebulb.