The investigation of post-thaw chilled storage and the applicability of large-scale cryopreservation in chub (Squalius cephalus) sperm.

Chub (reophillic cyprinids) is one of the most sensitive bioindicator fish of environmental changes following anthropogenic activities. The improvement of different biotechnological procedures could help support its conservation and strengthen the natural populations. The aim of this study was to compare the effects of two different hormonal agents (carp pituitary extract and Ovopel™) on various motility parameters (pMOT-%, DAP-µm, VCL µm s-1, VSL-µm s-1, LIN-%, ALH-µm, BCF-Hz) of fresh and cryopreserved/thawed sperm (stored at 4 °C for 6 h). Additionally, we sought to develop a novel, large-scale cryopreservation method for chub sperm, assessing freezing methods (Styrofoam box and a controlled-rate freezer) and different containers (0.5, 5 mL straw and 4 mL cryotube) for sperm cryopreservation. The results of this study indicated no difference between the carp pituitary extract and Ovopel treated groups in either the fresh or frozen/thawed sperm (at 0, 3, 6, hour post thawing, P = 0.4351). In contrast, the quality of the thawed chub sperm was negatively affected after 3 h chilled storage in both hormonal treatments (P = 0.0036, P < 0.0001). When assessing the motility parameters of the sperm between the 5 mL straw and 4 mL cryotube groups cryopreserved in a Styrofoam Box, no difference was observed (P = 0.103). Additionally, sperm loaded in 4 mL cryotubes showed no difference in motility when cryopreserved with either the Styrofoam box or controlled-rate freezer methods (P = 0.109). A similar hatching rate was observed in sperm preserved using the Styrofoam box (35 ± 7 %) and controlled rate freezer (25 ± 9 %) methods (P = 0.300). In a second fertilization trial, hatching rate was similar between control (72 ± 19 %) and cryopreserved (4 mL cryotube and Styrofoam box, 61 ± 5 %) groups. (P = 0.257). Based on our findings and its standard features (less species specific, precise dose calculation), Ovopel can be a good candidate for the stimulation of spermiation in chub sperm prior to cryopreservation. Furthermore, our study presents a novel and applicable method for the large-scale cryopreservation of chub sperm.

The systematic development and optimization of large-scale sperm cryopreservation in northern pike (Esox lucius).

In our study, a systematic development of a new large-scale sperm cryopreservation protocol was carried out in northern pike (Esox lucius). The effect of 2 sugar based (glucose and trehalose) extenders, 3 dilution ratios (1:3, 1:9 and 1:19) 2 vol straws (0.5 and 5 mL) and a 10 mL cryotube, 2 different cryopreservation methods (Polystyrene box-P. box and Controlled Rate Freezer-CRF), as well as 3 different thawing periods (3, 3.5 and 4 min) were investigated on the motility of thawed sperm. The glucose based extender showed significantly higher pMOT (1:3-18 ± 16%, 1:9-20 ± 13%, 1:19-16 ± 12%) at all dilution ratios than in the trehalose based extender (1:3-0.3 ± 1%, 1:9-1±1%, 1:19-4±2%). A similar tendency was recorded in VCL and STR at a ratio 1:3 and 1:9. No significant difference was measured in sperm movement between the P. box and CRF using the 0.5 mL straw. Similarly no significant difference was observed in all motility parameters with 10 mL cryotube frozen in CRF at a ratio 1:3-1:19. An effective and short thawing period (3 min) was experimentally specified for the 10 mL cryotube cryopreserved in the CRF. In all large-scale cryopreservation methods, high pMOT (straw CRF: 57 ± 10%, straw P. box: 50 ± 9%, cryotube CRF: 41 ± 10%), and STR were measured, and no significant difference was recorded in all motility parameters. Our results demonstrate the effectiveness of our newly developed extender and the applicability of 3 different large-scale cryopreservation methods in pike sperm. Our protocols could be new prospective candidates for future exploitation in hatchery practice.

The effects of different preservation methods on ide (Leuciscus idus) sperm and the longevity of sperm movement.

The present study investigated the effects of chilled storage and cryopreservation on ide sperm motility and fertilizing capacity alongside the longevity of sperm movement. The parameters of motility (progressive motility-pMOT, curvilinear velocity-VCL and straightness-STR) have been recorded during 48 h of chilled storage (4 °C) at 24-h intervals. The longevity of sperm movement was measured following activation for up to 120 s (in a range at 10-120 s) in freshly stripped and thawed sperm. A formerly established cryopreservation method was tested on ide sperm where motility parameters, hatching rate and larval malformation (according to 7 category groups) were investigated. Significant decrement of pMOT has already been observed after 24 h (6 ±â€¯5%) compared to the freshly stripped sperm (49 ±â€¯22%). pMOT and STR showed no significant changes for up to 120 s following activation in fresh sperm, whereas VCL showed significant difference between 10 (51 ±â€¯11 µm/s), 90 (33 ±â€¯3 µm/s) and 120 (31 ±â€¯4 µm/s) seconds as well as between 20 (48 ±â€¯12 µm/s), and 120 s. No negative effect of cryopreservation was recorded on pMOT (fresh: 49 ±â€¯19%, cryopreserved: 22 ±â€¯22%), VCL (fresh: 45 ±â€¯9 µm/s and cryopreserved: 57 ±â€¯5 µm/s), STR (fresh: 81 ±â€¯3% and cryopreserved: 92 ±â€¯1%) hatching rate (fresh: 22 ±â€¯15%, cryopreserved: 33 ±â€¯18%) or larval malformation (fresh: 12 ±â€¯4%, cryopreserved: 12 ±â€¯4%). No significant correlation was found between the three motility parameters and hatching rate. Cryopreservation had no effect on hatching and the prevalence of larval deformity. Furthermore craniofacial and eye deformities were characteristic in the group originating from fertilization with cryopreserved sperm, while edemas (pericardial, yolk) occurred more frequently in the control. The formerly developed cryopreservation protocol (method for cyprinids) was applicable to ide sperm.

Chilled and post-thaw storage of sperm in different goldfish types.

The effective storage time of sperm after stripping (for 48 hr in 6-hr intervals) and after thawing (for 6 hr in 2-hr intervals) in Black moor, Oranda and Calico goldfish types was investigated. Variations in sperm density were also measured in all lines. The efficiency of a sperm cryopreservation method formerly developed for common carp was recorded in all three goldfish lines. Motility parameters ((pMOT, %), curvilinear velocity (VCL, µm/s) and straightness (STR, %)) of Black moor sperm did not decrease significantly during 48 hr of storage. A significant reduction in the Oranda type compared to the fresh control was observed in pMOT after 42 (23 ± 2%) and VCL after 36 (94 ± 12 µm/s) hours (pMOT 84 ± 5%, VCL 150 ± 11 µm/s). In the Calico type, pMOT decreased significantly already after 18 (42 ± 26%) and VCL after 6 (105 ± 8 µm/s) hours (fresh: pMOT 92 ± 5%, VCL 151 ± 6 µm/s). A high pMOT immediately following thawing was measured in Oranda (46 ± 12%) and Calico (55 ± 15%) types, whereas a reduced pMOT was recorded in Black moor (24 ± 19%). In Calico, pMOT showed a significant reduction after 6 hr (19 ± 11%) in comparison with the initial value, with no changes observed in VCL and STR. None of the parameters changed in the Black moor and Oranda types. Evidence was found that different goldfish lines have different sperm quality and characteristics. Further studies can investigate the possible effects of chilled and post-thaw storage on the fertilizing capacity of sperm in the Black moor, Oranda and Calico goldfish types.

Commercial-scale out-of-season cryopreservation of Eurasian perch (Perca fluviatilis) sperm and its application for fertilization.

The quality and fertilizing capacity of perch (Perca fluviatilis) sperm collected outside of the spawning season (off-season) and cryopreserved at a commercial scale, were tested. Basic parameters (equilibration time, dilution ratio, sperm concentration, post-thaw motility duration) which can have a significant effect on cryopreservation success were systematically investigated for effects on sperm quality using computer assisted sperm analysis (CASA). No significant decrease in progressive motility (pMOT) and straightness (STR) of fresh-diluted sperm was recorded among groups equilibrated for 0, 30 or 60min in an extender with cryoprotectants. Curvilinear velocity (VCL) was reduced significantly after 30min (30min: 146±15µm/s, 60min: 124±18µm/s) of equilibration compared to the control (174±9µm/s). After thawing, no decrease in pMOT or VCL was observed at different equilibration times in any of the analyzed groups. No correlation was observed among progressive motility, dilution ratios (p=0.7) and cell concentrations (p=0.1). The use of different activating solutions resulted in similar pMOT and VCL in the first 120s post-thaw. Nevertheless, post-thaw sperm motility was reduced after 30s using all activators. Motility parameters with low variation were recorded after thawing of 57 straws (pMOT: 37±7%, VCL: 92±10µm/s, STR: 89±3%). Ten randomly selected straws from commercial-scale cryopreservation resulted in a high fertilization rate (cryopreserved sperm: 72±14%, fresh control: 94±2%). An optimized commercial-scale cryopreservation protocol was successfully developed for Eurasian perch. The applicability of the off-season collected perch sperm for cryopreservation and fertilization was demonstrated.

Comparison of two different methods in the cryopreservation of Eurasian perch (Perca fluviatilis) sperm.

Two different cryopreservation methods were compared and an optimal dilution ratio for the use of controlled-rate freezer (CRF) was established for Eurasian perch (Perca fluviatilis) sperm. Progressive motility (72 ± 15%) and curvilinear velocity (VCL, 146 ± 11 µm/s) of sperm cryopreserved with CRF did not reduce significantly compared to fresh sperm [progressive motility (90 ± 4%), VCL (173 ± 24 µm/s)]. On the other hand, progressive motility (62 ± 15%) and VCL (120 ± 21 µm/s) of sperm cryopreserved with the conventional floating frame technique were significantly lower when compared to the fresh control. Sperm in both cryopreserved groups showed significantly higher straightness [STR, CRF (84 ± 4%), frame (84 ± 2%)] than in the fresh control group (68 ± 4%). Perch sperm cryopreserved with CRF at a dilution ratio of 1:20 showed significantly higher progressive motility (49 ± 6%) than at a ratio of 1:5 (39 ± 6%) and showed significantly higher VCL (129 ± 11 µm/s) than at dilution ratios of 1:10 (112 ± 17 µm/s) and 1:5 (115 ± 9 µm/s).

Impact of environmental and genetic factors on the scale shape of zebrafish, Danio rerio (Hamilton 1822): a geometric morphometric study.

Intraspecific morphological variability may reflect either genetic divergence among groups of individuals or response of individuals to environmental circumstances within the frame of phenotypic plasticity. Several studies were able to discriminate wild fish populations based on their scale shape. Here we examine whether the variations in the scale shape in fish populations could be related to genetic or environmental factors, or to both of them. In the first experiment, two inbred lines of zebrafish, Danio rerio (Hamilton 1822) reared under identical environmental conditions were compared. Secondly, to find out what effect environmental factors might have, offsprings were divided into two groups and reared on different diets for 12 weeks. Potential recovery of scales from an environmental effect was also assessed. Experimental groups could successfully be distinguished according to the shape of scales in both experiments, and the results showed that both genetic and environmental factors may notably influence scale shape. It was concluded that scale shape analysis might be used as an explanatory tool to detect potential variability of environmental influences impacting genetically homogeneous groups of fish. However, due to its sensitivity to environmental heterogeneity, the applicability of this technique in identifying intraspecific stock membership of fish could be limited.

A note on the special fillet fatty acid composition of the dwarf carp (Cyprinus carpio carpio) living in thermal Lake Hévíz, Hungary.

Fatty acid (FA) composition of the fillet and the intestinal content of dwarf common carp (Cyprinus carpio carpio) living in Lake Hévíz was determined in wintertime collected samples and results were compared to widespread literature data on carp. Fillet FA profile of the thermally adapted (28 °C) Hévíz dwarf carps differed from profiles originated from divergent culture and feeding conditions in the overall level of saturation. Fillet myristic acid proportions largely exceeded all literature data in spite of poor dietary supply. Fillet fatty acid results indicate the effects of thermal adaptation (high saturation level) and the correlative effects of feed components rich in omega-3 fatty acids, with special respect to docosahexaenoic acid. With the application of discriminant factor analysis the Hévíz sample was accurately differentiated from the literature data on carp fillet fatty acid profile, mostly based on C14:0, C18:1 n9, C18:2 n6, C20:1 n9 and C20:4 n6 FAs. In summary, fillet FA profile suggested thermal adaptation, location specificity and the ingestion of algal and bacterial material.

In vivo tracking of maturation in male European eel, Anguilla anguilla (L.), by computed tomography.

The present study aimed in vivo tracking of maturation of male eel by computed tomography (CT). Additionally, individually monitored testes sizes were correlated with the conventionally used external maturity indicators (i.e. eye and nose indexes) in order to test and improve their usefulness at individual level. Testes could be clearly identified with the CT from the end of the third week of hCG administration routinely used to induce maturation in fish. The volume of testes increased exponentially during hormone treatment, and by the end of the sixth week of maturation procedure all males produced motilable spermatozoa. Present results prove that testes size can noninvasively be monitored with CT from maturity level where testes size rich 3000 mm3 volume. Eye and nose indexes are in close correlation with testes volume and thus can also be effectively used to monitor maturity level of male eel, but preferably only at stock level. However, due to their high individual variability, these indexes can be applied only with caution at individual level and should be supplemented with other noninvasive techniques such as CT.

Induced oogenesis of the European eel (Anguilla anguilla L.) in freshwater condition.

European eel is a catadromous fish species, which means that after living in freshwater premature individuals adapt to sea water, and migrate to the Sargasso Sea for spawning. Although male eel can be sexually matured even in freshwater, to date, it was believed that female eel can be matured only in seawater. Here we show that the process of sexual maturation may be induced in freshwater by treating female eels with carp pituitary (GSI = 9.87 ± 1.55%). It is thus proposed that seawater condition is not an obligatory environment for stimulating gametogenesis and for artificial maturation of the European eel in neither gender.

Investigation of potential stress parameters in rainbow trout (Oncorhynchus mykiss).

The transportation of rainbow trout in the presence of the anesthetic clove oil was investigated. Before the transportation tests, an acute experiment was conducted to verify that removal of the fish from the water for one minute does not significantly increase the glucose or cortisol concentration of the blood plasma. In the main experiment two different transportation conditions were compared: transport in water only and in water with anesthetic. During transportation without addition of clove oil, blood plasma glucose and cortisol concentrations changed significantly. The concentration of glucose increased from 4.92 mmol/L prior to transportation to 6.16 mmol/L and values similar to the initial ones (4.95 mmol/L) were observed 5 hours after transportation. Concentration of the stress hormone cortisol increased from the initial 37.2 ng/mL to 89.2 ng/mL and returned to a value of 36.1 ng/mL 3 hours post transportation. Respective values of glucose concentration have not changed significantly during transportation in the presence of clove oil (4.3; 4.4; 4.4 mmol/L), whereas those of cortisol showed a slight decrease with the passing of time (28.1; 26.7; 20.18 ng/mL). Results show that transportation stress can significantly be reduced by the use of anesthetics.

Cryopreservation of sperm of two European percid species, the pikeperch (Sander lucioperca) and the Volga pikeperch (S. volgensis).

Experiments were carried out on sperm cryopreservation of two European percid fish species, the pikeperch (Sander lucioperca) and the Volga pikeperch (S. volgensis). Two experiments were conducted on pikeperch sperm. In the first, the effects of three extenders (Glucose, KCl, Sucrose) and two cryoprotectants (dimethyl-sulfoxide: DMSO, methanol: MeOH) were tested on motility and fertilization. In the second, the effects of two dilution ratios (1 : 1, 1: 9) and two cryoprotectants (DMSO, MeOH) on hatching were investigated. In the experiment on Volga pikeperch the suitability of using cryopreservation for fertilization was investigated. In the first experiment on pikeperch the highest post-thaw motility (28 +/- 21%) and fertilization rate (43 +/- 12%) was found with DMSO as cryoprotectant in combination with Glucose extender. In the second, the highest hatch rate (41 +/- 22%) was observed with MeOH as cryoprotectant and 1 : 1 sperm dilution ratio, however no significant difference was found among the results. In the experiment on Volga pikeperch hatch rates with cryopreserved sperm (60 +/- 2%) did not significantly differ from the control (60 +/- 6%). Contamination of sperm with urine seems to be a key problem in the success of sperm cryopreservation of these species.

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 Gödöllö-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.

Cryopreservation of European eel (Anguilla anguilla) sperm using different extenders and cryoprotectants. Short communication.

Experiments were carried out on the sperm cryopreservation of artificially induced eels. The effects of several extenders and two cryoprotectants on the motility of spermatozoa were investigated. The highest post-thaw motility was observed with the combination of Tanaka's extender and DMSO as cryoprotectant. Further dilution after thawing resulted in complete loss of motility in samples frozen in presence of DMSO while sperm frozen with methanol as cryoprotectant retained its motility after further dilution.

Cryopreservation of spermatozoa in cyprinid fishes.

The present study investigated semen cryopreservation in cyprinid fish using computer-assisted sperm motility analysis for viability control. Spermatozoa of the bleak, Chalcalbumus chalcoides, were used as a basic model to describe the toxic and cryoprotective effects of internal and external cryoprotectants, their most effective concentrations and combinations, the freezing and thawing conditions, and the effects of equilibration. We also used these data to develop a cryopreservation protocol for Barbus barbus, Chondrostoma nasus, Ctenopharyngodon idella, Cyprinus cario, Hypohtalmichthys molitrix, Leuciscus cephalus, Rutilus meidingerii, and Vimba vimba. For all investigated species the optimal extender composition was a buffered physiological sperm motility-inhibiting saline solution containing 10% DMSO and 0.5% glycin. The optimal sperm equilibration period in the extender was < or = 5 min. Freezing was performed in an insulated box in liquid nitrogen vapor and it was optimal at 4 to 5 cm above the surface of the liquid, depending on the species. Thawing was optimal in a 25 degrees C water bath whereby the thawing time ranged depending on species from 15 to 45 sec. This cryopreservation protocol resulted in frozen-thawed semen with 35 to 65% motile and 5 to 25% locally motile spermatozoa depending on the quality of fresh semen.

Assessment of ovulation of common carp (Cyprinus carpio) females selected for induced spawning on the basis of external morphological characteristics.

Secondary sexual characteristics such as softening and rounding of the abdomen as well as reddening and protrusion of the anal papilla and vent can be of help to breeders in selecting common carp (Cyprinus carpio) females prepared for propagation. To assess the reliability of this method, long-term data obtained on induced spawning of common carp at a large-scale fish hatchery were evaluated. The average spawning ratio of 2,620 females receiving hormonal injections was 79.8%. The average pseudogonadosomatic index (PGSI) calculated from data on the egg production of 2,086 females was 16.3 +/- 5.87% (mean +/- SD) for the same period. There was a correlation between fish weight and the time of induction determined by the breeder on the basis of external morphological characteristics. The similarity of the responses of females, including both spawning ratio and PGSI, among the different weight categories proved the reliability of this method for identification.

[Spontaneous dissection of the coronary arteries: a rare cardiologic diagnosis]. / Spontandissektion der Koronararterien: Eine seltene kardiologische Diagnose.

A 55-year-old woman who was admitted to hospital with acute chest pain as a case of emergency suffered from an acute anteroseptal myocardial infarction. Four weeks later coronary angiography revealed a long dissection of the left anterior descending artery (LAD) as well as a significant stenosis of the left main and the proximal circumflex. Cardiovascular surgery was done subsequently. In addition to myocardial revascularization using coronary artery bypass grafts a readaptation of the dissecting artery walls and a proximal ligation of the LAD before anastomosis were performed. Clinical data, pathogenesis, and indications for medical and surgical treatment of spontaneous artery dissection are presented.

Chilling sensitivity of carp (Cyprinus carpio) embryos at different developmental stages in the presence or absence of cryoprotectants: work in progress.

The unsolved problem of cryopreservation of the yolk-rich teleost embryos may be related, in part, to their sensitivity to chilling and cryoprotective agents. The aim of this study was to gain data on the sensitivity of carp embryos to low temperatures at different developmental stages and on the possible protective and toxic effects of cryoprotectants. A total of 86,400 morulae, half-epiboly and heartbeat-stage embryos was selected and then placed in water or in 1 M methanol, dimethyl sulfoxide (Me2SO), glycerol or 0.1 M sucrose solution at 0, 4 or 24 degrees C for 5 min or 1 h. Following these treatments, the embryos were held in a 24 degrees C water bath until the evaluation of hatching rates. In every developmental stage a significant decrease of hatching rates following exposure to 4 or 0 degree C was detected. Sensitivity to chilling changed significantly with development (heartbeat < morula < half-epiboly). Half-epiboly stage embryos were less sensitive to a short period of exposure to cryoprotectants than morula and heartbeat stages. A 1-h exposure to cryoprotectants revealed a stage dependent sensitivity. Toxicity increased in the order of methanol < Me2SO < glycerol in morula and half-epiboly stages, and methanol < glycerol < Me2SO in the heartbeat stage. The results show morulae are partially protected against chilling in Me2SO and sucrose, half-epiboly in Me2SO, sucrose and methanol, and heartbeat-stage in methanol and glycerol. The results further suggest that carp embryos are sensitive to chilling and that toxicity and protective effects against chilling of cryoprotectants are stage-dependent. The finding on the low chilling sensitivity of heartbeat-stage embryos and the protective effect of certain cryoprotectants may be useful in designing cryopreservation protocols.