The growth performance of pond-reared common carp (Cyprinus carpio) larvae propagated using cryopreserved sperm.

The aim of our study was to determine the efficacy of utilizing cryopreserved common carp sperm (in comparison to fresh sperm) for propagation at a Hungarian aquaculture facility. The sperm was frozen in 5 mL straws using an extender method that was previously tested in common carp. Sperm motility was monitored using a computer-assisted sperm analysis system. The hatching and malformation rates among the specimens were recorded before the stocking of larvae in both groups. The growth (body weight, total length) and survival rates of the fish were measured during the pre-nursing (from May to June: between 1 and 26 days post hatching) and grow-out periods (from June to October: between 26 and 105 days post hatching) of the same year. The fresh sperm, which was collected and pooled prior to fertilization, showed high MOT (97%), pMOT (92%), VCL (106 µm s-1), LIN (75%), and ALH (1.84 µm). Prior to the fertilization trial of the cryopreserved sperm, low MOT (34%), pMOT (14%), and VCL (61 µm s-1) values were observed in frozen-thawed sperm. A significantly higher hatching rate was measured in the fresh sperm group (87%) when compared to the cryopreserved sperm group (42%). No significant difference in the overall malformation rate was observed in larvae originating from either the fresh or frozen sperm. A significant difference between the two test groups was observed in the incidence of deformed tails (fresh: 20%, cryopreserved: 55%). Except for one sampling period, no significant difference in the body weight and total length of the fish larvae was found between the two groups throughout the pre-nursing and grow-out periods. A significantly higher larvae survival rate was noted in the fresh sperm (72%) as compared to the cryopreserved group (43%) by the end of the pre-nursing stage. However, no significant difference in survival rate was observed for the cryopreserved sperm (96%) in comparison to the fresh sperm (95%) by the end of the grow-out stage. The results of this study showed, for the first time in large-scale pond culturing, an equal growth and viability in larvae propagated from cryopreserved sperm when compared to fresh sperm (despite the limited available rearing ponds provided by the commercial company).

Black Soldier Fly (Hermetia illucens) Meal as Direct Replacement of Complex Fish Feed for Rainbow Trout (Oncorhynchus mykiss) and African Catfish (Clarias gariepinus).

In the experiments, defatted black soldier fly meal reared on vegetable byproducts was used in the fry rearing of two economically important fish species, African catfish and rainbow trout. Both fish species were reared in a recirculation system and 0-33-66-100% of the complex fry feed was replaced by a defatted prepupae meal of black soldier flies during a 28-day feeding experiment. African catfish was reared at 25 ± 1 °C while rainbow trout was reared at 12 ± 1 °C. The results showed that the growth of African catfish was not significantly reduced when 66% of the feed was replaced by soldier fly meal (mean weight in the control fish group at the end of the experiment was 0.4632 ± 0.2469 g, while the 66% group resulted mean weights of 0.4150 ± 0.1886 g) and the survival did not show any statistically different results (mean survival in control group was 57.48 ± 13.76% while it was 56.6 ± 7.763% in the 66% group). In the case of rainbow trout, replacing the feed entirely with insect meal did not cause a decrease in weight gain (final mean weight in the control group was measured at 1.9640 ± 0.4154 g, while in the group consuming only insect meal, it was 1.9410 ± 0.4248 g) or in survival (in the control group 98.5%, while in the group consuming only insect meal 99.5%). All these preliminary results indicate that black soldier fly meal can be used directly as a nursery feed in fish farming as a partial or total replacement of complete feeds. The results showed that black soldier fly meal could replace 66% of the complex brood feed of African catfish and up to 100% of rainbow trout feed without deterioration of production results. Our experiments have therefore opened the way for further experiments on insect meal in larval rearing.

The effect of two different experimental rearing temperatures on the quality and the large-scale cryopreservation of Eurasian perch (Perca fluviatilis) sperm.

Eurasian perch is an important fish species for European aquaculture diversification, but the quality of reproduction still remains one of the main limitations for further industry development. In particular, the optimal condition to obtain the best quality of sperm is poorly understood. The aim of our study was to measure the possible effects of two experimental rearing temperatures (6 °C and the conventionally used 12 °C) and of hormonal stimulation, on the motility parameters (pMOT, VCL, VSL, LIN, ALH, BCF), osmolality and fertilizing capacity of Eurasian perch sperm at the end of the reproductive cycle. A prior untested, large-scale (5 mL cryotube and Polystyrene box) cryopreservation method was implemented using fresh sperm obtained from the two above mentioned temperature groups. Males were injected with 100 µg body weight kg-1 sGnRHa. No significant difference was recorded between the two rearing temperatures and between the saline control and sGnRHa treated groups on the different features of sperm quality. A similar fertilization rate was monitored in all sGnRHa treated (6 °C: 69 ± 13%, 12 °C: 81 ± 11%) and saline control groups (6 °C: 79 ± 10%, 12 °C: 87 ± 4%). Correspondingly, no significant difference in hatching rate was observed in the sGnRHa injected (6 °C: 27 ± 9%, 12 °C: 40 ± 20%) and saline control (6 °C: 35 ± 18%, 12 °C: 36 ± 7%) males. However, a notable negative effect of freezing process on sperm movement was observed following thawing in both temperature groups. No significant difference in the motility parameters was measured between the two temperature groups following large-scale cryopreservation. Furthermore, a similar result was observed in the fertilizing capacity (6 °C: 79 ± 10%, 12 °C: 75 ± 8) of thawed sperm as well as in the hatching rate (6 °C: 52 ± 13%, 12 °C: 46 ± 19%). Our results indicate that fresh Eurasian perch sperm can tolerate a reduced rearing temperature following hormonal treatment. The adopted large-scale cryopreservation method could be used efficiently in the future for the fertilization of large amounts of Eurasian perch eggs following a precise standardization process.

Repeated hormonal induction of spermiation affects the stress but not the immune response in pikeperch (Sander lucioperca).

Hormonal induction of spermiation, previously reported to be immunogenic in fishes, is a common hatchery practice in pikeperch, Sander lucioperca. The aim of the present study was to investigate the effects of repeated induction of spermiation in pikeperch, following application of either human chorionic gonadotropin (hCG) or salmon gonadoliberine analogue (sGnRHa) on sperm quality indices as well as on immune and stress response. Mature males of pikeperch (n = 7 per group) were stimulated twice with five days between injections of either hCG (hCG; 500 IU kg-1), sGnRHa (sGnRHa; 50 µg kg-1) or NaCl (control group; 1 ml kg-1) to assess spermatozoa motility with a computer-assisted sperm analysis (CASA) system. During second sampling, blood plasma was sampled for humoral innate immune (peroxidase and lysozyme activities, ACH50), stress (cortisol, glucose) and endocrine (testosterone) markers. In addition, the head kidney was dissected to assay the expression of several immune genes (such as il1, c3, hamp, tnf-α and lys genes). The results indicate that hormonal treatment significantly increased sperm production. Sperm sampled after the hormonal treatment maintained its quality throughout the study, regardless of the sampling time. However, it appears that the application of hCG induced elevated cortisol and glucose plasma levels compared to the control group. Almost all immune markers, except the relative expression of hepcidin (hamp gene), were unaffected by the two hormones applied. The results showed that the induction treatment of spermiation processes in pikeperch resulted in an important physiological stress response for which the intensity varied according to the hormonal agent used. However, this stress response (more profound following application of hCG) was weakly associated with innate immune functions. On the other hand, a significant negative correlation between the expression of several important immune markers (peroxidase activity, relative expression of c3 and il1 genes) and sperm quality indices indicates significant involvement of immune status on sperm quality. The results obtained shed light on immune-system-induced modifications to sperm quality. The data presented here highlight the need for careful revision of broodstock management and selection practices where welfare status as well as individual predispositions of fish to cope with the stress should be taken under the consideration.

The comparison of two different extenders for the improvement of large-scale sperm cryopreservation in common carp (Cyprinus carpio).

In our study, a traditionally used (Grayling, already used in cyprinid species) and a newly tested (Pike) extender was tested to avoid sperm agglutination phenomenon following thawing during carp sperm cryopreservation. A large-scale (elevated volume of sperm) freezing method in a controlled-rate freezer using 5 ml straw and 10 ml cryotube was also systematically established. In all experiments, the sperm cryopreserved in using Grayling extender (except only one sample) showed an agglutination phenomenon (damaged and intact cells adhered to each other) after thawing where Pike extender resulted the regular cell suspension. No significant difference was observed between the two cryopreserved groups (Pike and Grayling extender) in all motility parameters using the 0.5 ml straw and the polystyrene box. Similarly, motility parameters did not show a significant difference in the two frozen groups with the 5 ml straw, also in the polystyrene box. A significantly higher progressive motility (pMOT, Grayling: 54% ± 8%, Pike: 37% ± 5%), straight line velocity (VSL, Grayling: 50 ± 5 µm/s, Pike: 39 ± 4 µm/s) and beat cross frequency (BCF, Grayling: 20 ± 1 Hz, Pike: 17 ± 1 Hz) was observed in the case of the grayling extender by the 5 ml straw cryopreserved in a controlled-rate freezer (CRF) compare to the pike extender. A significantly higher VSL (Grayling: 45 ± 3 µm/s, Pike: 38 ± 4 µm/s) was observed by the grayling extender using the 10 ml cryotube than with the pike extender. Despite the randomly occurring differences in a few parameters, our new controlled freezing method using the newly tested Pike extender, the 5 ml straw or the 10 ml cryotube can be a good solution for the preservation of elevated volume of carp sperm.

Effect of urine contamination on semen quality variables in Eurasian perch Perca fluviatilis L.

The objectives of the present study were to determine values for semen quality variables in the Eurasian perch (i.e., osmolality of seminal plasma as well as sperm motility characteristics analyzed with CASA system) in response to (1) the method of milt collection (stripping or catheterization) and (2) experimental contamination of catheterized semen with urine (0%, 5%, 10%, 20%, 30% and 50% of contamination). Additionally, the effect of short-term chilled storage of experimentally contaminated semen (during the 24 h post semen collection period) on motility characteristics was investigated. Use of a typical stripping procedure resulted in about 5%-10% contamination of semen with urine, what is much less compared with other species. Markedly lesser values of straight line velocity (VSL) and consequently less linearity of spermatozoa movement (LIN) in perch semen, however, occurred as a result of stripping (46 ± 4 µm/s and 38 ± 4% for VSL and LIN, respectively), when compared to sperm collected by catheterization (87 ± 5 µm/s and 77 ± 2% for VSL and LIN, respectively), indicate that even a 10% contamination of semen with urine may have negative effects on quality. Exposure of semen to urine resulted in a significant dose-dependent decrease in the percentage of motile spermatozoa (MOT) and both velocity variables (VSL and VCL). Amount of urine contamination also affected MOT, VCL, VSL and LIN value during short-term storage. In conclusion, it is important to avoid semen contamination by urine when using the stripping procedure in the Eurasian perch, either for controlled reproduction or sperm preservation.

Development of sperm vitrification protocols for two endangered salmonid species: the Adriatic grayling, Thymallus thymallus, and the marble trout, Salmo marmoratus.

Vitrification was applied to the sperm of two endangered fish species of Soca River basin in Slovenia, the Adriatic grayling (Thymallus thymallus) and marble trout (Salmo marmoratus) following testing different cooling devices and vitrifying media. Sperm was collected, diluted in species-specific non-activating media containing cryoprotectants, and vitrified by plunging directly into liquid nitrogen without pre-cooling. Progressive motility, curvilinear velocity, and straightness of fresh and vitrified-warmed sperm were evaluated with computer-assisted sperm analysis (CASA). Fertilization trials were carried out to test the effectiveness of vitrification in the case of grayling. A protocol utilizing a glucose-based extender, 30% cryoprotectants (15% methanol + 15% propylene glycol), 1:1 dilution ratio, and droplets of 2 µl on a Cryotop as cooling device yielded the highest post-thaw motility values for both Adriatic grayling (7.5 ± 6.5%) and marble trout (26.6 ± 15.8%). Viable embryos were produced by fertilizing eggs with vitrified grayling sperm (hatching 13.1 ± 11.7%, control hatching 73.9 ± 10.4%). The vitrification protocol developed in this study can be utilized in the conservation efforts for the two species as an alternative to slow-rate freezing when working in field conditions or when specific equipment necessary for slow-rate freezing is not available.

Effects of hCG and salmon gonadoliberine analogue on spermiation in the Eurasian perch (Perca fluviatilis).

This study analysed (i) the effect of human chorionic gonadotropin (hCG) and salmon gonadoliberine analogue (sGnRHa) on the effectiveness of induction of spermiation and (ii) the effect of latency time following the application of those spawning agents on the quantity and quality of the sperm of Eurasian perch, Perca fluviatilis, obtained during out-of-season spawning. For this study, pond-reared fish were used which had been acclimated to the controlled conditions. Three groups were distinguished which were treated with either saline (0.9% NaCl; control group), hCG (500 IU kg-1) or sGnRHa (100 µg kg-1). The fish were kept in a recirculating system at 12 °C throughout the study, during which sperm was collected every two days between the 2nd and 10th day following hormonal treatment. During the study, quantitative (e.g. sperm volume, total sperm production) and qualitative (measured with a computer-assisted sperm analysis system - i.e. CASA) parameters were monitored. The results of the study indicate that the hormonal treatment had a highly beneficial effect on the spermiation rate (100% in experimental groups from day 6 following injection) as well as quantity, which increased 50% in experimental groups (over 2200 × 109 of spermatozoa per kg of body weight) by day 4 following injection. For the sperm quality, both spawning agents tested had a rather positive effect, although sperm motility rate (MOT) was seen to be significantly reduced on day 10 following the application of hCG (MOT = 72.8% ± 8.1), which was not observed after the application of sGnRHa (minimum mean MOT 81.7% ± 6.1). The results clearly indicate that hormonal treatment had a positive effect on spermiation in Eurasian perch, most apparent from day 6 following injection, regardless of the hormonal agent used. Though application of sGnRHa allowed a high volume of high quality sperm to be stripped for two days longer (up to day 10 post-injection) compared to the application of hCG.

Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos.

BACKGROUND: Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. METHODS: In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. RESULTS: Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90-120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. CONCLUSIONS: Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development.

Cryosurvival of isolated testicular cells and testicular tissue of tench Tinca tinca and goldfish Carassius auratus following slow-rate freezing.

Experiments were carried out to test the efficiency of cryopreservation of whole testicular tissue in tench Tinca tinca and goldfish Carassius auratus and compare it to cryopreservation of isolated testicular cells. Additionally, effects of three cryoprotectants (dimethyl sulphoxyde - Me2SO, methanol - MeOH and ethylene glycol - EG) at three concentrations (1M, 2M and 3M) on post-thaw cell viability were assessed. Tissue pieces/isolated testicular cells were diluted in cryomedia and cryopreserved by slow-rate freezing (1°C/min to -80°C followed by a plunge into the liquid nitrogen). In both species Me2SO and EG generally yielded higher cryosurvival of early-stage germ cells than MeOH, while spermatozoa of neither species displayed such a pattern. In most cases a 3M>2M>1M viability pattern emerged in both species for both sample types regardless of the cryoprotectant used. Sample type (dissociated testicular cells vs testicular tissue) did not seem to affect viability rates of tench early-stage germ cells and goldfish spermatozoa, while the opposite was observed for tench spermatozoa and goldfish early-stage germ cells. Additionally, through histological analysis we displayed that tissue structure mainly remained unaltered after thawing in goldfish. These results indicate that cryopreservation of whole testicular tissue is indeed a valid alternative method to cryopreservation of dissociated testicular cells. Early-stage germ cells obtained from cryopreserved testis can be further used in different purposes such as transplantation into suitable donors while viable sperm might be used for fertilization when feasible.

Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla).

Vitrification was successfully applied to the sperm of two fish species, the freshwater Eurasian perch (Perca fluviatilis) and marine European eel (Anguilla anguilla). Sperm was collected, diluted in species-specific non-activating media and cryoprotectants and vitrified by plunging directly into liquid nitrogen without pre-cooling in its vapor. Progressive motility of fresh and vitrified-thawed sperm was evaluated with computer-assisted sperm analysis (CASA). Additional sperm quality parameters such as sperm head morphometry parameters (in case of European eel) and fertilizing capacity (in case of Eurasian perch) were carried out to test the effectiveness of vitrification. The vitrification method for Eurasian perch sperm resulting the highest post-thaw motility (14±1.6%) was as follows: 1:5 dilution ratio, Tanaka extender, 30% cryoprotectant (15% methanol+15% propylene-glycol), cooling device: Cryotop, 2µl droplets, and for European eel sperm: dilution ratio 1:1, with 40% cryoprotectant (20% MeOH and 20% PG), and 10% FBS, cooling device: Cryotop, with 2µl of sperm suspension. Viable embryos were produced by fertilization with vitrified Eurasian perch sperm (neurulation: 2.54±1.67%). According to the ASMA analysis, no significant decrease in head area and perimeter of vitrified European eel spermatozoa were found when compared to fresh spermatozoa.

Improvement of common carp (Cyprinus carpio) sperm cryopreservation using a programable freezer.

The applicability of a programmable freezer for the increased-scale cryopreservation of common carp sperm was investigated. The effect of different equilibration times, cryopreservation methods, extenders, dilution ratios, activating solutions on the post-thaw motility of common carp sperm was investigated. The suitable post-thaw storage time-interval as well as fertilizing capacity of cryopreserved sperm was also examined. The motility, curvilinear velocity (VCL) and straightness (STR) values did not decrease significantly during 60min of equilibration neither in equilibrated nor thawed groups. Motility parameters of thawed sperm were similar using a conventional cryopreservation technique using a polystyrene box [motility (33%), VCL (47µm/s) and STR (88%)] and a programmable freezer: [motility (32%), VCL (54µm/s) and STR (89%)]. The highest motility and VCL was measured with a sugar based extender (grayling extender) at a ratio 1:9 (motility: 52%, VCL: 76µm/s) and 1:20 (motility: 49%, VCL: 76µm/s). The activating solution for cyprinids (ASC) could prolong sperm movement up for 2min. A storage time of six hours following thawing did not have a significant effect on the motility parameters of thawed carp sperm. Agglutination was observed during cryopreservation of an elevated volume of sperm whereas motility 47%, VCL 62µm/s and STR 91% were measured after thawing. Fertilization rate with thawed sperm (32%) was significantly lower compared to the control group (73%). According to our results, the developed method using a programmable freezer is suitable for the cryopreservation of elevated number of straws. However, carp sperm agglutination during freezing may have a negative effect on the fertilizing capacity.