Gergely Bernáth

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.

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.

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.

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 ± 5%) compared to the freshly stripped sperm (49 ± 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 ± 11 μm/s), 90 (33 ± 3 μm/s) and 120 (31 ± 4 μm/s) seconds as well as between 20 (48 ± 12 μm/s), and 120 s. No negative effect of cryopreservation was recorded on pMOT (fresh: 49 ± 19%, cryopreserved: 22 ± 22%), VCL (fresh: 45 ± 9 μm/s and cryopreserved: 57 ± 5 μm/s), STR (fresh: 81 ± 3% and cryopreserved: 92 ± 1%) hatching rate (fresh: 22 ± 15%, cryopreserved: 33 ± 18%) or larval malformation (fresh: 12 ± 4%, cryopreserved: 12 ± 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.

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.

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.

Collection of Gametes

Within this chapter, general information on the characteristics of Eurasian perch gametes is given. In addition, methods possible to apply for their collection for the purpose of controlled fertilization are briefly described. Practical advice on the recognition of moment of ovulation as well as sperm collection is given.

Harvest, Transport of Spawners, Prophylaxis

Within this chapter the theoretical background, as well as detailed practical advices of collection of the spawners of Eurasian perch from lakes and from earthen ponds are discussed. A special attention is paid to the time of collection and additionally, the methods of transportation as well as basic prophylactic methods are described.

Short- and Long-Term Storage of Gametes

This section characterizes the importance of short and long-term storage of gametes as well as the theoretical background of successful storage of eggs and sperm. Considering eggs, a brief explanation on the effect of storage time on egg quality was described which turned into practical recommendations in this aspect. A detailed protocol of short-term sperm storage as well as a commercially applicable cryopreservation protocol are given.