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Abstract: The aim of this study was to optimize the conditions for hypothermic storage of spermatogonial stem cells (SSCs) and oogonial stem cells (OSCs) of common carp Cyprinus carpio. This was conducted by storing gonadal tissue or isolated cells for 24 hr under hypothermic conditions in the first experiment and by testing two different storage media (L‐15 or DMEM supplemented with 10% FBS and 25 mM HEPES) and regular medium change (every 4 days) during two weeks of hypothermic storage in the second experiment. During the first 24 hr, isolated cells showed no decrease in viability, while cells obtained from hypothermically stored tissues displayed significantly lower viability after only 6 hr (Tukey's HSD, p < 0.01) indicating that hypothermic storage of isolated cells is superior to storing tissue pieces. The 2‐week trial demonstrated that storage media have a profound influence, while regular medium exchange does not have a positive effect on cell viability. Viability of SSCs and OSCs after two weeks was approximately 40% and 25%, respectively; however, survival of ~70% was obtained after 10 days of storage for SSCs and 7 days for OSCs. Hypothermic storage developed in this study has many practical applications during the development of surrogate broodstock technologies for common carp, but also in carp hatcheries and for the conservation of genetic resources of closely related cyprinid species. [ABSTRACT FROM AUTHOR]

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, 2 μl 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 2 μl 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. [ABSTRACT FROM AUTHOR]

The kinetics of dissolution of kaolinite in industrial spent liquor followed by the precipitation of the desilication product was studied by using calcined and dimethyl sulfoxide intercalated Eckalite1 as starting material. These studies were aimed at understanding the influence of the combination of different pre-treatments on the variations in the maximum silica concentrations and shift (or in some cases, the disappearance) of the trigger point in the Bayer desilication process. In addition to the quantitative characterization of the dissolution kinetics, a variety of physico-chemical parameters of the kaolinites, like the specific surface area, Lewis acidity, hydrodynamic diameter, zeta potential, optical band gap and thermogravimetric behaviour was also determined. From the experimental data, it can be concluded that the thermal/mechanochemical dehydroxylation and the formation of metakaolinite/mechanochemically amorphized kaolinite are responsible for the increase in the concentration of the dissolved silica in the supersaturated sodium aluminate solution. The variation in the position of the trigger point could be connected to the deformation, fragmentation/delamination, and amorphization of the kaolinite structure evolved during the mechanochemical pre-treatment. The joined application of pre-calcination and pre-milling verified the conclusions drawn. In addition, the combined treatment can be optimized to end up with a dissolution process that is carried out in a time and energy saving manner. The extent of the desilication products obtained at the end of the dissolution tests showed a considerable dependence on the applied pre-treatments: the formation of the metakaolinite phases was found to facilitate the evolution of the ball of yarn-type lepispherical structures with sizes ranging from 600 to 3000 nm. [Display omitted] • Dissolution behaviour of treated kaolinites in spent aluminate liquors was studied. • Heat and mechanical treatments of the solids alone and in combination were employed. • Intercalation induced delamination of the layers was achieved by DMSO incorporation. • Due to dehydroxylation, growth of maximum dissolved Si-concentration was observed. • Delamination/fragmentation/amorphization resulted in shift of the trigger points. [ABSTRACT FROM AUTHOR]

Using industrial spent Bayer-liquor solution and 95 °C temperature, the dissolution and desilication products formation kinetics of five raw kaolinite samples obtained from different sources were found to display profound differences. It was hypothesized that these differences were connected to the physico-chemical properties of the solids. The solids were characterized by powder X-ray diffractometry, N 2 adsorption-desorption isotherms, temperature-programmed NH 3 desorption, infrared and ultraviolet–visible diffuse reflectance spectroscopies, scanning electron microscopy connected with energy dispersive X-ray analysis, dynamic light scattering and thermal analysis. The detailed structural study of the as-received samples was supplemented by exploring the effect of mechanochemical treatment. It was found that beside specific surface area and pore volume, the amount of the Lewis acid sites proved to be the most important factors, and the picture was found to be modulated by the degree of aggregation of the primary particles. On mechanochemical treatment (60 min milling in a mixer mill), the number of the acid sites increased significantly (from 0.004–0.013 to 0.055–0.066 mmol/g), and largely as a result of this, the dissolution of all raw kaolinites became practically instantaneous and source-independent. [Display omitted] • Dissolution of kaolinite was studied in hot and highly alkaline aluminate solution. • Dissolution behavior of kaolinites from five different sources was compared. • Physico-chemical data of the initial, milled and calcined solids were determined. • Increased dissolved silica concentration is due to the formation of metakaolinite. • The number of Lewis-acid centres plays important role in the kaolinite dissolution. [ABSTRACT FROM AUTHOR]

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 60 min 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 (47 μm/s) and STR (88%)] and a programmable freezer: [motility (32%), VCL (54 μm/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: 76 μm/s) and 1:20 (motility: 49%, VCL: 76 μm/s). The activating solution for cyprinids (ASC) could prolong sperm movement up for 2 min. 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 62 μm/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. [ABSTRACT FROM AUTHOR]

The objective of our study was to investigate the effects of heavy metals on the fertilizing capacity of exposed zebrafish sperm, on embryonic survival, and on occurrence of embryonic deformities following fertilization with exposed sperm. It is important to test heavy metals because they are well-known pollutants. Sperm of externally fertilizing species can get in contact with pollutants found in aquatic environment. Zebrafish sperm, despite its advantages, has seldom been used in in vitro toxicological studies and no reports are available regarding the fertilizing capacity of exposed sperm. Zebrafish sperm was stripped and exposed to concentrations of the tested heavy metals (Zn2+, Cd2+, Cr3+, Cu2+, Ni2+, Hg2+, As3+) for 30 or 120 minutes. Calculated half-maximal effective concentration (EC50) values do not differ significantly from those calculated for motility for any of the tested heavy metals, which means fertilization rate can indicate the toxicity of the given substance following exposure of sperm. Thus, its application as in vitro toxicological end point is reasonable. The survival of embryos and embryonic development have not been affected by the exposure of spermatozoa, which means all alterations in spermatozoa caused by heavy metals have been expressed before 24 hours post fertilization. [ABSTRACT FROM AUTHOR]

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 (1 M, 2 M and 3 M) 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 3 M > 2 M > 1 M 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. [ABSTRACT FROM AUTHOR]

Due to a lack of cryopreservation protocols for fish eggs and embryos, alternative techniques which will enable storage of female genetic resources are crucial for future development of reproduction management in conservation biology and aquaculture. Experiments were conducted to develop an optimal vitrification protocol for cryopreservation of brown trout Salmo trutta juvenile ovarian tissue. Needle immersed vitrification (NIV) method was used where ovaries were pinned on an acupuncture needle, passaged through equilibration and vitrification solutions containing different combinations and concentrations of methanol (MeOH), propylene glycol (PG) and dimethyl sulfoxide (Me 2 SO) and subsequently plunged into liquid nitrogen. Vitrification solutions containing equal cryoprotectant concentrations (3M Me 2 SO and 3M PG) yielded the highest oogonia survival rates (up to 40%) and qualitatively and quantitatively unaltered perinucleolar follicles. The method developed for brown trout could be applied to the conservation of female genetic resources of other salmonid species, including endangered and endemic species or populations. [ABSTRACT FROM AUTHOR]