New paper accepted in Reproduction in Domestic Animals

A new paper of our group was accepted for publication in the journal `Reproduction in Domestic Animals`.

Lujić J., Marinović Z., Kása E., Šćekić I., Urbányi B., Horváth Á. 2018. Preservation of common carp germ cells under hypothermic conditions: whole tissue vs isolated cells. Reproduction in Domestic Animals. 

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 h 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 h, isolated cells showed no decrease in viability while cells obtained from hypothermically stored tissues displayed significantly lower viability after only 6 h (Tukey’s HSD, p < 0.01) indicating that hypothermic storage of isolated cells is superior to storing tissue pieces. The two-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 different carp hatcheries and for the conservation of genetic resources of closely related cyprinid species.

New paper accepted in Fish Physiology and Biochemistry

A new paper in collaboration with our colleagues from the Angling Club of Tolmin (Slovenia) was accepted for publication in the journal `Fish Physiology and Biochemistry`.

Kása E., Lujić J., Marinović Z., Kollár T., Bernáth G., Bokor Z., Urbányi B., Lefler K., K., Jesenšek D., Horváth Á. 2018. Development of sperm vitrification protocols for two endangered salmonid species: the Adriatic grayling, Thymallus thymallus and the Marble trout, Salmo marmoratus. Fish Physiology and Biochemistry.

Abstract:
Vitrification was successfully applied to the sperm of two endangered fish species of Soča river basin in Slovenia, the Adriatic grayling (Thymallus thymallus) and marble trout (Salmo marmoratus). Sperm was collected, diluted in species-specific nonactivating media containing cryoprotectants and vitrified by plunging directly into liquid nitrogen without pre-cooling in its vapor. Progressive motility, curvilinear velocity and straightness of fresh and vitrified-warmed sperm were evaluated with computerassisted 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 (hatch after vitrification/warming: 13.1±11.7%, control hatch: 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.

New paper accepted in Fish Physiology and Biochemistry

A new paper in collaboration with our colleagues from University of Ljubljana, Slovenia, was accepted for publication in the journal `Fish Physiology and Biochemistry`.

Lujić J.,Marinović Z., Sušnik Bajec S., Djurdjevič I., Urbányi B., Horváth Á. 2018. Interspecific germ cell transplantation: A new light in the conservation of valuable Balkan trout genetic resources? Fish Physiology and Biochemistry

Abstract: Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhychus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings, however additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labelled brown trout and grayling germ cells were intraperitoneally transplanted into three to five days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation,fluorescently labelled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia - 27%; grayling spermatogonia - 28%; grayling oogonia - 23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell ransplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or pop ulations of salmonid fish and thus give rise to new or improved management strategies for such species.

New paper accepted in Science of the Total Environment

A new paper in collaboration with our colleagues from University of Novi Sad, Serbia (Department of Biology and Ecology, Faculty of Science) was accepted for publication in Science of the Total Environment. It was long and complex, but finally successful scientific mission. We would  like to thank to all coauthors, but especially to dr Nada Tokodi and dr Damjana Drobac for all their effort. 

Tokodi N., Drobac D., Meriluoto J., Lujić J., Marinović Z., Važić T., Nybom S., Simeunović J., Dulić T., Lazić G., Petrović T., Vuković-Gačić B., Sunjog K., Klarević S., Kračun-Kolarević M., Subakov-Simić G., Miljanović B., Codd G.A., Svirčev Z. 2018. Cyanobacterial effects in Lake Ludoš, Serbia - is preservation of a degraded aquatic ecosystem justified? Science of the Total Environment

Abstract - Cyanobacteria are present in many aquatic ecosystems 51 in Serbia. Lake Ludoš, a wetland area of international significance and an important habitat for waterbirds, has become the subject of intense research interest because of practically continuous blooming of cyanobacteria. Analyses of water samples indicated a deterioration of ecological condition and water quality, and the presence of toxin-producing cyanobacteria (the most abundant Limnothrix redekei, Pseudanabaena limnetica, Planktothrix agardhii and Microcystis spp.). Furthermore, microcystins were detected in plants and animals from the lake: in macrophyte rhizomes (Phragmites communis, Typha latifolia and Nymphaea elegans), and in the muscle, intestines, kidneys, gonads and gills of fish (Carassius gibelio). Moreover, histopathological deleterious effects (liver, kidney, gills and intestines) and DNA damage (liver and gills) were observed in fish. A potential treatment for the reduction of cyanobacterial populations employing hydrogen peroxide was tested during this study. The treatment was not effective in laboratory tests although further in-lake trials are needed to make final conclusions about the applicability of the method. Based on our observations of the cyanobacterial populations and cyanotoxins in the water and other aquatic organisms and, a survey of historical data on Lake Ludoš, it can be concluded that the lake is continuously in a poor ecological state. Conservation of the lake in order to protect the waterbirds (without urgent control of eutrophication) actually endangers them and the rest of the biota in this wetland habitat, and possibly other ecosystems. Thus, urgent measures for restoration are required, so that the preservation of this Ramsar site would be meaningful.

New paper accepted in Fish Physiology and Biochemistry

A new paper in collaboration with our collegues from the Polish Academy of Sciences was accepted for publication in Fish Physiology and Biochemistry, entitled 'Optimisation of sodium and potassium concentrations and pH in the artificial seminal plasma of common carp Cyprinus carpio L.'.


Beata Irena Cejko, Ákos Horváth, Tímea Kollár, Eszter Kása, Jelena Lujić, Zoran Marinović, Béla Urbányi, Radosław Kajetan Kowalski
DOI: https://link.springer.com/article/10.1007%2Fs10695-018-0491-3



Abstract
The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during shortterm storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n = 5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/ 110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg−1 . Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0–9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.

New paper accepted in The Journal of Visualized Experiments (JoVE)

A new paper in collaboration with our collegues from the Polytechnic University of Valencia was accepted for publication in The Journal of Visualized Experiments (JoVE), entitled 'Handling and treatment of male European eels (Anguilla anguilla) for hormonal maturation and sperm cryopreservation'

J.G. Herranz-Jusdado, E. Kása, T. Kollár, V. Gallego, D.S. Peñaranda, C. Rozenfeld, L. Pérez, Á. Horváth, J.F. Asturiano The Journal of Visualized Experiments (JoVE). In press.

Abstract
During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka´s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Video protocol for zebrafish testis vitrification

During the previous period, we have prepared a video protocol for the video journal JoVE https://www.jove.com/ regarding vitrification of zebrafish testis. This publication is supported by the COST Action FA 1205 AQUAGAMETE https://aquagamete.webs.upv.es/ It was a really nice and new experience for the whole team. 

New paper accepted in Cryobiology

A new paper with the authorship of Jelena Lujić was accepted for publication in Cryobiology, entitled `First successful vitrification of salmonid ovarian tissue`

LujićJ., Marinović Z., Sušnik Bajec S., Djurdjevič I., Kása E., Urbányi B., HorváthÁ. 2017. First successful vitrification of salmonid ovarian tissue.Cryobiology, in press. DOI: 10.1016/j.cryobiol.2017.04.005

Abstract

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 (Me2SO) and subsequently plunged into liquid nitrogen. Vitrification solutions containing equal cryoprotectant concentrations (3M Me2SO and 3M PG) yielded the highest oogonia survival rates (up to 40%) and qualitatively and quantitatively un-altered 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.

New paper accepted in Aquaculture

A new paper with the authorship of Ákos Horváth was accepted for publication in Aquaculture, entitled Cryobanking of aquatic species.

Sonia Martínez-Páramo, Ákos Horváth, Catherine Labbé, Tiantian Zhang, Vanesa Robles, Paz Herráez, Marc Suquet, Serean Adams, Ana Viveiros, Terrence R. Tiersch, Elsa Cabrita- 2017. Cryobanking of aquatic species. Aquaculture, Volume 472, 1 April 2017, Pages 156–177. http://doi.org/10.1016/j.aquaculture.2016.05.042
Abstract

This review is focused on the applications of genome cryobanking of aquatic species including freshwater and marine fish, as well as invertebrates. It also reviews the latest advances in cryobanking of model species, widely used by the scientific community worldwide, because of their applications in several fields. The state of the art of cryopreservation of different cellular types (sperm, oocytes, embryos, somatic cells and primordial germ cells or early spermatogonia) is discussed focusing on the advantages and disadvantages of each procedure according to different applications. A special review on the need of standardization of protocols has also been carried out. In summary, this comprehensive review provides information on the practical details of applications of genome cryobanking in a range of aquatic species worldwide, including the cryobanks established in Europe, USA, Brazil, Australia and New Zealand, the species and type of cells that constitute these banks and the utilization of the samples preserved.

New paper accepted in Reproduction in Domestic Animals

A new paper with the first authorship of Gergely Bernáth was accepted for publication in Reproduction in Domestic Animals:

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

The effective storage time of sperm after stripping (for 48 hours in 6 hours intervals) and after thawing (for 6 hours in 2 hours 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 hours 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±8µm/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 hours (19±11%) in comparison to 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.

New paper accepted in Zoological Studies

A new paper with the first authorship of Jelena was accepted for publication in Zoological Studies, entitled "Phylogeographic Identification of Tench Tinca tinca (L., 1758) (Actinopterygii: Cyprinidae) from the Northern Balkans and Adjacent Regions and its Implications for Conservation"


Abstract
The tench, Tinca tinca, is an endangered freshwater fish species in the Balkans. However, there are no management and conservation strategies developed for this species so far. In order to be able to develop such strategies, we first determined the phylogeographic identity of 70 tench individuals from four countries (Serbia, FYRO Macedonia, Hungary and Croatia) by PCR-RFLP analyses of two nuclear markers (Act and RpS7) and one mitochondrial marker (Cytb). All makers enabled the identification of two major geographic clades of tench (Western and Eastern), while nuclear markers additionally enabled the identification of hybrids between the two clades. Based on the mitochondrial marker Cytb, tench populations can be separated into two distinct areas: areas north of the Danube River with the dominant Western origin, and areas south of the Danube River with the dominant Eastern origin. Data obtained for the Act gene demonstrated Eastern origin for most individuals (88.23%) while data obtained for the RpS7 gene demonstrated mixed origin with a high percentage of hybrids. The presence of high numbers of individuals with Western alleles for the RpS7 gene in areas south of the Danube may indicate a natural invasion of this phylogroup. According to these results, areas north and south of the Danube are identified as two main management units. Additionally, we identified the rare western haplotype W2 based on the Cytb marker which clearly indicated human-aided dispersals of tench in the investigated region and since some individuals with W2 origin were cultured, attention must be given to the genetic structure and identity of the introduced individuals, whether during introduction or reintroduction since biological and ecological consequences of the hybridization between the two major clades are still unknown. Finally, we propose and discuss management and conservation strategies for tench of both management areas.