Hungarian Molecular Life Sciences 2017

Jelena and Zoran have participated at the conference Hungarian Molecular Life Sciences 2017, organized by Hungarian Biochemical Society and Hungarian Genetical Society in Eger, Hungary, from 31/3/2017 – 2/4/2017.

The new results of our research group were presented by oral presentation:

Lujić J., Marinović Z., Sušnik-Bajec S., Yoshizaki G., Kása E., Urbányi B., Horváth Á. 2017. Germline stem cell cryopreservation and transplantation. [Abstract] Hungarian Molecular Life Sciences 2017, Hungary, 31.3 – 2.4.2017; Eger, pp. 91.

Congress hall 1

Congress hall 2 and poster section

Professional exhibition

Presentation

Questions at the end

Work in Slovenia and farewell to Nataša and Jovana

Between April 3rd-8th, we had our annual trip to Tolmin, Slovenia do conduct field work on grayling (Thymallus thymallus) sperm cryopreservation. As a reminder: the Angling Club of Tolmin uses cryopreserved sperm and molecular markers to increase the percentage of the autochthonous Adriatic genotype of the grayling in its waters. Sperm is taken and frozen from wild individuals during the spawning season while fin clips of the same fish are sent to Ljubljana for genetic analysis. Once the results of the genetic analysis arrive (2-3 weeks) people of the Angling Club can use the sperm of those individuals that have the highest percentage of the Adriatic genotype. Unfortunately, this year only one male individual was caught...
Also, we carried out isolation and cryopreservation of marble trout (Salmo marmoratus) and grayling testes and ovaries. The objective of these studies is to use these gonads later for isolation of spermatogonia and oogonia. These can then be transplanted into suitable recipients, in this case triploid rainbow trout larvae. At the moment we are waiting for a shipment of mixed-sex triploid rainbow trout embryos, thus, the gonads will have to stay frozen until these arrive.
Finally, after a one-month stay, we had to say goodbye to our two students from the University of Novi Sad, Nataša Cvetković and Jovana Lovren. They endured an incredibly tough one month of intensive training in cryopreservation, transplantation, histology and immuno-histochemistry in our lab and did a very good job. Thank you, Nataša and Jovana, and hope to see you soon in Hungary for new adventures!
We would also like to express our gratitude to Mr. Dušan Jesenšek, manager of aquaculture activities of the Angling Club of Tolmin for his help and guidance, as well as to Dr. Simona Sušnik-Bajec, Dr. Aleš Snoj and Ida Djurdjevič of the University of Ljubljana for their participation in the experiments.

Dissection of grayling juveniles for the isolation of gonads

Fish collection

Sample collection for genetic analysis and cryopreservation

The first grayling male

Sperm stripping

Cryopreservation on the field

Parallel experiments

Transplantation in Lillafüred vol. 2

On March 9-10th, we continued our transplantation work at the Lillafüred Trout Farm. As previously, spermatogonia and oogonia were isolated from rainbow trout and transplanted into newly hatched tiger trout recipients. What made this occasion special was that this time we enjoyed the company of two students from the University of Novi Sad in Serbia, Nataša Cvetković and Jovana Lovren. They participate in a training stay in Gödöllő and came with us to Lillafüred. Both were very enthusiastic to learn about trout and transplantation. As always, our gratitude goes to Mr. György Hoitsy, the manager of the Lillafüred Trout Farm whose hospitality was overwhelming.

Teamwork in the isolation of rainbow trout gonads

Jovana and...

...Nataša during transplantation

Carp spawning

Our common carp broodstock has finally matured. The first batch of eggs was stripped from the fish on March 7th. The eggs were fertilized with freshly stripped sperm and the first larvae hatched on March 10th. The broodstock is now ready for experiments – at least a year ahead of its sexual maturation in pond farms. It can be stimulated to spawn year-round and larvae can be produced for experiments whenever we want. Thanks to Balázs Csorbai for his assistance in the induced spawning.

Eggs are stripped from a female common carp of the broodstock

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.

Practical class in our fish system

Master students of our University had part of practical classes of the subject `Thesis preparation, experimental design and scientific writing` in our recirculating fish rearing system. 

Transplantation in Lillafüred

On February 15-16th, Jelena, Zoran and Ákos spent two days at the Lillafüred Trout Farm conducting experiments on the isolation of rainbow trout spermatogonia and their transplantation into tiger trout recipients. Tiger trout is a hybrid of brown trout (Salmo trutta m. fario) and brook trout (Salvelinus fontinalis) and is supposed to be sterile, making it an ideal recipient for transplantation experiments. The transplantation experiments went according to plans, spermatogonia were injected into 205 tiger trout larvae. We are grateful to the manager of the trout farm, Mr. György (Gyuri) Hoitsy and his son Márton (Marci) Hoitsy for their hospitality and assistance during the work and for rearing the fish for our experiments.

Dissection of rainbow trout for isolation of gonads

Obvious difference between ovaries (left) and testes (right) of juvenile rainbow trout

Team work: Zoran is checking viability of isolated cells while Jelena is preparing the next sample

Transplantation of spermatogonia into tiger trout recipients

And now it is Marci's turn to try transplantation

Tiger trout larvae in anesthesia

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.

Kick-off meeting of the new GOODFISH project

The kick-off meeting of the GINOP-2.3.2.-15-2016-00025 GOODFISH project was held in Gödöllő on February 6th, 2017 with the attendance of all Consortium Members as well as prospective subcontractors. Attendees discussed the scientific activities that lie ahead, issues related the the budget and consortium agreement. The project is an unprecedented collaborative effort by the leading aquaculture research and education institutions in Hungary and targets the complex development of the culture conditions and management of genetic resources of three autochthonous farmed fish species, the wels catfish, common carp and the pikeperch. For more details, please consult the Projects menu of this blog. We are looking forward to a successful collaboration with our partners!

Attendees of the meeting

New paper accepted in PLOS ONE

A new paper with the shared first authorship of Tímea Kollár was accepted for publication in PLOS ONE, entitled Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos.

Abstract

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.

Student recruitment trip to Novi Sad

On Monday, January 23rd, Eszti, Jelena, Zoran and Ákos visited Dr. Željko Popović at the Department of Biology and Ecology of the Faculty of Sciences at the University of Novi Sad in Serbia. The purpose of the visit was to meet with two candidates for the Hungarian state scholarship "Stipendium Hungaricum" and discuss with them the possibilities of pursuing their PhD in Hungary. It was agreed that the two candidates (Nataša Cvetković and Jovana Lovren) will visit our lab in Hungary in March for a one-month training in fish biotechnology. We enjoyed the wonderful hospitality of our Serbian colleagues and looking forward to a continued collaboration.

Meeting with the candidates at the Department of Biology and Ecology (from left to right: Dr. Željko Popović, Jelena, Nataša Cvetković, Jovana Lovren, Zoran, Eszti and Ákos)

Air spawning trial in Tolmin

Ákos has participated in a trial of air spawning of marble trout (Salmo marmoratus) at the fish farm of the Angling Club of Tolmin conducted by Dr. Radosław Kowalski and his team – Beata Cejko, Sylwia Judycka and Michał Blitek – from the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn, Poland on January 15-18th, 2017. Dr. Kowalski has applied and perfected this technology in many salmonid species previously and it worked fine on the marble trout, as well (see the video below). Air spawning is claimed to be less stressful for the fish than manual stripping, results in higher egg quality and more complete removal of eggs from the body cavity.
In addition to the air spawning trial, experiments were conducted on the cryopreservation of marble trout sperm. Information was exchanged by the Slovenian and Polish partners on the production of fish products such as trout caviar and trout pâté. Finally, we enjoyed the company of our Polish partners at our department in Gödöllő, too. We conducted experiments on the cryopreservation of common carp sperm which was also a good test of our carp broodstock for sexual maturity.
We would like to thank our partners in Slovenia and Poland for this truly international cooperation. We very much hope to continue this work in the future.

Radek preparing marble trout caviar while Robi from the Angling Club of Tolmin is taking notes

Happy customers tasting marble trout caviar

The team on the famous Napoleon bridge in Kobarid 

High expectations from cryopreserved common carp sperm in Gödöllő

Radek showing their well-equipped laboratory-van

our collegues from the Department of Aquaculture listening to Radek's presentation

sightseeing in Budapest (Michał, Eszti, Radek, Sylwia, Beata, Jelena, with the statue of the Hungarian poet Attila József)

Happy Holidays!

The end of the year is nearing. It has been a very exciting one for our group with successes, exciting travels, lots of work, nice publications but also sad losses. We are looking forward to the new year with hope for more interesting discoveries, meetings with fellow scientists and friends and new collaborations. We wish everyone Merry Christmas and a very Happy New Year of 2017!