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.

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.