First successful vitrification of salmonid ovarian tissue

Lujić, Jelena; Marinović, Zoran; Bajec, Simona Sušnik; Djurdjevič, Ida; Kása, Eszter; Urbányi, Béla; Horváth, Ákos

doi:10.1016/j.cryobiol.2017.04.005

Cited by 34 publications

(21 citation statements)

References 8 publications

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“…Furthermore attention should be given to the concentrations used as well. The present study and the study conducted on brown trout Salmo trutta ovaries 15 demonstrate that the best results are obtained by using the same concentration of two cryoprotectants, however this may vary in other species. Lastly, zebrafish testes are very small, and it is even possible to pin several testes on one needle.…”

Section: Protocolsupporting

confidence: 55%

“…Most of the previous studies regarding cryopreservation of zebrafish genetic resources were primarily focused on cryopreservation of zebrafish sperm 2,3,4 . However, protocols for cryopreservation of mature oocytes and embryos have not been developed yet, even though some recent studies demonstrate that cryopreservation of early-stage oocytes is plausible 14,15 . In this study we demonstrated successful cryopreservation of zebrafish spermatogonia which offers new possibilities for the storage of valuable genetic resources, especially since after transplantation they can mature into both male and female gametes 5 .…”

Section: Discussionmentioning

confidence: 99%

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Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Marinović

Lujić

Kása

et al. 2018

JoVE

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Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

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Section: Protocolsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Marinović

Lujić

Kása

et al. 2018

JoVE

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“…Vitrification as an ultra-fast cryopreservation method has been largely overlooked until recently. The studies of Lujić et al [26], Seki et al [27], Higaki et al [28], Marinović et al [29] pioneered in developing vitrification methods for fish germ cells as this method has several advantages over the traditional slow-rate freezing: (1) cost efficiency as it does not require special and costly equipment; (2) time-effectiveness as the sample preparation is quicker; (3) low volumes of liquid nitrogen are needed. As high cryoprotectant concentrations are needed to reach the amorphous glassy state of the tissue during vitrification, optimization of the tradeoff between the cryoprotectant combinations, their concentrations and attaining the highest possible cooling rates is of utmost importance.…”

Section: Introductionmentioning

confidence: 99%

Cryopreservation and transplantation of common carp spermatogonia

et al. 2019

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Common carp ( Cyprinus carpio ) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me 2 SO). In the next experiment, a wide range of cooling rates (0.5–10°C/min) and different concentrations of Me 2 SO were tested resulting in the highest survival achieved using 2 M Me 2 SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.

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“…Early stages of the germ line, specifically the oogonia and spermatogonia, are large, round cells found in the gonads of female and male fish, respectively. These cells have been successfully cryopreserved in various fish species including tench (Tinca tinca) 11 , tiger puffer (Takifugu rubripes) 12 , brown trout (Salmo trutta) 13 , rainbow trout (Oncorhynchus mykiss) 14 , manchurian trout (Brachymystax lenok) 15 , marine goby (Asterropteryx semipuctata) 16 , Siberian sturgeon (Acipenser baerii) 17 , sterlet (Acipenser rethenus) 18 and Chinese rosy bitterling (Rhodeus ocellatus ocellatus) 19 . Spermatogonia are a flexible target for biobanking compared to spermatozoa, as these cells can be collected from a fish at any life stage, without having to target adult fish during spawning periods.…”

mentioning

confidence: 99%

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis

Rivers

Daly

Jones³

et al. 2020

Sci Rep

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Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia’s Murray–Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 μm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through “A” (> 9 μm) and “B” gates (2–5 μm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the “B” gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for “large cell” (> 9 μm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants (p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.

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First successful vitrification of salmonid ovarian tissue

Cited by 34 publications

References 8 publications

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Cryopreservation and transplantation of common carp spermatogonia

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis

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