Introduction
The need for conservation of genetic resources of fish leads to a continuous development of methods in reproduction. Cryopreservation of gametes offers a solution for the long-term preservation of these resources. In fish, however, only sperm can be cryopreserved which puts the application of these methods in disadvantage. To overcome this problem, a set of methodologies have been developed for the cryopreservation and transplantation of germline stem cells (GSCs) such as spermatogonia and oogonia for the surrogate production of gametes (Yoshizaki et al., 2011). In the present work, we describe the results of 7 years of experiments on the cryopreservation and interspecific transplantation of salmonid GSCs.
Materials and Methods
The studies have been carried out in Slovenia and Hungary. Brown trout (Salmo trutta m. fario), grayling (Thymallus thymallus) and rainbow trout (Oncorhynchus mykiss) were used as donors of GSCs whereas the hybrid tiger trout (S. trutta m. fario × Salvelinus fontinalis) as well as diploid and triploid rainbow trout were used as recipients.
Brown trout ovarian tissue was vitrified in varying concentrations of methanol, dimethyl-sulfoxide (DMSO) and propylene glycol (PG). Isolated GSCs of brown trout and grayling were transplanted into diploid and triploid rainbow trout larvae whereas those of the rainbow trout were transplanted into larvae of the hybrid tiger trout. Colonization of the recipient gonads as well as incorporation of donor-derived GSC-s was verified using fluorescent microscopy, molecular biology and histology.
Results
Vitrification of salmonid ovarian tissue using needle-immersed vitrification (NIV) on acupuncture needles using 3 M DMSO and 3 M PG as cryoprotectants resulted in up to 40% survival of cells and qualitatively and quantitatively unaltered follicles (Lujić et al. 2017). Transplantation of brown trout and grayling GSCs into diploid rainbow trout recipients resulted in successful colonization of 26-28% of the recipient gonads. This was verified by fluorescent microscopy and the presence of donor-specific molecular markers 2 months post-transplantation (Lujić et al. 2018). In the trials using the hybrid tiger trout as a recipient, one male individual has reached sexual maturity 22 months post-transplantation and produced sperm that was used to fertilize rainbow trout eggs. The resulting progeny was verified to be 100% rainbow trout using sequencing of species-specific DNA fragments. On the other hand, hybridization resulted in very high mortalities (up to 95%).
Transplantation of brown trout and grayling spermatogonia into triploid rainbow trout recipients was conducted in the form of 3 independent trials in Slovenia and Hungary. The recipients failed to reach sexual maturity and were sacrificed 3 years post-transplantation. Their gonads were underdeveloped and typical of triploid fish. Brown trout- and grayling-specific fragments of the mitochondrial DNA control region, however, were amplified in all three trials in the gonads of several recipients.
Discussion
The results of our studies have shown the importance of finding the appropriate combination of donor and recipient in salmonids. It is clear that brown trout and grayling GSCs were capable of colonizing the gonads of rainbow trout recipients, however, in triploid individuals they remained in a dormant or semi-dormant state and did not undergo gametogenesis. Our findings are in contrast with those of Hattori et al. (2019) who were able to obtain donor-derived sperm and eggs from triploid rainbow trout recipients following transplantation of Atlantic salmon (Salmo salar) spermatogonia. The hybrid tiger trout was able to produce donor-derived sperm, however, its use as a recipient was compromised by the high mortality of the hybrids. In addition, some tiger trout males are known to produce sperm which further complicates their utility as recipients.
Acknowledgements
This research was supported by the Ministry of Innovation and Technology of Hungary within the framework of the Thematic Excellence Programme 2020, National Challenges Subprogramme (TKP2020-NKA-16). The research was further supported by the project NKFIH K138425.
References
Hattori, R.S., Yoshinaga, T.T., Katayama, N., Hattori-Ihara, S., Tsukamoto, R.Y., Takahashi, N.S., Tabata, Y.A., 2019. Surrogate production of Salmo salar oocytes and sperm in triploid Oncorhynchus mykiss by germ cell transplantation technology. Aquaculture 506, 238–245.
Lujić, J., Marinović, Z., Sušnik Bajec, S., Djurdjevič, I., Kása, E., Urbányi, B., Horváth, Á., 2017. First successful vitrification of salmonid ovarian tissue. Cryobiology 76, 154–157.
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 Physiol. Biochem. 44, 1487–1498.
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