The conservation of endangered fish is of critical importance. Cryobanking could provide an effective backup measure for use in conjunction with the conservation of natural populations; however, methodology for cryopreservation of fish eggs and embryos has not yet been developed. The present study established a methodology capable of deriving functional eggs and sperm from frozen type A spermatogonia (ASGs). Whole testes taken from rainbow trout were slowly frozen in a cryomedium, and the viability of ASGs within these testes did not decrease over a 728-d freezing period. Frozen-thawed ASGs that were intraperitoneally transplanted into sterile triploid hatchlings migrated toward, and were incorporated into, recipient genital ridges. Transplantability of ASGs did not decrease after as much as 939 d of cryopreservation. Nearly half of triploid recipients produced functional eggs or sperm derived from the frozen ASGs and displayed high fecundity. Fertilization of resultant gametes resulted in the successful production of normal, frozen ASG-derived offspring. Feasibility and simplicity of this methodology will call for an immediate application for real conservation of endangered wild salmonids.

The conservation of endangered fish is an urgent issue. Although cryo-banking of fish gametes might ultimately help conserve endangered fish, cryopreservation of fish eggs or embryos is still not possible due to their large size and high yolk content. Therefore, as an alternative, we focused on undifferentiated germ cells, such as primordial germ cells, spermatogonia, and oogonia, as materials for cryopreservation. Transplantation of cryopreserved germ cells into the body cavity of allogeneic or xenogeneic recipients sterilized by triploidization or endogenous germ cell ablation resulted in migration of the transplanted germ cells toward the recipient genital ridges, where they were eventually incorporated. The donor-derived germ cells initiated either spermatogenesis or oogenesis in the recipient gonads, depending on the sex of the recipient. Furthermore, by mating the male and female recipients, viable offspring derived from the frozen germ cells were produced. Although this technology was established using salmonid fish, we found that it is applicable to a wide range of fish species. Thus, this method might represent a "silver bullet" for preserving the valuable genetic resources of endangered fish species.

Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish.

The medaka (Oryzias latipes) is a teleost model distinguished from other model organisms by the presence of inbred strains, wild stocks, and related species. Cryopreservation guarantees preservation of these unique biological resources. However, because of their large size, cryopreservation techniques for their eggs and embryos have not been established. In the present study, we established a methodology to produce functional gametes from cryopreserved testicular cells (TCs). Whole testes taken from medaka were cryopreserved by vitrification. After thawing, the cells dissociated from cryopreserved testicular tissues were intraperitoneally transplanted into sterile triploid hatchlings. Some cells, presumably spermatogonial stem cells, migrated into the genital ridges of recipients and resulted in the production of eggs or sperm, based on sex of the recipient. Mating of recipients resulted in successful production of cryopreserved TC-derived offspring. We successfully produced individuals from the Kaga inbred line, an endangered wild population in Tokyo, and a sub-fertile mutant (wnt4b^−/−) from cryopreserved their TCs. This methodology facilitates semi-permanent preservation of various medaka strains.

Because of the lack of cryopreservation techniques for fish eggs and embryos, cryopreservation of fish spermatogonia and subsequent generation of eggs and sperm would be an exit strategy for the long-term preservation of genetic resources. This study aimed to optimize cryoprotectants, cooling rates, and thawing temperatures for slow freezing of spermatogonia from endangered Manchurian trout (Brachymystax lenok). Whole testes were frozen with a cryomedium containing 1.3 M methanol, 0.2 M trehalose, and 10% egg yolk at a cooling rate of -1 °C/min and then stored in liquid nitrogen for 2 days. After thawing at 30 °C in a water bath, testicular cells from thawed testes were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted spermatogonia migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency did not significantly differ between frozen and fresh testes, demonstrating that Manchurian trout spermatogonia can be successfully cryopreserved in liquid nitrogen.

Although sperm cryopreservation is a powerful tool widely applicable in biodiversity conservation and broodstock management, cryopreservation of teleost eggs and embryos remains challenging. In the present study, we demonstrated that spermatogonia of rainbow trout (Oncorhynchus mykiss) cryopreserved for 5 years possessed the ability to differentiate into functional eggs or sperm in the gonads of triploid recipient masu salmon (Oncorhynchus masou). After cryopreservation for 5 years in liquid nitrogen, intraperitoneally transplanted spermatogonia migrated toward, and incorporated into, the gonads of xenogeneic recipients. The transplanted spermatogonia resumed spermatogenesis and oogenesis in male and female recipients, respectively, and differentiated into sperm or eggs within the gonads of male and female recipients at 2 years posttransplantation. The differentiated sperm and eggs generated normal rainbow trout representative of donor phenotypes. Thus, cryopreservation of spermatogonia is a powerful and reliable method for long-term preservation of fish genetic resources.