Cryopreservation of the Germplasm of Animals Used in Biological and Medical Research: Importance, Impact, Status, and Future Directions

Abstract

Molecular genetics and developmental biology have created thousands of new strains of laboratory animals, including rodents, Drosophila, and zebrafish. This process will accelerate. A decreasing fraction can be maintained as breeding colonies; hence, the others will be lost irretrievably unless their germplasm can be cryopreserved. Because of the increasingly critical role of cryopreservation, and because of wide differences in the success with which various forms of germplasm can be cryopreserved in various species, the National Institutes of Health National Center for Research Resources held a workshop on April 10-11, 2007, titled "Achieving High-Throughput Repositories for Biomedical Germplasm Preservation." The species of concern were mouse, rat, domestic swine, rhesus monkey, and zebrafish. Our review/commentary has several purposes. The first is to summarize the status of the cryopreservation of germplasm from these species as assessed in the workshop. The second is to discuss the nature of the major underlying problems when survivals are poor or highly variable and possible ways of addressing them. Third is to emphasize the importance of a balance between fundamental and applied research in the process. Finally, we assess and comment on the factors to be considered in transferring from a base of scientific information to maximally cost-effective processes for the preservation of this germplasm in repositories. With respect to the first purpose, we discuss the three methods of preservation in use: slow equilibrium freezing, rapid nonequilibrium vitrification, and the use of intracytoplasmic sperm injection to achieve fertilization with sperm rendered nonviable by other preservation treatments. With respect to the last purpose, we comment on and concur with the workshop's recommendations that cryopreservation largely be conducted by large, centralized repositories, and that both sperm (low front-end but high rederivation costs) and embryos (high front-end but modest rederivation costs) be preserved.

Generation of functional eggs and sperm from cryopreserved whole testes

“…Cryobanking allows for the long-term preservation (6) of fish species and their genetic diversity, and could therefore be a valid alternative to, or represent an enhancement of, captive breeding programs. Despite its potential applicability, cryopreservation methods for eggs and embryos have not previously been developed for any fish species (8). In the present study, it was demonstrated that ASGs taken from frozen testes possessed the ability to differentiate into oocytes when transplanted into female recipients, which suggested that at least some frozen ASGs possessed high levels of sexual plasticity.…”

“…Cryobanking of fish gametes to semipermanently (6) store genetic resources could serve as an alternative approach to traditional conservation methods (7); however, past attempts at cryopreservation of fish embryos and mature oocytes have been unsuccessful (8), as a result of their large size, high yolk content, and low membrane permeability (9,10). Although androgenesis performed by using frozen sperm and γ-ray-inactivated xenogenic eggs can regenerate live fish, their survival rate is extremely low, and resulting offspring become nuclear-cytoplasmic hybrids (11).…”

Toxicity of glycerol for the stallion spermatozoa: Effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential

“…In the near future, the probable introduction in the market of both refrigerated and frozen sexed sperm, stresses the need of further research in stallion sperm cryobiology. Is remarkable that procedures to freeze mammalian spermatozoa developed quasiempirically nearly 60 years ago are essentially used today [5][6][7]. Damage during cryopreservation occurs according to the two factors hypothesis [8], this theory states that cells cooled too rapidly are killed by intracellular ice formation, while cells cooled too slowly are killed by long exposure to concentrated solutions resulting from the progressive conversion of water to ice [9].…”

Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

“…In contrast, many animal species lack a practical germline storage form. For example, Drosophila needs to be maintained continuously in breeding populations because attempts to store them frozen in a permanent repository have met with only limited success (Steponkus et al 1990;Mazur et al 2008). In practice, long-term storage is unfeasible for large numbers of mutagenized lines.…”