Yue Liu and Terrence R. Tiersch

Imagine waking up after being dormant or asleep for 42,000 years. It sounds like something from science fiction movies. However, this story turned out to be real in 2018 when worms that had been frozen for thousands of years under the ice of Siberia returned to life moving and feeding in petri dishes with the help of scientists, demonstrating the capability for long-term preservation of cells and organisms by cryopreservation.

Using similar principles of “cell time travel,” researchers at the LSU AgCenter Aquatic Germplasm and Genetic Resources Center are developing sperm banks using cryopreservation to assist in the conservation of aquatic species including endangered fishes. Such banks are also referred to as germplasm repositories and include: cryopreserved germplasm, such as sperm, eggs or embryos; a comprehensive database that contains information related to the germplasm donor, such as genetics, physical characteristics, quality of male and female cells, and habitat information; sample preparation methods; and inventory.

With appropriate freezing and thawing procedures, metabolic and molecular activities can be paused at cryogenic temperatures of minus 196 degrees Celsius (minus 320 degrees Fahrenheit) in liquid nitrogen for thousands of years and be revived with normal cellular functions. In humans, cryopreservation has been used to bank sperm and embryos and is a multi-billion-dollar industry to assist reproduction. In agriculture, sperm cryopreservation has been applied for decades for genetic improvement of livestock. For example, in the 1950s the LSU Dairy Improvement Center began to cryopreserve bull sperm containing specific genetic features relevant to dairy production. In 2015, the Aquatic Germplasm and Genetic Resources Center was started at the former Dairy Improvement Center and adopted approaches used with livestock to develop germplasm repositories to assist breeding and genetic improvement of economically important aquatic species in Louisiana, such as the eastern oyster, blue catfish and red snapper.

In addition to human reproduction and agriculture, germplasm repositories have been used to assist genetic management in conservation programs for endangered land animals, such as the giant panda, black-footed ferret and pheasants. Valuable genetic resources of imperiled animals can be preserved as cryopreserved germplasm that can be used in the future to produce live young for integration into conservation programs, such as habitat restoration, captive breeding and translocations. These activities are used to compensate for genetic losses or negative changes that would otherwise be permanent. For example, in the recovery program for the black-footed ferret, sperm were cryopreserved from six of the last 18 survivors, and 20 years later the offspring produced from thawed sperm were incorporated into the captive breeding program, enhancing genetic diversity and reducing inbreeding. In addition, cryopreservation of germplasm can be relatively safe, low-cost, fast and efficiently managed, allowing for easy transport and administration, especially compared to maintaining live animals.

The United States has about 250 threatened species of fishes listed by the International Union for Conservation of Nature. There is urgent need to save from extinction these and thousands of other imperiled fishes around the world. Unfortunately, little attention has been given to using germplasm repositories to assist conservation programs of imperiled fishes. For example, in about 86 current recovery plans of endangered or threatened fishes developed by the U.S. Fish and Wildlife Service, only two mention cryopreservation, and those are limited to research purposes. AgCenter researchers have recognized the technical and strategic difficulties impeding the use of germplasm repositories in fish conservation programs and are developing techniques, approaches and facilities to address these challenges.

In the AgCenter, fishes of the family Goodeidae (considered to be among the most imperiled fishes in the world) were used as models for developing germplasm repositories for imperiled live-bearing fishes. This is especially challenging because of their unusual reproductive traits, such as bundled sperm, internal fertilization and bearing live young. Standardized methods were established for the first time to evaluate quality-related attributes of sperm bundles. In collaboration with the LSU School of Veterinary Medicine, important characteristics of sperm within bundles were identified using techniques such as computer-assisted sperm analysis and fluorescent cell imaging (Figure 1). A generalized cryopreservation protocol was developed for live-bearing goodeids, and live young of the endangered redtail splitfin were produced with artificial insemination using cryopreserved sperm (Figure 2).

AgCenter researchers also proposed a strategy to integrate germplasm repositories into a comprehensive recovery program. In the genetic banking process, sperm of wild populations are collected, cryopreserved, stored and genetically characterized. Fish, testes and fresh sperm can be transported to well-equipped central facilities, such as the Aquatic Germplasm and Genetic Resources Center, followed by processing and freezing. Another option is on-site cryopreservation that can be performed by use of a mobile laboratory developed by the AgCenter, avoiding the loss of sperm quality during shipment. The frozen sperm and related database information are maintained within germplasm repositories and can be used for routine genetic enhancement and long-term backup or to address specific needs identified by genetic analysis. Should genetic diversity of wild populations decline in the future, previously stored genetic resources can be used through artificial insemination with thawed sperm. Offspring produced with thawed sperm can be used for future breeding purposes or be incorporated into wild populations to enhance genetic diversity.

Beyond technical expertise, one difficulty in integrating germplasm repositories with conservation programs is developing collaborations among diverse sectors and people with different objectives. An ideal conservation or recovery program for endangered fishes should include a comprehensive approach, combining major concerns such as habitat restoration; population propagation and maintenance by captive breeding or translocation projects; and preservation of genetic diversity by germplasm repository projects. Lack of any of these can render a conservation or recovery program ineffective. Comprehensive programs require strong collaborations. For example, ecologists and legislators are needed for habitat restoration projects; reproductive biologists and hatchery managers are needed for captive breeding; cryobiologists and data specialists are needed for establishing central germplasm repositories; and conservation geneticists and administrative agencies are needed for comprehensive planning and implementation. All of these activities require dedicated funding, but such programs can better offer a chance for the future maintenance of endangered fishes.

Yue Liu is a postdoctoral researcher and Terrence R. Tiersch is a professor in the Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources.

(This article appears in the fall 2018 issue of Louisiana Agriculture.)

Read the article, "Expanding cryopreservation to improve genetics in aquatic species," also in this issue.
Watch a 2-min video about the Aquatic Germplasm and Genetic Resources Center.

Postdoctoral researcher Yue Liu opens a tank of liquid nitrogen to remove a sample of fish genetic material at the LSU AgCenter Aquatic Germplasm and Genetic Resources Center. Photo by Olivia McClure

Fish, including endangered fish, used for research sit in tanks at the Aquatic Germplasm and Genetic Resources Center on the Baton Rouge campus. Photo by Olivia McClure

Figure 1. Sperm of live-bearing fishes are found in the male, packaged into bundles (electron microscope), at left. This challenges development of cryopreservation protocols. Cell imaging techniques were used to investigate ionic signals, indicated by fluorescent colors (fluorescent microscope), at right, of sperm cells within bundles. Photos by Yue Liu

Figure 2. Live young of the endangered live-bearing redtail splitfin produced by artificial insemination of cryopreserved sperm. The ribbon-like structures (trophotaenia) are special features of live-bearing fishes, which continue to maternal-fetal nutrient delivery and will disappear in a few days after birth. Photo by Yue Liu