Development of germplasm repositories to assist conservation of endangered fishes: Examples from small-bodied livebearing fishes

Abstract

Germplasm repositories are a necessary tool for comprehensive conservation programs to fully preserve valuable genetic resources of imperiled animals. Cryopreserved germplasm can be used in the future to produce live young for integration into other conservation projects, such as habitat restoration, captive breeding, and translocations; thus compensating for genetic losses or negative changes that would otherwise be permanent. Although hundreds of cryopreservation protocols for various aquatic species have been published, there are great difficulties in moving such research forward into applied conservation projects. Successful freezing of sperm in laboratories for research does not guarantee successful management and incorporation of genetic resources into conservation programs in reality. The goal of the present review is to provide insights and practical strategies to apply germplasm repositories as a real-world tool to assist conservation of imperiled aquatic species. Live-bearing (viviparous) fishes are used as models herein to help explain concepts because they are good examples for aquatic species in general, especially small-bodied fishes. Small live-bearing fishes are among the most at-risk fish groups in the world, and need urgent conservation attention. However, development of germplasm repositories for small live-bearing fishes is challenged by their unusual reproductive characteristics, such as formation of sperm bundles, initiation of spermatozoa motility in an isotonic environment, internal fertilization and gestation, and the bearing of live young. The development of germplasm repositories for goodeids and Xiphophorus species can provide examples for addressing these challenges. Germplasm repositories must contain multiple basic components, including frozen samples, genetic assessment and information systems. Standardization and process generalization are important strategies to help develop reliable and efficient repositories. An ideal conservation or recovery program for imperiled species should include a comprehensive approach, that combines major concerns such as habitat (by restoration projects), population propagation and maintenance (by captive breeding or translocation projects), and preservation of genetic diversity (by repository projects). In this context, strong collaboration among different sectors and people with different expertise is a key to the success of such comprehensive programs.

Keywords: Conservation; Endangered aquatic species; Germplasm repositories; Live-bearing fishes; Sperm cryopreservation.

Fig. 1.

Sperm bundles (spermatozeugmata) of live-bearing fishes. The organization of sperm bundles (scanning electron micrographs) are different between (A) goodeids (Xenotoca eiseni) and (B) poeciliids (Xiphophorus helleri). The bundle periphery is composed of spermatozoa tails in goodeids but with spermatozoa heads in poeciliids. This is an example of variation in shared traits arising from the independent evolution of viviparity. Intracellular signaling of spermatozoa within bundles of (C) X. eiseni and (D) Xiphophorus maculatus were evaluated with cell imaging techniques [80]. Using fluorescence microscopy, concentrations of intracellular Ca²⁺ were indicated with colors and quantified by use of a CCD camera and associated software. Spermatozoa within bundles were loaded with the dye Fura-2 AM and multiple regions of interest were selected within bundles. Each region of interest represented multiple spermatozoa. The levels of intercellular Ca2+ were evaluated by fluorescence intensity (green and yellow colorations indicate elevations in intracellular Ca2+ whereas blue coloration indicate baseline levels. The colorations were produced by a cell imaging program for imaging purposes, and thus were not equivalent to actual colors of fluorescent emissions).

Fig. 2.

Evaluation scheme for the five activation phases of spermatozoa within bundles by categorizing sperm bundles distributed in a viewing area (within the dashed circle) [62]. The dashed straight arrows indicate free-swimming spermatozoa released from a sperm bundle and the curved double-arrows indicate sperm vibrating in place but not swimming. In the dashed circle, there are ten sperm bundles including six at P₀, one at P₁, one at P₂, one at P₃, and one at P₄, thus the frequency of activation phases (FAPs) are estimated as 60% FAP₀, 10% FAPi, 10% FAP₂, 10% FAP₃, and 10% FAP₄. For demonstration purposes, the sizes of spermatozeugmata, spermatozoa, and viewing area do not reflect actual scale. The FAP can be used for evaluation of quality of spermatozoa within bundles [64].

Fig. 3.

Representative mechanisms for spermatozoa motility activation in fishes with different spawning strategies. Freshwater egg-laying (oviparous) fishes release sperm into hypotonic environments, which induce water influx and K⁺ efflux, leading to Ca²⁺ influx as an intracellular signal that triggers spermatozoa motility activation. Seawater egg-laying fishes release sperm into hypertonic environments, which induce water efflux, leading to increases in intracellular ions that trigger spermatozoa motility activation. Live-bearing fishes (such as goodeids and poeciliids) transfer sperm from males into females across isotonic environments.

Fig. 4.

Schematic of the major steps involved in the journey of male gametes from testis to ovary of internally fertilized fish in the Cyprinodontiformes [72]. Sperm bundles are intact and spermatozoa are immotile in testes. It is unclear whether spermatozoa within bundles acquire motility potential during their passage through the male reproductive tract. Bundles are transferred into females by copulatory organs, such as gonopodia (the intromittent type) of poeciliids or andropodia (the non-intromittent type) of goodeids. Upon the arrival within the female reproductive tract, the dissociation of bundles may result from physical pressure, or dissolution by elevated pH of presumptive substances that bind spermatozoa within bundles. The dissociation could also be triggered by activation of the spermatozoa within bundles. Activation of spermatozoa can be affected by osmotic pressure, ion concentration or pH. After activation, free spermatozoa need to travel through the tract before fertilizing oocytes in the ovary. However, sometimes there are no available oocytes to fertilize immediately. Thus, before fertilization free spermatozoa may persist for a short term (several d) as in goodeids or longer term (several months) as in poeciliids. The long motility duration (several d) of spermatozoa from internally fertilized species is likely required for efficient transit and fertilization.

Fig. 5.

Evaluation of genetic differences among seven Louisiana coastal management areas where four subpopulations (various colors) were identified using STRUCTURE software with a Bayesian model, whereas x-axis represents the seven locations sampled and the y-axis represents the proportional assignment of the four identified clusters.

Fig. 6.

Mating schemes for reconstitution of a breed or selected population using sperm in the repository with (A) a standard backcrossing plan and (B) an alternative plan [107, 110]. Each box represents a new generation of animal in the backcrossing scheme and the increasing proportion of the genome from the population being reconstituted.

Fig. 7.

A strategy to integrate germplasm repositories into a comprehensive recovery program. In the genetic banking process, sperm of wild populations (or offspring of wild broodstock) are collected, cryopreserved, stored, and genetically characterized. Fish, testes, or fresh sperm (diluted in buffer solutions) can be transported to well-equipped central facilities (e.g., AGGRC), followed by sample processing and freezing. If the distance is relatively close (within several hundred miles), on-site cryopreservation can be performed by use of mobile facilities, avoiding reduction of sperm quality caused by shipment. The frozen sperm and related database information are maintained within germplasm repositories, and can be used for routine genetic enhancement, 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 utilized by artificial insemination with thawed sperm. Offspring produced with thawed sperm can be used for breeding purpose or be incorporated into wild populations to enhance genetic diversity.

Fig. 8.

A representative strategy of application of research to production by optimization or generalization. A generalized development process can be repeated for a single species (e.g., 3 species) as initial research to establish cryopreservation protocols. These protocols can be refined for each species and used in production as a primary applied protocol. However, when new protocols are needed for multiple (e.g., 50) closely related species, it is not necessary to repeat the research pathway. In practice, a primary applied protocol can be chosen as a foundational protocol to be adopted as a secondary applied protocol in production. After repeated evaluation a universal foundational protocol for multiple new species can be developed. Eventually, sufficient knowledge can lead to a future universal applied protocol in production.

Fig. 9.

A diagrammatic example showing how innovation can diverge with differential application and later be converged into standardization. An innovation is usually diverged into modified methods by individuals within a research community based on different applications. It is important to integrate and converge the modified methods into a standardized approach at the community level to enable direct comparison of research results and to foster technology application such as in germplasm repositories.

Fig. 10.

An idealized comprehensive conservation or recovery program (indicated by the central star) is a combination of projects (grey circles), such as habitat restoration, captive breeding, and germplasm repositories. To achieve such programs, strong collaborations (white circles) are needed among people, agencies, and facilities with different specialized expertise and function.