Ecological Civilisation and Amphibian Sustainability through Reproduction Biotechnologies, Biobanking, and Conservation Breeding Programs (RBCs).

Intergenerational justice entitles the maximum retention of Earth's biodiversity. The 2022 United Nations COP 15, "Ecological Civilisation: Building a Shared Future for All Life on Earth", is committed to protecting 30% of Earth's terrestrial environments and, through COP 28, to mitigate the effects of the climate catastrophe on the biosphere. We focused this review on three core themes: the need and potential of reproduction biotechnologies, biobanks, and conservation breeding programs (RBCs) to satisfy sustainability goals; the technical state and current application of RBCs; and how to achieve the future potentials of RBCs in a rapidly evolving environmental and cultural landscape. RBCs include the hormonal stimulation of reproduction, the collection and storage of sperm and oocytes, and artificial fertilisation. Emerging technologies promise the perpetuation of species solely from biobanked biomaterials stored for perpetuity. Despite significant global declines and extinctions of amphibians, and predictions of a disastrous future for most biodiversity, practical support for amphibian RBCs remains limited mainly to a few limited projects in wealthy Western countries. We discuss the potential of amphibian RBCs to perpetuate amphibian diversity and prevent extinctions within multipolar geopolitical, cultural, and economic frameworks. We argue that a democratic, globally inclusive organisation is needed to focus RBCs on regions with the highest amphibian diversity. Prioritisation should include regional and international collaborations, community engagement, and support for RBC facilities ranging from zoos and other institutions to those of private carers. We tabulate a standard terminology for field programs associated with RBCs for publication and media consistency.

Establishing Cell Lines from Fresh or Cryopreserved Tissue from the Great Crested Newt (Triturus cristatus):A Preliminary Protocol.

This study describes a successful protocol for establishing cell lines from the threatened Triturus cristatus in terms of collection, preparing, establishing, cryopreserving, thawing and quality checking. Different parameters such as media, media change, fresh vs. cryopreserved tissue and seeding density were tested to optimize culture conditions for this species. With fresh tissue, no considerable differences in the use of two different media were found, but with cryopreserved tissue, a combination of ITS (insulin/transferrin/selenite) and 2-mercaptoethanol had a positive effect on growth. Real-time measurements on the cell lines were used, for the first time in amphibian cells, to investigate the effect of different treatments such as media change with or without washing. Media change had a positive impact on the cells, whereas the effect was negative when combined with washing. It is concluded that establishment of cell lines is possible from the great crested newt, especially when using fresh tissue, but much more challenging if the tissue has been cryopreserved. Real-time measurement during cell culture is a useful tool to visualize the sensitivity of amphibian cells during different culture treatments.

Strong Heterogeneity in Advances in Cryopreservation Techniques in the Mammalian Orders.

Between 1970 and 2012, vertebrate abundance has declined by 58% with an average annual decline of 2%, calling for serious action to prevent a mass extinction and an irreversible loss of biodiversity. Cryobanks and cryopreservation have the potential to assist and improve ex situ and in situ conservation strategies by storing valuable genetic material. A great deal of studies concerning cryopreservation have been performed within the class Mammalia, although no systematic overview has previously been presented. The objective of this study is therefore to evaluate the status, pattern and future of cryopreservation within Mammalia. A strong disproportional distribution of studies in examined orders is displayed. For the majority of examined orders less than 10% of species has been examined. However, the cryopreservation of germplasm has in several cases been successful and resulted in successful applications of assisted reproductive techniques (ARTs). Various obstacles are associated with the development of cryopreservation protocols, and among them the most prominent is interspecific differences in cryotolerance. Extrapolation of protocols in closely related species is considered the most applicable procedure, and a future supplement to overcome this problem is the examination and comparison of cryobiological traits. Successful protocols have been developed for the vast majority of domesticated mammals, which gives incentive for the further extrapolation of protocols in threatened species.

Substituting mouse transcription factor Pou4f2 with a sea urchin orthologue restores retinal ganglion cell development.

Pou domain transcription factor Pou4f2 is essential for the development of retinal ganglion cells (RGCs) in the vertebrate retina. A distant orthologue of Pou4f2 exists in the genome of the sea urchin (class Echinoidea) Strongylocentrotus purpuratus (SpPou4f1/2), yet the photosensory structure of sea urchins is strikingly different from that of the mammalian retina. Sea urchins have no obvious eyes, but have photoreceptors clustered around their tube feet disc. The mechanisms that are associated with the development and function of photoreception in sea urchins are largely unexplored. As an initial approach to better understand the sea urchin photosensory structure and relate it to the mammalian retina, we asked whether SpPou4f1/2 could support RGC development in the absence of Pou4f2. To answer this question, we replaced genomic Pou4f2 with an SpPou4f1/2 cDNA. In Pou4f2-null mice, retinas expressing SpPou4f1/2 were outwardly identical to those of wild-type mice. SpPou4f1/2 retinas exhibited dark-adapted electroretinogram scotopic threshold responses, indicating functionally active RGCs. During retinal development, SpPou4f1/2 activated RGC-specific genes and in S. purpuratus, SpPou4f2 was expressed in photoreceptor cells of tube feet in a pattern distinct from Opsin4 and Pax6. Our results suggest that SpPou4f1/2 and Pou4f2 share conserved components of a gene network for photosensory development and they maintain their conserved intrinsic functions despite vast morphological differences in mouse and sea urchin photosensory structures.

Effects of post-mortem storage conditions of bovine epididymides on sperm characteristics: investigating a tool for preservation of sperm from endangered species.

The aim of this study was to establish and validate a reliable and efficient protocol for the recovery and cryopreservation of epididymal spermatozoa used for in vitro fertilization, using bulls of two different age classes. Testicles from 26 (37-51 weeks old, group 1) and 19 (52-115 weeks old, group 2) Danish Holstein bulls were collected after slaughter and stored at 5°C. After 0, 24 or 48 h, epididymides were isolated and spermatozoa collected. Assessments included spermatozoal motility, viability and morphology before and after cryopreservation and in vitro embryo production. Results showed that live spermatozoa can be collected from epididymides of bulls after their death. Storage of the testicles at 5°C for 24 h followed by cryopreservation of recovered epididymal spermatozoa resulted in 21% (group 1) and 31% (group 2) blastocysts produced in vitro. These results illustrate that epididymal spermatozoa recovered from testicles kept in specific conditions can be used to preserve genetic material from endangered and threatened species or populations in nature as well as in domestic and zoo animals.