The perils of poly- and perfluorinated chemicals on the reproductive health of humans, livestock, and wildlife.

Poly- and perfluoroalkyl substances (PFAS) are a prominent class of persistent synthetic compound. The widespread use of these substances in various industrial applications has resulted in their pervasive contamination on a global scale. It is therefore concerning that PFAS have a propensity to accumulate in bodily tissues whereupon they have been linked with a range of adverse health outcomes. Despite this, the true extent of the risk posed by PFAS to humans, domestic animals, and wildlife remains unclear. Addressing these questions requires a multidisciplinary approach, combining the fields of chemistry, biology, and policy to enable meaningful investigation and develop innovative remediation strategies. This article combines the perspectives of chemists, soil scientists, reproductive biologists, and health policy researchers, to contextualise the issue of PFAS contamination and its specific impact on reproductive health. The purpose of this article is to describe the challenges associated with remediating PFAS-contaminated soils and waters and explore the consequences of PFAS contamination on health and reproduction. Furthermore, current actions to promote planetary health and protect ecosystems are presented to instigate positive social change among the scientific community.

Reproductive biology research down under: highlights from the Australian and New Zealand Annual Meeting of the Society for Reproductive Biology, 2021.

Against the backdrop of a global pandemic, the Society for Reproductive Biology (SRB) 2021 meeting reunited the Australian and New Zealand reproductive research community for the first time since 2019 and was the first virtual SRB meeting. Despite the recent global research disruptions, the conference revealed significant advancements in reproductive research, the importance of which span human health, agriculture, and conservation. A core theme was novel technologies, including the use of medical microrobots for therapeutic and sperm delivery, diagnostic hyperspectral imaging, and hydrogel condoms with potential beyond contraception. The importance of challenging the contraceptive status quo was further highlighted with innovations in gene therapies, non-hormonal female contraceptives, epigenetic semen analysis, and in applying evolutionary theory to suppress pest population reproduction. How best to support pregnancies, particularly in the context of global trends of increasing maternal age, was also discussed, with several promising therapies for improved outcomes in assisted reproductive technology, pre-eclampsia, and pre-term birth prevention. The unique insights gained via non-model species was another key focus and presented research emphasised the importance of studying diverse systems to understand fundamental aspects of reproductive biology and evolution. Finally, the meeting highlighted how to effectively translate reproductive research into policy and industry practice.

Cryopreservation and Flow Cytometric Analysis of Ovarian Tissue in Murray River Rainbowfish, Melanotaenia fluviatilis.

Freshwater fish populations are declining with many small, Australian fish species at risk of extinction within the next twenty-years. Cryopreservation of reproductive cells and tissues makes it possible to reproduce individuals from a species even after they are extinct in the wild. We describe the successful cryopreservation of ovarian tissue in the Murray River Rainbowfish, Melanotaenia fluviatilis (Order: Atheriniformes). Histology showed that oogonia are 13.70 µm ± 1.75 µm in size, stain positive for germ-line marker Vasa, and represent approximately 2.29 ± 0.81% of cells in the ovary. Flow cytometry was used to analyse ovarian cell suspensions, requiring an optimised tissue digestion protocol. We found that 0.25% trypsin with 1.13 mM EDTA produced cell suspensions with the highest viability (76.28 ± 4.64%) and the highest number of cells recovered per gram of tissue (1.2 × 108 ± 4.4 × 107 cells/g). Subsequent sorting of ovarian cell suspensions by flow cytometry increased oogonial cells in suspension from 2.53 ± 1.31% in an unsorted sample to 5.85 ± 4.01% in a sorted sample (p = 0.0346). Cryopreservation of ovarian tissue showed DMSO-treated samples had higher cell viability post-thaw (63.5 ± 18.2%) which was comparable to fresh samples (82.5 ± 7.1%; p = 0.36). Tissue cryopreserved in 2.0 M DMSO had the highest cell viability overall (76.07 ± 3.89%). This protocol could be applied to bio-banking programs for other species in the Melanotaeniidae, and perhaps species in other families and orders of Australian fish.

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis.

Globally, fish populations are in decline from overfishing, habitat destruction and poor water quality. Recent mass fish deaths in Australia's Murray-Darling Basin highlight the need for improved conservation methods for endangered fish species. Cryopreservation of testicular tissue allows storage of early sperm precursor cells for use in generating new individuals via surrogacy. We describe successful isolation and cryopreservation of spermatogonia in an Australian rainbowfish. Testis histology showed rainbowfish spermatogonia are large (> 10 µm) and stain positive for Vasa, an early germ line-specific protein. Using size-based flow cytometry, testis cell suspensions were sorted through "A" (> 9 µm) and "B" gates (2-5 µm); the A gate produced significantly more Vasa-positive cells (45.0% ± 15.2%) than the "B" gate (0.0% ± 0.0%) and an unsorted control (22.9% ± 9.5%, p < 0.0001). The most successful cryoprotectant for "large cell" (> 9 µm) viability (72.6% ± 10.5%) comprised 1.3 M DMSO, 0.1 M trehalose and 1.5% BSA; cell viability was similar to fresh controls (78.8% ± 10.5%) and significantly better than other cryoprotectants (p < 0.0006). We have developed a protocol to cryopreserve rainbowfish testicular tissue and recover an enriched population of viable spermatogonia. This is the first step in developing a biobank of reproductive tissues for this family, and other Australian fish species, in the Australian Frozen Zoo.

New directions in assisted breeding techniques for fish conservation.

Fish populations continue to decline globally, signalling the need for new initiatives to conserve endangered species. Over the past two decades, with advances in our understanding of fish germ line biology, new exsitu management strategies for fish genetics and reproduction have focused on the use of germ line cells. The development of germ cell transplantation techniques for the purposes of propagating fish species, most commonly farmed species such as salmonids, has been gaining interest among conservation scientists as a means of regenerating endangered species. Previously, exsitu conservation methods in fish have been restricted to the cryopreservation of gametes or maintaining captive breeding colonies, both of which face significant challenges that have restricted their widespread implementation. However, advances in germ cell transplantation techniques have made its application in endangered species tangible. Using this approach, it is possible to preserve the genetics of fish species at any stage in their reproductive cycle regardless of sexual maturity or the limitations of brief annual spawning periods. Combining cryopreservation and germ cell transplantation will greatly expand our ability to preserve functional genetic samples from threatened species, to secure fish biodiversity and to produce new individuals to enhance or restore native populations.

Production of Germ-Line Chimeras in Zebrafish.

The induction of germ-line chimerism in fish is a strategy for the reproduction of endangered or genetically valuable fish species. Chimeras can be created by transplanting a single primordial germ cell or multiple blastomeres from a donor into a sterile host embryo. When the host reaches sexual maturity, it will produce donor-originating gametes throughout its reproductive life span. This technique provides unique experimental conditions for basic biology research in model fish species like zebrafish. The success of cell transplantation relies on the effective sterilization of host embryos, the correct identification of developing germ cells, and the synchronization of migratory cues between the host and the transplanted cells. Developments in non-transgenic methods of germ cell ablation and identification have made germ cell transplantation more applicable to use in conservation and aquaculture. In this chapter, we provide a protocol for germ cell labeling by injection of chimeric RNA or FITC-dextran, the sterilization of host embryos using an antisense morpholino oligonucleotide, and two methods for producing germ-line chimeras in zebrafish: single primordial germ cell transplant and blastomere transplant.

Genetic population structure of Natterer's bats explained by mating at swarming sites and philopatry.

During autumn 'swarming', large numbers of temperate bats chase each other in and around underground sites. Swarming has been proposed to be a mating event, allowing interbreeding between bats from otherwise isolated summer colonies. We studied the population structure of the Natterer's bat (Myotis nattereri), a swarming species in northern England, by sampling bats at seven sites in two swarming areas and at 11 summer colonies. Analysis of molecular variance (amova) and genetic assignment analyses showed that the swarming areas (60 km apart) support significantly different populations. A negative correlation was found between the distance of a summer colony from a swarming area and the assignment of bats to that area. High gene diversity was found at all sites (HE = 0.79) suggesting high gene flow. This was supported by a low FST (0.017) among summer colonies and the absence of isolation by distance or substructure among colonies which visit one swarming area. The FST, although low, was significantly different from zero, which could be explained by a combination of female philopatry and male-mediated gene flow through mating at swarming sites with bats from other colonies. Modelling suggested that if effective size of the summer colonies (Ne) was low to moderate (10-30), all mating must occur at the swarming sites to account for the observed FST. If the Ne was higher (50), in addition to random mating during swarming, there may be nonrandom mating at swarming sites or some within-colony mating. Conservation of swarming sites that support potentially large populations is discussed.