Optimizing genetic resource conservation and semen cryobanking in aquatic and avian species with a focus on investigating the impact of ovarian fluid on fertilization success in native Mediterranean trout

In Italy, over the years, there has been a progressive decrease in animal biodiversity, mainly due to the combined effect of harmful human actions, continuous loss and fragmentation of habitats, pollution, and climate change. In this regard, the preservation of animal biodiversity has become increasingly important over the years, not only from the perspective of livestock breeding, which benefits from the preservation of different species, but also for animals living in the wild environment. Their survival is crucial to protect and recover the precious natural heritage at the national and international levels, through the maintenance of high genetic variability able to ensure greater ecological stability, enabling populations to adapt to climate and environmental changes. According to the Food and Agriculture Organization (FAO) of the United Nations, the biodiversity conservation can be achieved through the use of various techniques such as in situ and ex situ. The ex situ technique can be further divided into in vivo and in vitro. The in situ technique involves the maintenance of live animals within the livestock production system in which they were developed, while in the ex situ in vivo technique, the maintenance of live animals takes place outside their original habitat. The ex situ in vitro technique requires cryopreservation of genetic material in haploid form (sperm and oocyte), diploid form (embryos, somatic cells), or DNA sequences (FAO 2012, 2015). In the last two decades, at the European level, safeguarding animal genetic resources has played a central role in important conservation programmes that envisaged the adoption of both in situ and ex situ strategies. Among these, of particular interest is our European LIFE project entitled 'Recovery of S. Macrostigma: Application of Innovative Techniques and Participatory Governance Tools in the rivers of Molise' (Acronym Nat.Sal.Mo). Salmo Macrostigma syn. Salmo cettii is an endemic species of the Mediterranean area, currently classified as 'vulnerable' in Europe and 'endangered' in Italy. The main threats to the species include genetic introgression through crossbreeding with zootechnical strains, loss of natural habitats, and unregulated fishing. The primary objective of the project is to ensure the recovery and conservation of native Mediterranean trout in the basins of the Biferno and Volturno rivers (Molise region), using two innovative techniques: sperm freezing with relative creation of the first European cryobank, and the installation of semi-natural nests. In the Nat.Sal.Mo project, the use of doses of frozen semen from the cryobank, combined with appropriate fertilization schemes, has proven to be an effective strategy for restoring the genetic integrity of this species, allowing the maintenance of a high genetic variability in offspring and population fitness. Another innovative technique involves the construction of semi-natural nests, which emulate the behaviour of females in nature. This approach ensures greater success by minimizing non-adaptive selective pressure, as eggs are seeded close to hatching, avoiding exposure of larvae and early life stages to an artificial environment. In Italy, even the drastic decrease in native poultry breeds constitutes an emergency, mainly resulting from the widespread use of high-performance hybrids in the national poultry sector. To address this issue, a significant project funded by Masaf (Ministry of Agriculture, Food and Forestry) titled "Protection of Biodiversity in Italian Poultry Breeds: Insights and Monitoring (TUBAvI-2)" has been launched. The aim of this project is to recover and preserve the genetic resources of Italian poultry species Gallus gallus and Meleagris gallopavo at risk of extinction, through targeted actions to increase population size while maintaining genetic variability. The research activities described in this doctoral thesis are part of the two aforementioned projects, which results have been published in four articles, divided into two sections. The first section comprises two studies focused on Mediterranean trout conservation, with the goal of improving both the success of fertilization rate in artificial breeding and the understanding of the mechanisms involved in natural reproduction. This was achieved through in vitro studies of the effect of ovarian fluid (OF) on both cryopreserved and fresh semen. The first study investigated the impact of OF as a natural sperm activation agent on Mediterranean trout sperm swimming performance post-thawing. The OF efficacy was compared with an activating solution (D-532) and a mixture of them (50% D-532, 50% OF). The results demonstrated that the presence of OF, either alone or combined with D-532, significantly enhanced fertilization rates with frozen sperm. Notably, 100% OF was found to be a particularly effective medium, simplifying and expediting operations in the field along riverbanks. The second study aimed to assess the impact of native trout OF versus non-native trout OF (S. trutta) on the swimming performance of native males in Molise rivers. OF is crucial for fertilization success in externally fertilized fish, with interactions being species- and/or population-specific. These interactions can mitigate genetic introgression from non-native individuals. The study compared OF from native S. cettii females to zootechnic S. trutta females, evaluating their effects on sperm motility in males from the Biferno River. Our results indicate that ovarian fluids from native females (diluted at 20%) significantly improve the performance of native male sperm, unlike those from zootechnic S. trutta. These preliminary results suggest that OF/spermatozoa interactions could potentially influence or regulate hybridization mechanisms involving the native Mediterranean trout population in the Biferno River and the introduced domestic line of brown trout. Session 2 includes two studies aimed at safeguarding the biodiversity of chicken and turkey breeds. The first study aims to highlight the importance of cryobanks for the semen of native breeds and their crucial role in connecting in situ and ex situ techniques to improve the effectiveness of conservation programs. In our work, activities aimed at implementing the national cryobank for chicken and turkey breeds are described, with particular attention to the identification of specific reference freezing protocols for the respective species. The creation of the first Italian semen cryobank for native chicken and turkey breeds represents a valuable tool for the protection and conservation of biodiversity within Italian poultry populations still present in agricultural systems. The use of NMR technique in our second study has proven to be a valuable tool to deepen the understanding of the biological bases of turkey semen cryopreservation and to identify the metabolites involved in the freezing process of turkey donor sperm. This suggested the potential integration of specific metabolites in both the diet and freezing medium to enhance sperm resilience during the cryopreservation process. This discovery represents a significant contribution to the scientific community, considering the crucial importance of semen cryopreservation in the conservation and safeguarding of genetic resources. This is particularly relevant in the face of the growing threat of population decline in some breeds/species and the urgent need to counteract the risks of inbreeding to improve animal biodiversity.