Improving Atlantic Salmon (Salmo salar) conservation and farming sustainability, through the gamete level

Graziano, Marco (2022) Improving Atlantic Salmon (Salmo salar) conservation and farming sustainability, through the gamete level. Doctoral thesis, University of East Anglia.

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This Ph.D. project focused on reproductive biology in Atlantic salmon (Salmo salar) with the aim to address pure and applied questions about sperm form and function, fertilisation compatibility, inbreeding, gamete plasticity and epigenetic effects. Using gametes from wild and farmed salmon strains, I generated key data on 1) hatchery methods of gamete handling to optimise fertility, 2) gamete function, fertility and reproductive potential of farm salmon escapes 3) compatibility and fitness consequences of reproduction between genetic siblings 4) impacts of and responses to thermal variation at the gamete level and 5) effect of the physical properties of salmon ovarian fluid in sexual and natural selective mechanisms. On one side, conducted experiments aimed to delineate causes and effects of artificial coexistence between escaped farm salmon and native populations, generating reproductive potential data to inform management of wild fish facing farm introgression, and the sustainability of salmon aquaculture under rising global demand for animal protein. On the other one, gamete interactions and fertility across different levels advance our understanding of gamete biology, sperm-egg interaction, sexual selection, local adaptation, hybridisation, speciation, phenotypic plasticity, and cryptic female choice.

In the first chapter of my thesis, I review the scientific literature across two main, but related, research areas: 1) Salmon farming sustainability and wild Atlantic salmon conservation, and 2) gamete evolution and sperm and egg biology. Following, I delineate four experimental Work Packages and chapters that respectively examine: 1) dry versus wet fertilization techniques in the hatchery and their relevance to fertility, 2) inbreeding avoidance at the gamete level and offspring fitness consequences, 3) thermal plasticity in gametes and adaptive epigenetic inheritance, and 4) influence of the non-Newtonian viscoelastic properties of salmon ovarian fluid on natural and sexual selective mechanisms.

This body of research has allowed me to produce results that shine a light on sexual selection at the gamete level in an externally fertilising animal model, in a variety of contexts, and that could be applied to improve salmon conservation and sustainability in the aquaculture sector. Specifically, in the first experimental Chapter (Chapter 3), we explored the effects of two in vitro fertilisation methods on gamete fertility, compatibility, embryo development and reproductive outcomes in farmed and wild strains of Atlantic salmon; with the aim to improve hatcheries’ sustainability and wild salmon reintroduction efforts. We revealed that farmed salmon strains were far more resilient to different fertilisation techniques, their embryonic development was faster, and they manifested a higher degree of chromosomal abnormalities as compared to wild fish. When wild and farmed males competed over fertilisation of eggs from wild and farmed females, farmed male paternity rates ranged from 70 to 100% in both types of females, suggesting a clear threat to wild genetic pools following escapes. Our results stand in contrast with previous findings and present important insights that can help improving sustainability and reintroduction efforts in conservation aquaculture.

In Chapter 4, we tested the presence of inbreeding avoidance mechanisms at the gamete level that could arise from the philopatric return of salmon to their natal streams to spawn, in a context of polyandry. We hypothesised that in salmon, inbreeding avoidance would have to evolve at the gamete level and could be based on the interaction between sperm and the ovarian fluid released with eggs at oviposition, which may play a key role in determining fertilisation success. We compared sperm motility parameters in sibling and non-sibling ovarian fluid, and assessed fertilisation and hatching success, growth rate and paternity in sperm competition trials between sibling and non-sibling males. We found that sperm activated in ovarian fluid of sibling females showed lower values of motility-related parameters and led to an average of 36% reduction in fertilisation rates in the resulting crosses. Furthermore, offspring from sibling crosses were smaller before the onset of sexual maturation, but we found no difference in survival rates between sibling and non-sibling cross. When sperm from sibling and non-sibling males were competing simultaneously for the same egg batch, we found no influence of this on paternity, but surprisingly, sibling-sired offspring showed consistently higher degrees of multi-locus heterozygosity across the five microsatellite loci analysed. Our findings indicate that post-mating inbreeding avoidance mechanisms have evolved at the gamete level in salmon, but that this does not necessarily affect the sperm competitiveness of sibling males. Our results have direct implications for conservation aquaculture and salmon farming sustainability due to the effect that inbreeding could have in these sectors.

We applied the acquired knowledge on sexual selection at the gamete level, gamete and embryo performance and survival rates to establish in Chapter 5 the presence of mechanisms of thermal plasticity of gametes and/or thermal selection which could affect egg and sperm function and competitiveness. We tested the hypothesis that gametes primed to a specific temperature positively influence hatching success and embryonic development in the same temperature environment and compared sperm and eggs. We incubated half of the eggs and sperm collected from wild Atlantic salmon at two temperatures (cold and warm) and performed in vitro fertilisations where we crossed warm and cold-incubated gametes for each of the mating pairs and reared one half of each clutch in cold temperature and the other in warm temperature. We monitored hatching success, hatching time, embryo survival before and after the eyed stage and presence of developmental abnormalities. We found that when the temperature for eggs, sperm and embryo development matched, embryos hatched earlier than in scenarios where gamete incubation and development temperature did not match Warm temperature exposure during embryo development generally caused increased rates of deaths after the eyed stage. Interestingly, we observed opposite effects of gamete incubation temperature on offspring fitness between eggs and sperm, where warm incubation was beneficial for eggs but detrimental for sperm which in turn negatively affected hatching success. Overall, we showed that gamete plasticity did not significantly improve offspring fitness, suggesting that these stages are particularly vulnerable to a changing environmental temperature.

In the last experimental section, Chapter 6, we gained insights on the physical structure of this fluid and potential impacts on reproduction; while its biochemical effects in relation to sperm energetics have been investigated, the influence of the physical environment in which sperm compete remains poorly explored. Using soft-matter physics approaches of steady-state and oscillatory viscosity measurements, we simulated the frequencies resembling those exerted by sperm swimming through the fluid near eggs. We demonstrated that this fluid, which in its relaxed state is a gel-like substance, displays a non-Newtonian viscoelastic and shear-thinning profile, where the viscosity decreases with increasing shear rates. We concurrently found that the ovarian fluid can also display a shear-thickening phase at high frequencies , provided it is probed gently enough. We highlight the presence of a unique frequency-dependant structural network with important implications on sperm energetics and fertilisation and that could forunish the basis for a novel, bio-physically mediated, sexual selection mechanism.

We suggest how ovarian fluid physical properties deserve more attention when studying processes of sexual selection and that mechanisms enabled by non-Newtonian reproductive fluids within female internal genital tracts, like lubrication, facilitation and capacitation, should also be applied to the external fertilization environment. This opens new avenues into the study of cryptic female choice with important implications for understanding the evolution of sexual traits and exploring the underestimated role of physical properties of the fertilization environment that surrounds the gametes.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 31 May 2023 10:43
Last Modified: 31 May 2023 10:53


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