Nutritional and environmental effects on triploid Atlantic salmon skeletal deformity, growth and smoltification

The Atlantic salmon (Salmo salar) is an iconic species that dominates the global finfish production sector with increasing market demand. The Scottish industry and government alone aspires for expansion of the sector to 210,000 t by 2020 with 154, 000 t produced in 2013. As such, there are pressures to improve sustainable development in particular to minimise the genetic impact of escapees on wild populations and reduce sea lice infection which are required for the granting of “green licenses” in Norway. The use of triploidy has been tested in the 1980’s with little success owing to suboptimal rearing conditions leading to elevated mortalities, poorer growth and a higher prevalence of deformities, in particular of the skeleton. Collectively: recent success of triploid trout farming, expansion to the salmon production sector and potential resulting pressure on wild stocks through escapee increases have reinstated interest to implement artificially induced triploid Atlantic salmon in commercial production. As diploid Atlantic salmon have undertaken extensive domestication to achieve the high quality production and welfare standards observed to date, triploid conspecifics too require husbandry optimisation to realise potential. In particular, industrialisation requires that higher observations of deformities and inconsistent growth trajectories during seawater ongrowing be resolved through optimisation of rearing regimes and subsequent standardization of husbandry protocols. Triploids possess additional genomic material and increased cell size yet reduced frequency that reflects known differences in physiology and supports that, in effect, triploids should be considered as a new species relative to diploid conspecifics. Therefore, this doctoral thesis aimed to study nutrition and temperature effects on triploid Atlantic salmon traits throughout the production cycle from ‘egg to plate’. Nutrition trials aimed to improve growth potential and mitigate skeletal deformities both in freshwater (FW) and saltwater (SW) whilst attempts were made to define a window of smoltification to ensure optimal ongrowing performance. Finally, impacts of embryonic temperature regimes that are known to impact long term performance and deformity development in triploids, were examined in relation to DNA regulation and yolk composition in an attempt to underpin potential mechanisms for the environmental impact of temperature on developmental phenotype. One of the main restrictions to triploid Atlantic salmon implementation is the increased prevalence and severity of skeletal deformities, particularly after the maring phase. The work performed in this thesis first demonstrated that protein and / or phosphorous (P) supplementation throughout SW ongrowing not only reduced the level of severely deformed (≥ 10 deformed vertebrae observable by x-radiography) individuals by 30 % but also sustained 6.8 % faster growth and improved harvest grade compared to triploids fed a standard grower diet (chapter 2). Comparison of x-radiography and severely deformed individuals between harvest and sea transfer highlighted that protein and P supplementation arrested deformity development whereas prevalence increased in triploids fed a standard grower diet. This implied that severe deformities were of FW origin and strongly suggest requirement for improved nutrition in FW to optimise SW performance. Therefore investigation of higher dietary P inclusion in FW was investigated and results showed significantly reduced number of deformed vertebrae and no severely deformed individuals in those fed 19.7 g total P Kg-1 compared with those fed 13.0 & 16.7 g total P Kg-1 (chapter 3). Most deformities were localised in the central (vertebrae 27 – 31) and caudal (vertebrae 52 – 57) regions for all treatments. However, triploids fed lower dietary P displayed a particular increase in prevalence within the tail region (vertebrae 32- 47) which is consistent with SW ongrowing reports and results from chapter 2, further highlighting FW origin of higher vertebral deformities reported in SW ongrowing in triploids. Higher P supplementation in FW also significantly improved growth in triploid parr compared to diploids and lower supplementation. However, this effect did not transpire in later FW smolt stages where weights were significantly higher in triploids fed lower compared to higher P supplementation. Expression of target genes involved in osteogenesis and bone P homeostasis in vertebrates were then analysed and a ploidy effect of osteogenic genes alp, igf1r and opn as well as a dietary effect on P homeostasis gene fgf23 was apparent in the parr stages but not smolt. In addition, stronger ploidy-diet effects were also observed in parr stages for whole body mineral concentrations. Collectively, growth, gene expression and whole body mineral content results indicate these earlier parr life stages may be more sensitive to P supplementation. This pronounced effect may be a consequence of seasonal accelerated growth associated with this period, where higher temperatures were also observed. The potential for shorter P supplementation windows in commercial production was addressed in chapter 4 with hope to cut economic cost to raw mineral inclusion in feed and also mitigate potential anthropogenic eutrophication on the environment that may be induced by P leached through uneaten feed and faeces. Triploids were fed higher dietary P (17.4 g total P Kg-1) until either early (5 g) or later (20 g) parr stages, or smolt (83 g) and monitored for performance throughout freshwater (FW) development. During later parr development (30 g), x-radiography assessment demonstrated that increased dietary P reduced the number of deformities and severely deformed individuals with no indication that feeding P for shorter windows improved skeletal integrity. Hence, P supplementation may be required throughout FW development for optimal skeletal performance. In addition, no differences in deformities were observed between triploid treatments at smolt. An effect of dietary P supplementation on whole body mineral concentration was observed in the early and later parr stages that was not as pronounced as smolt, which is consistent with results in chapter 3. Together, these results indicate that skeletal assessment during early developmental stages may not reflect smolt performance most likely as a consequence of seasonal effects of improved linear growth in the cooler winter temperatures prior to smolt where reversible deformities observed at parr may also be alleviated. In the same study (chapter 4), the inclusion of the probiotic Pediococcus acidilactici (Bactocell™) was also tested as a means to enhance gut assimilation as suggested in previous studies and therefore reduce the levels of P supplementation. Results clearly indicate superior skeletal performance in parr (30 g) as well as significantly less deformed vertebrae and no severely deformed individuals. However, at smolt (~83g), no effects of the dietary probiotic treatment were observed which may also be attributed to seasonal effects. Overall, nutritional research clearly indicate triploids require higher dietary P for optimal growth and skeletal development, which although is not consistent between life stages, is ultimately required throughout FW for optimal skeletal development at smolt. The use of probiotics offer a promising avenue for reduced P requirement in FW feed and further research should verify results and assess long-term performance. Timing of SW transfer according to correct parr-smolt transformation (PST) is essential for survival and growth performance in ongrowing where feeding and growth rate accelerate post-transfer. So far, SW transfer regimes and in particular the smoltification ‘window’ remains loosely defined in triploid Atlantic salmon and it is crucial that this be addressed to ensure optimal ongrowing survival and performance. Results in chapter 5 show that triploid Atlantic salmon reared under an ambient photo-thermal regime (S1+) have a wider smoltification window within 155 – 365 degree days as well as an earlier onset by 48 degree days. This was confirmed through raised Na+, K+ - ATPase (NKA) activity that was maintained for a longer duration and earlier skin silvering compared to diploid siblings. In addition, reduced plasma chloride (Cl-) levels alongside improved survival following SW challenge compared with diploid siblings strongly suggest that triploids had improved hypo-osmoregulatory capacity and a wider smolt window. Although other studies have demonstrated that triploid salmonids may have earlier onset of PST none to date have investigated the window duration. Results in this study need to be verified against other photo-thermal regimes; a wider smolt window may be of great benefit to industry as there is potential for reduced FW rearing periods, earlier onset of ongrowing and increased sea-transfer flexibility compared to diploid conspecifics. Suboptimal egg incubation conditions, in particular higher temperatures, are one of the primary causes of deformity in triploid Atlantic salmon. This may be associated with embryogenesis being stenothermal and also where the critical process of somitogenesis and the underlying changes in DNA regulation occur. Hence, diploid and triploid embryos were reared at temperature regimes known to be optimal and suboptimal for development (5.9, 7.9 and 10.7 ºC) from fertilisation until the eyeing stages and then at 7.8 ºC until hatch. Temperature / ploidy associated mechanisms that may induce phenotypic variation were analysed comprising of global DNA methylation (DNAme), as an indicator of DNA regulation, as well as changes in Nitrogenous metabolites (NM) including Free Amino Acid (FAA) concentrations. Differences in genomic weight between diploids and triploids may potentially impact DNA regulation and the availability of maternally provided resources such as NMs for the dramatic process of reorganisation of the methylome during embryogenesis. Although changes in NM utilisation were apparent between life stages and influenced by temperature, no impact of ploidy was evident. In addition, no impact of temperature was observed on DNAme levels. This indicates availability of maternally provided NMs and DNA programming may not necessarily be a factor in temperature induced deformities in triploids and phenotype assessment in later life-stages would verify this conclusion. In addition sequence specific DNAme results and analysis of other epigenetics process such as hitstone modification would verify or reveal other epigenetic effects. However, results did reveal interesting ploidy differences in DNAme levels post gastrulation where triploids maintained lower DNAme levels relative to diploids throughout somitogenesis indicating a delay in the DNA remethylation or reprogramming process. This is the first study to identify potential triploid specific differences in DNA reprogramming in salmonids and so verification as well as an understanding of the impact on epigenetics and long-term phenotype must be assessed. This doctoral work adds significantly to existing knowledge on improved husbandry practice of triploid Atlantic salmon through: improved nutritional regimes and understanding of PST that have potential for improved production traits including growth, reduction of skeletal deformities and reduced rearing periods. This work also pioneers study of DNA regulation in triploid embryogenesis that pose important future questions to explain fundamental differences associated with altered cellular and genomic make-up in triploids. This research will assist in enabling triploid salmon as a tool for sustainability in global aquaculture production of Atlantic salmon as demonstrated by the development of patented triploid feed in relation to these trials and optimised protocols for SW transfer. Ultimately, this additional knowledge highlights the potential for triploids to perform equally well if not better than diploid conspecifics