ADVANCES IN THE EARLY LARVAL STAGES OF SIBERIAN STURGEON: MUSCLE DEVELOPMENT AND STRUCTURE UNDER DIFFERENT REARING CONDITIONS

Fish larval stages are particularly sensitive to environmental conditions, as these can severely affect survival and potential growth. In fish species destined for production and commercialisation, a suitable early environment is of great importance, for what concerns growth efficiency and quality at harvest. Temperature and stocking density are among the most important factors affecting the early stages of development, regarding somatic and muscle growth of fish. Moreover, environmental enrichment is considered to improve biological functioning of captive animals, including fish, by improving their psychological and behavioral needs. Siberian sturgeon (Acipenser baerii) is a species at risk of extinction and, therefore, production of this species in aquaculture is of vital importance, not only to provide the market with caviar and meat, but also for repopulation purposes. Taking into account that little is known regarding the impact of environmental conditions in early life stages of the Siberian sturgeon, the aim of this Thesis is to provide new knowledge on the influence on muscle growth and development during the endogenous feeding phase. This was performed by addressing morphological, physiological and molecular aspects of muscle growth structure and development. This Thesis may contribute to identify and select the most suitable environmental conditions to improve Siberian sturgeon larval rearing in aquaculture production. Three trials have been performed, one in each year of my PhD, where 1) rearing temperature, 2) stocking density and 3) environmental enrichment were examined in important timepoints of Siberian sturgeon larvae development: hatching, schooling and complete yolk-sac absorption stage. In Chapter 1, is presented a general overview of Siberian sturgeon biology and development, a brief description of the muscle morphology and of the methods used to study muscle growth. Following, the types of sturgeon muscle growth are described, as well as the mechanisms involved in muscle growth regulation. Finally, the environmental effects on growth are presented along with a short overview of the plasticity of the myogenic phenotype. In Chapters 2, 3 and 4, the effects of three different rearing temperatures (16, 19 or 21 °C) applied from hatching until the yolk-sac full absorption were assessed. Siberian sturgeon fertilized eggs were all incubated at 16°C and newly-hatched larvae were then subjected to three different temperatures throughout the endogenous feeding phase. All the three experimental rearing temperatures were congruent with a correct development of farmed A. baerii (Chapter 2, 3 and 4), but a rearing temperature of 22°C lead to a higher developmental rate, which could be advantageous in commercial hatcheries, as it shortens the endogenous feeding phase and allows to feed the larvae sooner with exogenous feed. In Chapter 2, histometrical, histochemical and immunohistochemical analyses were performed in order to characterize muscle growth (total muscle area, TMA; slow muscle area, SMA; fast muscle area, FMA), development (anti proliferating cell nuclear antigen –PCNA) as well as stress status by specific stress biomarkers (heat shock protein 70 or 90; HSP70 or HSP90). Histometry revealed that both TMA and FMA were larger in the schooling stage at 19°C while no differences were observed in the SMA at any of the tested rearing temperatures. PCNA quantification revealed a significantly higher number of proliferating cells in the yolk-sac absorption phase at 22°C than at 16°C. HSP90 immunopositivity seems to be particularly evident at 19°C. HPS70-immunopositivity was never observed in the developing lateral muscle. It was possible to conclude from this study that a temperature of 19°C could be taken into account by commercial hatcheries, as it supported larger size in developing larvae and suggesting a higher growth potential. The aim of Chapter 3 was to investigate the fatty acid composition of yolk‐stage Siberian sturgeon larvae reared at three different temperatures. The fatty acid composition of larvae was affected by temperature. The larvae reared at lower temperatures (16°C) showed adaptive behavior compared to the larvae reared at two higher temperatures (19°C and 22°C), conserving a greater quantity of polyunsaturated fatty acids and consuming saturated fatty acids for energetic purposes. Chapter 3 study suggests that at a lower temperature sturgeon shows an adaptation to the variation of the temperature, carried out with the purpose to conserve the fatty acids that guarantee a greater fluidity to the cell membranes at the lower temperatures. The aim of Chapter 4 was to assess growth, muscle development and stress status in Siberian sturgeon larvae at different rearing temperatures. Immunofluorescence localization of myogenin and Igf1, the expression of genes involved in muscle development and growth (myog and Igf-1) and in the stress status (Hsp70, Hsp90α, Hsp90β) and iv) were performed as well as whole body cortisol. Both at the schooling stage and at the end of the trial, there were no significant differences regarding larval weight among temperatures. Both at schooling and at the end of the trial, larvae reared at 16°C showed a lower level of cortisol than those reared at 19°C or 22°C. Igf-1 immunopositivity was particularly evident in red muscle at the schooling stage at all temperatures and it was similarly expressed in white muscle at both schooling and yolk-sac absorption stage at all tested temperatures. Myogenin immunopositive cells were detected in the cytoplasm of undifferentiated cells at all stages and at all temperatures considered. At schooling, all of the analysed genes resulted significantly more expressed in larvae reared at 16°C compared to the larvae reared in the other two rearing temperatures (Hsp70, hsp90α, hsp90β, myog, Igf1). No differences were found in the expression of the analysed genes between larvae reared at 19°C and 22°C. Conversely, at the end of the trial, no significant differences were found in the expression of all genes among rearing temperatures. From Chapters 2, 3 and 4, it would appear that, taking into account all analysed variables, it would seem that a temperature of ranging from 16 to 19°C would be a good compromise between growth potential and stress status. In Chapters 5 and 6, the effects of different rearing stocking densities were examined during the pre-larval phase. After hatching, Siberian sturgeon newly-hatched larvae were subjected to three different stocking densities (30, 80 or 150 larvae/litre) until the yolk-sac was fully absorbed. In Chapter 5, muscle growth and development were evaluated by assessing body weight and length, muscle histometrical analyses, qualitative morphological study analyses and fatty acid profile. At the end of the trial, larvae reared at the lower density were heavier and longer and presented a higher proliferation rate of the muscle fibres. Total muscle area was lower for larvae reared at the highest density at schooling, which, from a morphological point of view, showed an acceleration in muscle development that may be detrimental at a medium-long term. Fatty acids profile revealed no differences between densities while, during development, there was a selective consumption: sparing or increasing of essential fatty acids to the detriment of their precursors. Results of Chapter 5 suggest that lower densities appear to be more suitable to rear Siberian sturgeon in this particular stage of development. In Chapter 6, growth (Specific Growth Rate, SGR and Condition Factor, K), muscle development and stress status were evaluated, through immunofluorescence, whole body cortisol and Real-Time PCR. SGR was significantly improved at the yolk-sac absorption stage for larvae reared at the lower density, while no differences were found regarding K. Alpha skeletal muscle actin (ACTA1) immunofluorescence was detected in the cytoplasm of muscle cells in all developmental stages and no morphological differences among densities were detected regarding muscle structure at all stages. The levels of relative expression of different genes involved in the growth process (igf1 and igf2), in the myogenesis process (myog) and in the regulation of cellular stress (glut1, glut2 and hsp70) were analysed. All the genes examined have shown an up-regulation in both development stages at all the rearing densities considered, with the exception of the myog gene which was, instead, always down-regulated. This down-regulation is significantly greater in larvae reared in high-density in the phase of the complete absorption of the yolk sac. Cortisol levels did not differ significantly, both in time and across densities. To conclude, taking into account the SGR and the gene expression results of Chapter 6, we suggest that lower densities are used in these stages of development, as these showed a higher growth potential and lower stress levels. From Chapter 5 and 6, it is evident that the lowest density tested was the most favourable one but, it is not applicable in a commercial hatchery as it is space consuming and, thus, not economically feasible. Therefore, we would suggest that an intermediate density could be a good compromise from a commercial hatchery point of view. In Chapter 7 the morpho-functional and behavioural responses of early life phases in Siberian sturgeon (Acipenser baerii) towards two types of substrate (Bioballs type 1: 35mm; Bioballs type 2: 38mm BB1 and BB2 respectively) vs. no substrate (CTR), were examined, from hatching and during the endogenous feeding period. Number of larvae swimming in the water column were analysed, larval growth in terms of weight and length and histometrical analyses were also performed in order to evaluate muscle development. Larvae reared with BB1 substrate were present in a lower number in the water column, were heavier and longer at the end of the trial, and showed larger areas of total muscle area, slow muscle area and fast muscle area. Moreover, larvae reared with no substrate showed an acceleration of muscle differentiation, which can have negative consequences on its growth potential. It would seem more favourable to provide a substrate rather than a bare bottom for Siberian sturgeon in these early phases of development, in particular a substrate with characteristics similar to those of BB1. This Thesis ends with a general discussion in Chapter 8. The effects of temperature, stocking density and environmental enrichment during larval phases in relation to muscle growth and development are discussed and the main conclusions of the present work and future research direction are also presented. Overall, the present Thesis shows that Siberian sturgeon, in the early stages of development, does not appear to be particularly sensitive to temperature variations, even if the results may suggest a range temperature of 16-19°C. The discourse concerning density and environmental enrichment is different: the obtained results suggest a medium rearing density and the presence of a substrate in the tanks.