Spotted Wolffish Broodstock Management and Egg Production: Retrospective, Current Status, and Research Priorities.

The first artificially fertilized spotted wolffish (Anarhichas minor) eggs hatched in Norway in the mid-1990s as this species was considered by Norwegian authorities to be a top candidate species for cold-water aquaculture in the North Atlantic regions. Previous research conducted in Norway (since 1992) and Canada (since 2000), focused on identifying key biological parameters for spotted wolffish cultivation which led, respectively, to the rapid establishment of a full commercial production line in northern Norway, while Québec (Canada) is witnessing its first privately driven initiative to establish commercial production of spotted wolffish on its territory. The control of reproduction can be viewed as a major requirement to achieve the development of performant strains using genetic selection tools and/or all-year-round production to bring about maximal productivity and synchronization among a given captive population. Although the basic reproduction aspects are more understood and controlled there are still some challenges remaining involving broodstock and upscaling of operations that limit the achievement of a standardized production at the commercial level. Quality of gametes is still considered a major constraint and it can be affected by multiple factors including nutrition, environmental conditions, handling practices, and welfare status. Internal insemination/fertilization and the protracted incubation period are challenging as well as the establishment of a health monitoring program to secure large-scale operations. The profound progress achieved in the control of reproduction, sperm handling, and cryopreservation methods for this species is presented and discussed. In this review, we also go into detail over the full range of up-to-date cultivation practices involving broodstock and identify areas that could benefit from additional research efforts (i.e., broodstock nutrition, health and welfare, scaling-up egg and larval production, genetics, and development of selective breeding programs).

Production of Fatty Acids and Protein by Nannochloropsis in Flat-Plate Photobioreactors.

Nannochloropsis is an industrially-promising microalga that may be cultivated for alternative sources of nutrition due to its high productivity, protein content and lipid composition. We studied the growth and biochemical profile of Nannochloropsis 211/78 (CCAP) in optimized flat-plate photobioreactors. Eighteen cultivations were performed at two nutrient concentrations. The fatty acid, protein content and calorific values were analyzed after 8, 12 and 16 days. Neutral lipids were separated and the changes in fatty acids in triglycerides (TAGs) during nutrient depletion were recorded. The maximum cell density reached 4.7 g∙L-1 and the maximum productivity was 0.51 g∙L-1∙d-1. During nutrient-replete conditions, eicosapentaneoic acid (EPA) and total protein concentrations measured 4.2-4.9% and 50-55% of the dry mass, respectively. Nutrient starvation induced the accumulation of fatty acids up to 28.3% of the cell dry weight, largely due to the incorporation of C16:0 and C16:1n-7 fatty acyl chains into neutral lipids. During nutrient starvation the total EPA content did not detectibly change, but up to 37% was transferred from polar membrane lipids to the neutral lipid fraction.

Quantitative characteristics of Atlantic halibut (Hippoglossus hippoglossus L.) egg quality throughout the reproductive season.

Assessment of egg quality is an important aspect in finfish hatchery management, but guidelines for such assessment are scarce, especially for marine fish production. In the present work, potential indicators of egg and larval quality were measured in 39 batches of eggs of Atlantic halibut (Hippoglossus hippoglossus L.) throughout the reproductive season. The paternal influence on offspring was minimized by using the same cryopreserved semen for all fertilizations; consequently, maternal effects were emphasized. The progression of the spawning season and ovarian fluid parameters, including pH, electrical conductivity (EC), osmolality, and the amount of fluid were registered. The behavior of the broodfish at egg collection was registered and the eggs were analyzed for cortisol content. Fertilization and hatching rates as well as larvae survival were calculated, and larval standard length and myotome height were measured. Furthermore, the occurrence of major types of larval deformities was registered. The myotome height was significantly (P < 0.05) affected by the spawning season progress. Cortisol content in the eggs was decreasing with the progressing spawning season and correlated positively with the occurrence of yolk-sac edema. The ovarian fluid pH and EC were significantly related to fertilization and hatching rates. High fertilization and hatching success was associated with pH greater than 7.9 and EC less than 2.5 mS/cm. Low fertilization rates (< 50%) resulted in further low hatchability from such egg batches. Ovarian fluid EC was significantly and positively related to increased occurrence of yolk-sac edema. High quantity of ovarian fluid in egg batches was associated with reduced egg quality in terms of fertilization and hatching rates and occurrence of yolk-sac edema. A cumulative effect of ovarian fluid pH, EC, osmolality, and quantity explained up to 62% of the total variation in fertilization rates. The findings from the present study indicate that parameters measurable at the initial phase of production, in particular ovarian fluid pH and EC, might have a potential for future use as egg quality indicators in hatchery management.

Nucleotide enrichment of live feed: a promising protocol for rearing of Atlantic cod Gadus morhua larvae.

We investigated the effect of two commercial nucleotide products (NT1 and NT2), administered through live feed, on growth and stress tolerance of Atlantic cod larvae. Expression of genes related to muscle growth (igf-1, igf1r, igf-2, fst, fgf6, myod, and myhc) and nucleotide metabolism (uox, hprt, ndk, and uck) was evaluated during larval development. In addition, the expression of genes related to stress (hif-1α, hif-2α, hif-3α, and mb) was studied after an air exposure stress test. The enrichment of rotifers with nucleotides did not reveal any difference in nucleotide profiles, the exception being the RNA level of the NT1-enriched group that was significantly higher than the unenriched rotifer. Unenriched Artemia showed poor nucleotide profiles compared to enriched Artemia since 5' UMP, 5' GMP, and 5' AMP were observed only in the nucleotide groups. At 38 days post-hatch (dph), NT1 group had significantly higher dry weight (3.1 ± 0.1 mg) than the control (CON; 2.3 ± 0.1 mg). The treatments did not produce any significant differences in the expression of the key myogenic genes. Among the genes associated with nucleotide metabolism, ndk was down-regulated in NT1 at 38 dph. In the air exposure test, survival was significantly higher in the CON (77 ± 6 %) than in NT1 (48 ± 3 %) and NT2 (50 ± 3 %). After air exposure, mb was expressed at lower levels in NT2 group, hif-2α was induced in NT1 group, and hif-3α was upregulated in all groups. Our findings indicate that the improvement in the nucleotide profile of Artemia upon nucleotide enrichment could eventuate in the rapid growth of larvae.

Induction of meiotic gynogenesis in Atlantic cod (Gadus morhua L.) through pressure shock.

The Atlantic cod, Gadus morhua, is one of the most important species for commercial fisheries and a promising candidate for aquaculture. Precocious sexual maturation of males is one of the major issues compromising large scale production. The potential approaches to this problem include production of all female populations. Consequently, the objective of this study was to develop an effective protocol to induce meiotic gynogenesis in the Atlantic cod by using hydrostatic pressure shock. Our first experiment tested the relevance of gamete quality on achievement of chromosome manipulation and identified the best time interval between fertilization and pressure shock. Our second experiment was designed to determine the optimal pressure value and duration of the pressure shock. Eight combinations of pressure values and durations were tested. Among them, the 34.47 MPa/6 min combination gave the best survival rate (23.6 ± 3.9%), the highest percentage of normal larvae (15.7 ± 3.6%), and the highest percentage of meiotic diploids (88.89%). In both experiments, haploid controls served as an indirect reference for paternal DNA inactivation. Chromosome counting confirmed the restoration of diploidy in gynogenetic fish. The present study optimizes a procedure for the induction of meiotic gynogenesis in the Atlantic cod, thus laying the basis for further applications towards producing monosex and defining the sex determination system.

Maternal gene expression in Atlantic halibut (Hippoglossus hippoglossus L.) and its relation to egg quality.

BACKGROUND: The commercial production of Atlantic halibut (Hippoglossus hippoglossus L.) suffers from a major bottleneck due to the low success of producing juveniles for on-growing. Atlantic halibut females are routinely hand-stripped and incorrect timing of stripping can result in low quality eggs due to post-ovulatory aging. Post-ovulatory aging leads to compositional changes in eggs that include maternally provided proteins and RNAs. There have been few studies of the maternally provided mRNA transcripts that control early development in commercially important fish species. The present study aimed to study maternal gene expression in Atlantic halibut and its relation to egg quality parameters including blastomere symmetry and hatching success. RESULTS: A maternal EST library containing 2341 sequences was constructed by suppressive subtractive hybridisation. Thirty genes were selected for expression studies; 23 novel genes and 7 genes with documented roles in early development. The expressions of twenty-one selected genes were measured by qPCR from fertilization to the 10-somite stage. Three genes were identified as strictly maternal genes that were expressed until the start of gastrulation; askopos (kop), si:dkey-30j22.9 (Tudor family member), and Tudor 5 protein (Tdrd5). The expressions of 18 genes at the 8-cell stage were correlated with egg quality parameters. The majority of genes showed either no or very minor correlations with egg quality parameter. However, two genes correlated positively with hatching success (r> 0.50, HHC00353: r = 0.58, p < 0.01; HHC01517: r = 0.56, p < 0.01) and one gene (HHC00255) was negatively correlated with the percentage of normal blastomeres (r = -0.62, p < 0.05). CONCLUSIONS: During this study we have related maternal levels of gene expression to hatching success in fish. Poor hatching success was not correlated with a general decrease in transcript abundance but with low transcript levels of some specific genes. Thus, the molecular mechanisms leading to low Atlantic halibut egg quality cannot be entirely explained by post-ovulatory aging.