Anarhichas Minor Olafsen Research Articles

Step by step optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772)

Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, low sperm volume with relatively low cell concentration and the lack of gametes synchronization (simultaneous availability of mature eggs and sperm) represent a challenge for the industry. Thus, the development of protocols for sperm storage are crucial. Four sequential experiments were conducted to optimize a sperm cryopreservation protocol for this species. First, three different cryoprotectants (DMSO; 1, 2-propanediol; and methanol) at different concentrations (5, 10, and 20%) were tested for their toxicity. No significant differences (p > 0.05) were detected between the control samples and cryoprotectants at concentration up to 10% DMSO, 10% propanediol, and 20% methanol in terms of motility parameters. Second, using the highest non-toxic concentrations of cryoprotectants, sperm was cryopreserved in 0.5 mL straws, at different distances from the liquid nitrogen (1.5, 2.5, 4.5, and 7.5 cm) that correspond to different freezing rates. Motility parameters after freezing/thawing decreased for all the cryoprotectants (p < 0.001), however, methanol had the lowest protective capacity while DMSO the highest. Afterwards, two different thawing rates (1 min at 5 °C; and 25 s at 10 °C) were tested using only 10% DMSO and 10% propanediol. Both for the DMSO and propanediol, there were no significant differences (p > 0.05) between the two thawing rates. The best results were obtained using 10% DMSO. Finally, the fertilization capacity of cryopreserved sperm (10% DMSO and thawed at 5 °C for 1 min) was tested against fresh sperm using two spermatozoa:egg ratios and 4 h gametes contact time. The ratio of eggs with normal cell cleavage, abnormal cleavage or undeveloped were counted at the 2–4 cell stage. Cryopreserved sperm showed lower fertilization capacity at a concentration of 5 × 104 spermatozoa:egg compared with fresh sperm (p < 0.001). At a concentration of 5 × 105 spermatozoa:egg, similar fertilizations rates to the fresh sperm were obtained. The presence of the cryoprotectant DMSO during the 4 h contact time did not affect the fertilization rate or the percentage of embryos with abnormal cleavage (p > 0.05). To cryopreserve spotted wolffish sperm it is recommended to use 10% DMSO, loaded in 0.5 mL straws, freeze at a height between 4.5 (−14.05 °C/min) and 7.5 cm (−5.9 °C/min) from liquid nitrogen for 10 min and thaw for 1 min at 5 °C (177.9 °C/min). In vitro fertilization with cryopreserved sperm should be performed with a concentration of at least 5 × 105 spermatozoa per egg.

Metabolic and digestive enzyme activity profiles of newly hatched spotted wolffish (Anarhichas minor Olafsen): effect of temperature

Three groups of newly hatched spotted wolffish (Anarhichas minor) were held at three different temperatures in order to determine relationships between metabolic, digestive and growth response in rapidly developing larvae. Growth rates were successfully modulated by temperature (5, 8 and 12 °C). Activity levels of trypsin and lactate dehydrogenase (LDH) were positively linked to specific growth rates at all temperatures. Trypsin showed a positive compensation (higher activity at lower temperature) whereas glycolytic enzymes (pyruvate kinase and Lactate dehydrogenase) and aspartate aminotransferase (AST) showed a negative compensation (lower activity at lower temperature). Citrate synthase was not affected by growth rate, indicating that the level of aerobic capacity was adequate in sustaining the high energetic needs associated with rapid growth early in the life of the spotted wolffish. In light of our results, it is suggested that protein digestion, as demonstrated by the activity profile of trypsin in relation to growth rate and temperature, is likely a key growth-limiting agent during the early-life stages of wolffishes. Our results are discussed in comparison with A. lupus, a closely related species displaying different temperature preferences and growth capacities.

Biodiversity genomics for species at risk: patterns of DNA sequence variation within and among complete mitochondrial genomes of three species of wolffish (Anarhichas spp.)

The first marine fish species to be listed under the Canadian Species At Risk Act as Threatened with extinction are the spotted wolffish ( Anarhichas minor Olafsen, 1774) and the broadhead wolffish ( Anarhichas denticulatus Krøyer, 1844); a third species, the striped wolffish ( Anarhichas lupus L., 1758), is listed as a species of special concern. As part of the recovery plan for wolffish, we determined the complete mitochondrial DNA (mtDNA) genome sequences of all three species to identify the most variable gene regions for population analysis. Anarhichas genomes comprise either 16 519 or 16 520 base pairs (bp), among which there are 449 single nucleotide polymorphisms (SNPs). The most variable protein-coding loci are ND4, CYTB, and ND2, with 4.40, 4.22, and 4.19 SNPs / 100 bp, respectively. Comparisons of rates of synonymous and nonsynonymous nucleotide substitutions indicate no evidence of selection. The control region, characterized in many species as hypervariable, was less variable than 9 of 13 protein-coding loci (2.45 SNPs / 100 bp). Phylogenetic analysis shows that A. lupus and A. minor are more closely related to each other than either is to A. denticulatus. Amplification and sequence analysis of a contiguous block of 6392 bp that spans the ND4, ND5, ND6, and CYTB loci is an efficient strategy for evaluating patterns of intraspecific mtDNA variability.

Cage culture of spotted wolffish ( Anarhichas minor Olafsen)

Flat-bottom sea cages offer a cost-effective alternative for culture of bottom-dwelling fish. In an experiment lasting 428 days, spotted wolffish ( Anarhichas minor Olafsen) grew equally fast when kept in tanks on land and in a flat-bottom sea cage at the same stocking density (increasing from 7.5 to 42 kg m − 2 during the experiment) and same water temperature. A lag phase of 1 month with negative growth was observed following transfer from tanks to the sea cage. The results confirm that flat-bottom sea cages may be an option for culture of this species.

Endocytosis of indigenous bacteria and cell damage caused by Vibrio anguillarum in the foregut and hindgut of spotted wolffish (Anarhichas minor Olafsen) fry: an electron microscopical study

Endocytosis of indigenous bacteria and cell damage caused by Vibrio anguillarum in the foregut and hindgut of spotted wolffish (Anarhichas minor Olafsen) fry: an electron microscopical study

Influence of high-M alginate on the growth and survival of Atlantic cod ( Gadus morhua L.) and spotted wolffish ( Anarhichas minor Olafsen) fry

Atlantic cod and spotted wolffish fry were fed high-M alginate containing feed for 59 and 55 days, respectively. During this period the fry showed a higher specific growth rate compared to controls. Uptake and distribution of alginate was studied by inclusion of the 125I-labelled molecule in the feed. The stomach and intestine contained the highest amount while the kidney, liver and spleen contained some, indicating that the alginate was taken up by the gut and transported to internal organs. Cod fry fed 0.06% and 0.1% high-M alginate showed a death rate of 51.4% and 53.3%, respectively. The lowest mortality, 48.1%, was found in fry fed 0.01% high-M alginate. Controls showed a mortality rate of 49.0%. Differences were, however, not statistically significant. Challenge of the immunostimulated fry (fed 0.02% and 0.06% alginate for 62 days) with atypical Aeromonas salmonicida bacteria resulted in accumulated mortalities of 56% and 49%, respectively, 47 days after infection. The group that received 0.06% alginate for a shorter period (47 days) and then control feed until challenged, and the group that received alginate by bath reached a cumulative mortality of 59% and 60%, respectively. Lowest mortality (44%) was seen in the control group. Numerous microabscesses were found in both immunostimulated and control fish in secondary lamellae of the gills, haematopoietic tissues of the kidneys, the submucosa and mucosa of the intestine, the spleen, the liver and the myocardium of the heart.

Effects of incubation temperature on development and yolk sac conversion efficiencies of spotted wolffish (Anarhichas minor Olafsen) embryos until hatch

Two egg batches of spotted wolffish, Anarhichas minor Olafsen, were incubated at 4, 6 and 8°C. Embryo samples were fixed and compared on each 100th daydegree until hatching (up to 1000 daydegrees). Embryos, yolk sacs and chorions were dissected and the sizes, wet and dry weights were recorded separately. Comparisons of gross morphologies and measured parameters showed increasing and generally significant differences with time between the incubation temperatures. Lower temperatures produced longer and more differentiated larvae at hatch with a smaller yolk sac. Even though some unexpected deviations were registered among batches and experimental groups, it was clear that temperature affected embryo survival and time of hatching. Overall survival was best at 6°C, in agreement with results from earlier studies. Yolk conversion efficiencies measured around the hatching point were generally high, ranging from 60% to 78%, varied between the two batches and probably reflected the developmental variations between embryos and larvae at the respective ages (daydegrees). The hatching process was apparently an energy-demanding period; yolk conversion efficiencies of unhatched embryos of similar age at each temperature were always higher. Temperature is one environmental factor that can be manipulated in hatcheries to induce hatching of viable larvae at an optimal stage of differentiation with respect to first-feeding success and early survival.

The ontogeny of complement component C3 in the spotted wolffish ( Anarhichas minor Olafsen)

Eggs and larvae from the spotted wolffish ( Anarhichas minor Olafsen) were analysed to study the ontogeny of the complement component C3. By Western blotting experiments, the results revealed that C3 was present already in unfertilised eggs and that there was a decrease in C3 content towards hatch, whereas the results using reverse transcription–polymerase chain-reaction showed a continuous increase of C3 mRNA content towards hatch. These findings indicate a transfer of maternal complement component C3 to the offspring where the content of C3 decreases and the level of C3 mRNA increases towards hatch. Immunohistochemical experiments revealed that only the liver of spotted wolffish larvae contained complement component C3, suggesting that the liver is the main site of production.

Isolation and characterisation of spotted wolffish ( Anarhichas minor Olafsen) macrophages

Non-specific mechanisms are important in the defence of all multicellular animals against pathogenic microorganisms. Macrophages and granulocytes play a central role in this respect. It is thus pertinent to develop methods for obtaining and cultivation of macrophages and assessing their functions in the spotted wolffish, a cold water species of current interest for the aquaculture industry. Kidney macrophages from the spotted wolffish ( Anarhichas minor Olafsen) were isolated by density sedimentation using Percoll. The cells were highly phagocytic and possessed typical macrophage morphology evaluated by transmission and scanning electron microscopy. Using electron microscopic analysis, the size of the macrophages, collected from the Percoll density interface, was 5–9 μm. The viability in vitro was highest (87.1%) when the cells were kept at 13 °C with the addition of synthetic serum replacement (SSR-2) when measured 24 h after seeding. One day old cells were not significantly activated by addition of bacterial lipopolysaccharide (LPS) for 24 h when measured by reduction of nitroblue tetrazolium compared to control cells. The cells were negative in respect to synthesis and contents of complement component C3.

A review of the culture potential of spotted wolffish Anarhichas minor Olafsen

The first articially fertilized spotted wolffish eggs hatched only 10 years ago, and today the species is considered a very promising candidate species for cold water aquaculture in the North Atlantic. Recent research has focused on identifying key biological parameters in spotted wolffish aquaculture in order to establish a full production line for the species, and basic aspects of reproduction and larval development are now understood, controlled, and no longer limiting production.

Influence of egg lipids and fatty acids on egg viability, and their utilization during embryonic development of spotted wolf-fish, Anarhichas minor Olafsen

The changes in egg lipids and fatty acid compositions that occur during embryonic development of spotted wolf-fish, Anarhichas minor, were examined by monitoring individual egg batches from the time of spawning (egg stripping) until hatching. The lipids, present as 3.7±0.1% of the wet mass of the freshly stripped eggs, contained high percentages of monoenes (monounsaturated fatty acids (MUFAs), ca. 33%) and polyenes (ca. 43%) and approximately 20% saturated fatty acids (SFAs). The fatty acid profiles were dominated by a small number of fatty acids. The major SFA was 16:0 (ca. 14%), the dominant MUFA was 18:1 n-9 (ca. 21%), and among the polyenes, the n-3 highly unsaturated fatty acids (HUFAs) 22:6 n-3 docosahexaenoic acid (DHA) and 20:5 n-3 eicosapentaenoic acid (EPA) were present in the highest concentrations (EPA, ca. 16%; DHA, ca. 19%). The n-6 HUFA 20:4 n-6 arachidonic acid (AA) was present as ca. 1% of the total fatty acids in the freshly stripped eggs. This resulted in an AA:EPA of ca. 0.07, which is lower than reported for eggs of many other fish species. As embryonic development progressed, the percentage contribution of AA to the total fatty acids almost doubled. There were also increases in the relative proportions of SFAs (due mainly to an increase in the percentage of 16:0 to ca. 16% at hatch) and DHA (to ca. 23%), and there was a corresponding decrease in the percentage of MUFAs (mostly brought about by a decrease in the percentage of 18:1 n-9 to ca. 18% at hatch). The most marked changes occurred towards the end of incubation. The percentage of EPA changed little during incubation. This implies that there was selective retention of DHA, 16:0 and AA, and these fatty acids were probably incorporated into cell membranes. MUFAs, particularly 18:1 n-9, seem to have been catabolized to provide energy for the developing embryo, and some EPA also seems to have been utilized as an energy source. Survival of eggs to the eyed stage (range ca. 10–80%) and to hatch (ca. 5–75%) was negatively correlated with the %AA, %EPA and AA:DHA of the freshly stripped eggs. There was also a negative correlation between AA:EPA and egg survival, which implies that there is not a universal requirement for a high AA:EPA to ensure high rates of survival of fish eggs.

Early ontogeny of the spotted wolffish (Anarhichas minor Olafsen)

This study illustrates the embryo development of the spotted wolffish (Anarhichas minor Olafsen), an interesting candidate for cold-water aquaculture. The egg morphology (semitransparent, yellow-white with numerous oil droplets in the yolk), size (5.4–6.5 mm) and long embryogenesis (c. 800–1000 d°, depending on temperature) of A. minor are very similar to Anarhichas lupus. Cleavage is slow, and the first cell divisions take place at 12 h at 8°C. After 12 days the 2-mm embryo with the first somites is laid down and the blastopore starts closing. The fat globules in the yolk fuse into one after 22 days, and after 30 days eye pigmentation is noticeable. After 44 days, eye pigmentation is strong, the digestive tract folded and a green gall bladder can be noted in the now 11-mm-long embryo. One week later the blood is brightly red, the intestine is pigmented and the lower jaw is well developed. Premature hatching may occur from this stage. After 58 days vascularization of the yolk is complete, capillaries are noted in the fin fold, the first ray rudiments are established in the tail and pectoral fins, and the four gill arches are covered by the operculum. The preanal finfold is reduced after 72 days, stomach and gill filaments are formed, and six pigmented rows are noted on the 17-mm-long embryo body. After 86 days all fin rays are seen and the digestive tract is intensely pigmented and folded. Hatching (normal) starts after 110 days and may last for 2–3 weeks. Late embryos and early larvae of A. minor have more distinct bands of pigment along the body compared with the closely related A. lupus. An increase in both length and weight of the embryos in individual batches occurs during the hatching period.

Vaccination and immune responses against atypical Aeromonas salmonicida in spotted wolffish ( Anarhichas minor Olafsen) juveniles

To study the effect of early vaccination, wolffish juveniles of size 50 and 90 mm, respectively, were vaccinated with an oil-adjuvanted atypical A. salmonicida bacterin. Vaccination resulted in significant protection after challenge with the homologous bacterial strain and specific antibody responses were demonstrated against whole bacteria as well as purified A-layer protein and LPS by ELISA and Western blotting but individual variation in immune responses was apparent. The A-protein was the most immunogenic bacterial component. In addition, higher numbers of immunoglobulin producing cells were detected by in situ hybridisation in kidney and spleen of vaccinated fish compared to non-vaccinated fish. Plasma cells were also present in gut and gills in equal numbers irrespective of treatment. No plasma cells were found in the skin. Finally, the frequencies of expressed V Hfamilies and C Lisotypes of wolffish immunoglobulins were shown by PCR. The relative expression of the three variable regions of the Ig heavy chain and the three isotypes of the Ig light chain in the spotted wolffish spleen seemed to be unaffected by immunisation with a complex antigen like the A. salmonicida bacterin.

Mitogenic responses of leukocytes from the spotted wolffish ( Anarhichas minor Olafsen)

Mitogenic responses of leukocytes from the spotted wolffish ( Anarhichas minor Olafsen)

Effects of chronic ammonia exposure on growth and food conversion efficiency in juvenile spotted wolffish

The effects of chronic un-ionised ammonia (UIA) exposure on growth and food conversion efficiency (FCE) in spotted wolffish, Anarhichas minor Olafsen, were examined. Fish with a mean (S.D.) initial weight 14.2 (5.0) g were reared in shallow raceways (initial stocking density of 3.9 kg m −2) and exposed to four concentrations of UIA [0.0006 (control), 0.13 (low), 0.25 (medium) and 0.39 mg l −1 (high)] for 69 days at 8.3 °C, 33‰ and pH 8.1. Growth rates were significantly influenced by UIA concentrations, as the control and low-concentration group performed better than both the medium- and high-concentration group, and the medium group in turn displayed a higher growth rate than the high-concentration group throughout the experimental period. While experiencing negative growth rates in the first period, growth rates increased and were positive in fish from both the medium and high group in the second and third period, indicating an adaptation to ambient UIA. At the end of the experiment, mean weights were 13% and 40% lower at UIA concentrations of 0.13 and 0.25 mg l −1, respectively, when compared to the control group, whereas an overall decrease in mean weight was observed at 0.39 mg UIA l −1 in the experimental period investigated. The difference in growth performance is mainly attributed to variations in daily feed consumption, which varied significantly between all treatments throughout the experiment and decreased with increasing concentrations of UIA, and to a lesser extent by a decrease in food conversion efficiency.

Immunoglobulin V Hfamilies and light chain isotypes in the spotted wolffish ( Anarhichas minor Olafsen)

The spotted wolffish ( Anarhichas minor Olafsen) is a species of the Perciformes, the most diverse and numerous order of all fish. A cDNA library from head kidney tissue was screened for immunoglobulin (Ig) heavy and light chain transcripts, and showed highest identity to Ig sequences from other perciform species. So far only one Ig class is described in spotted wolffish, but three V Hfamilies were identified among the heavy chain transcripts. Highest diversity was located at the CDR3 region and demonstrates the importance of this gene element to the antibody repertoire. Two V Lfamilies were identified among the light chain clones and three distinct isotypes were present. Use of polymerase chain reaction and in situ hybridisation techniques revealed individual variations in the relative expression of the three isotypes of light chains.

Growth and oxygen consumption in normal and O2 supersaturated water, and interactive effects of O2 saturation and ammonia on growth in spotted wolffish (Anarhichas minor Olafsen)

The effects of dissolved oxygen (DO) concentration combined with sublethal doses of ammonia on growth and food conversion efficiency (FCE) were investigated in juvenile spotted wolffish. Fish with a mean (S.D.) initial weight of 95.4 (18.0) g were reared in shallow raceways (initial stocking density 10 kg m −2 ) at normoxic (9.6 mg l −1 ) and hyperoxic (14.5 mg l −1 ) conditions, and also under both normoxia and hyperoxia with an additional sublethal concentration of unionised ammonia (0.17 mg NH 3 l −1 ) added, for 8 weeks at 8 °C. There was an interacting effect of oxygen saturation and ammonia level on growth, as growth rate was significantly higher in the hyperoxic/NH 3 group compared to the normoxic/NH 3 group ( P <0.001), suggesting that hyperoxic conditions may increase tolerance to unionised ammonia in spotted wolffish. At the end of the experiment, no difference in mean weight was found between fish reared at normoxic and hyperoxic conditions without added ammonia, whereas mean weight was reduced under normoxia with added ammonia as compared to normoxia without added ammonia. Mean daily oxygen consumption was investigated in the non-ammonia groups at both normoxic and hyperoxic conditions, and O 2 consumption was significantly higher ( P <0.001) under hyperoxic (100.5 mg O 2 kg −1 h −1 ) compared to normoxic (79.9 mg O 2 kg −1 h −1 ) conditions. A clear diurnal rhythm in the O 2 consumption pattern was seen at both O 2 saturations, with the most striking observation being a dramatic increase in the morning when the light was turned on. Overall, our results support earlier findings of a high capacity to adapt to variations in environmental water quality parameters, and to restore normal growth rate in the spotted wolffish.

The spotted wolffish ( Anarhichas minor Olafsen) complement component C3: isolation, characterisation and tissue distribution

The complement component C3 was isolated from spotted wolffish ( Anarhichas minor Olafsen) serum by polyethylene glycol precipitation, anion exchange chromatography and gel filtration. Silver staining in SDS-PAGE and rabbit anti-wolffish C3 antiserum used in Western blotting revealed that spotted wolffish C3 contains two polypeptide chains, M r65 and 115 kDa, respectively. The high molecular weight α-chain of the C3 incorporated 14C-methylamine suggesting that it contained a reactive thioester group. The deduced amino acid sequence, after screening a liver cDNA expression library, showed that the wolffish C3 contained key amino acids for binding C3 convertase, factor H, I and properdin. Also, high degree of homology to other vertebrate C3 was found in the β–α junction site. Phylogenetic tree analysis indicated that the Japanese flounder and spotted wolffish that belong to order pleuronectiformes and perciformes, respectively, are phylogenetically close species. Immunohistochemical experiments showed that liver hepatocytes and blood contained C3, and in situ hybridisation experiments revealed that liver hepatocytes expressed C3.

Immunoglobulin producing cells in the spotted wolffish ( Anarhichas minor Olafsen): localization in adults and during juvenile development

The presence of immunocompetent cells was studied in the larval and adult stages of the spotted wolffish, Anarhichas minor. In situ hybridization with a probe complementary to the secretory Igμ-chain was used to localize immunoglobulin producing cells or plasma cells in organs from adult fish and the appearance of these cells in lymphoid tissues during juvenile development. Plasma cells were located in pronephros, spleen, gut, gills and skin of adult wolffish. In juveniles, the first plasma cells were detected in the kidney 1 week post-hatching and the appearance in other lymphoid organs was in the order spleen, gut and thymus. No plasma cells were detected in skin and gills during the sampling period of juveniles (<10 cm). Our study confirmed that plasma cells are present in both the systemic and mucosal compartments of adult fish but during ontogeny there is an earlier appearance of plasma cells in the gut compared to gill and skin compartments.

Graded environmental hypercapnia in juvenile spotted wolffish (Anarhichas minor Olafsen): effects on growth, food conversion efficiency and nephrocalcinosis

Abstract Growth performance and food conversion efficiency (FCE) were investigated in juvenile spotted wolffish (Anarhichas minor Olafsen), mean (S.D.) initial weight 15.7 (4.8) g, reared at four levels of carbon dioxide (CO2(aq)) for 10 weeks at 6 °C and 33‰. CO2 levels averaged 1.1 (control), 18.1 (low), 33.5 (medium) and 59.4 (high) mg l−1, with corresponding pH values of 8.10, 6.98, 6.71 and 6.45, respectively. In addition, kidneys from sampled fish were examined macroscopically for gross signs of calcareous deposits, i.e. nephrocalcinosis, at the start and end of the experiment. Growth was significantly reduced at the highest concentration (P