Ørjan Hagen

Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development.

Gene expression studies are fundamental to understand the molecular basis of severe malformations in fish development, particularly under aquaculture conditions. Real-time PCR (qPCR) is the most accurate method of quantifying gene expression, provided that suitable endogenous controls are used to normalize the data. To date, no reference genes have been validated for developmental gene expression studies in Atlantic halibut (Hippoglossus hippoglossus). We have determined the expression profiles of 6 candidate reference genes (Actb, Eef2, Fau, Gapdh, Tubb2 and 18S rRNA) in 6 embryonic and 5 larval stages of Atlantic halibut development. There were significant changes in expression levels throughout development, which stress the importance and complexity of finding appropriate reference genes. The three software applications (BestKeeper, geNorm and NormFinder) used to evaluate the stability of potential reference genes produced comparable results. Tubb2 and Actb were the most stable genes across the different developmental stages, whereas 18S rRNA and Gapdh were the most variable genes and thus inappropriate to use as reference genes. According to geNorm and NormFinder, the best two-gene normalization factors corresponded to the geometric average of Tubb2/Actb and Tbb2/Fau, respectively. We believe that either of these normalization factors can be used for future developmental gene expression studies in Atlantic halibut.

Expression of growth-related genes in muscle during fasting and refeeding of juvenile Atlantic halibut, Hippoglossus hippoglossus L.

The aim of this study was to establish a fasting-refeeding protocol to investigate the expression of growth-related genes during the transition between catabolic and anabolic states in Atlantic halibut (Hippoglossus hippoglossus L.). Juveniles of approximately 950 g were maintained at ambient temperature (5-8 degrees C) until the 1st of May, then fasted for two months and refed for two months at 7.7-8.0 degrees C under continuous low light. Fast epaxial myotomal muscle was sampled at -64 d (days), -38 d, 0 d (start of refeeding), 3 d, 7 d, 14 d, 30 d and 60 d. Average body mass was unchanged over the fasting period but increased by 24.4% following 60 d refeeding. qPCR was used to analyse the stability of expression of five potential reference genes (Eef2, Fau, 18SrRNA, Actb and Tubb2) with GeNorm and Normfinder. Expression of the growth-related genes, cathepsin B (ctsb), cathepsin D (ctsd), insulin-like growth factor-I and II (IGF-I and II) and insulin-like growth factor-I receptor 1a (IGF-IRa) was normalised using the geometric average of the two most stable housekeeping genes, Fau and 18SrRNA. IGF-I mRNA showed a transient 2.6-fold increase in abundance with refeeding at 7 d whilst transcripts for IGF-II and IGF-IRa were elevated during fasting and decreased 3.8-fold and 3-fold between the 0 d and 3 d samples respectively. Ctsb expression increased between -64 d and 0 d and then decreased approximately 10-fold by 14 d refeeding. In contrast, ctsd was relatively unaffected by the fasting-refeeding cycle, showing a modest (approximately 35%) transient decrease in expression between the 0 d and 30 d refeeding samples. It was concluded that the experimental protocol adopted and housekeeping genes identified were suitable for investigating the catabolic-anabolic transition in halibut skeletal muscle.

Activity of aspargate (cathepsin D), cysteine proteases (cathepsins B, B + L, and H), and matrix metallopeptidase (collagenase) and their influence on protein and water-holding capacity of muscle in commercially farmed atlantic halibut (Hippoglossus hippoglossus L.).

Atlantic halibut (Hippoglossus hippoglossus L.) were commercially farmed in Helgeland, Norway (May 2004-May 2005). The average weight (Mb) of fish increased over the 12 month production cycle by approximately 73% for females and approximately 50% for males, although during the winter months (November-early May) Mb was unchanged in females and declined by 18% in males because of sexual maturation and sperm release. Periods of zero or negative growth were associated with up to 5.7% (females) and 17.9% (males) decline in fast muscle protein content. The activities of cathepsins B, B + L, H, and D showed a reciprocal relationship and were highly correlated with the changes in protein content. Water-holding capacity was measured as the liquid loss increased from 3-5% in November to 11-13% in May. Two general additive models (GAMs) showed that cathepsin B + L, cathepsin D, and collagenase explained 73.1% of the total variance in protein content, while cathepsin H was the largest contributor to liquid loss, explaining approximately 48.8% of the total variance. The results indicate that to obtain the best flesh quality Atlantic halibut should be harvested in the fall or early winter when the liquid loss and cathepsin activities are low and less likely to cause problems during secondary processing and storage.

Flesh quality and biochemistry of light-manipulated Atlantic cod (Gadus morhua) and the significance of collagen cross-links on fillet firmness and gaping.

The aim of our study was to investigate whether light-manipulation, causing accelerated or delayed maturation, could be used as a tool to improve fillet gaping score and texture of farmed cod (Gadus morhua L.) harvested during summer. Control and accelerated groups had completed spawning by the time of harvest, and expressed sexual dimorphism in various biometric and flesh quality parameters. Pyridinoline cross-links, a key biochemical parameter investigated, were positively correlated with texture (r(2) = 0.4), as also were cathepsin B&L and sex. Gaping was also impacted by pyridinoline cross-links, together with body mass and maturation. Interestingly, the level of gaping was significantly reduced with a pyridinoline cross-link concentration of >2500 pmols g(-1) dry mass. In conclusion, light-manipulation can be used as a tool to improve flesh texture of farmed cod with a low gaping score, but with compromise of increased dress out percentage for the accelerated group.

Growth and development of skeletal anomalies in diploid and triploid Atlantic salmon (Salmo salar) fed phosphorus-rich diets with fish meal and hydrolyzed fish protein

Diploid and triploid Atlantic salmon, Salmo salar were fed high-protein, phosphorus-rich diets (56–60% protein; ca 18g phosphorus kg-1 diet) whilst being reared at low temperature from start-feeding until parr-smolt transformation. Performances of salmon fed diets based on fish meal (STD) or a mix of fishmeal and hydrolysed fish proteins (HFM) as the major protein sources were compared in terms of mortality, diet digestibility, growth and skeletal deformities. Separate groups of diploids and triploids were reared in triplicate tanks (initially 3000 fish per tank; tank biomass ca. 620 g) from 0–2745 degree-days post-start feeding (ddPSF). Growth metrics (weight, length, condition factor) were recorded at ca. 4 week intervals, external signs of deformities to the operculum, jaws and spinal column were examined in parr sampled at 1390 ddPSF, and external signs of deformity and vertebral anomalies (by radiography) were examined in fish sampled at the end of the trial (2745 ddPSF). The triploid salmon generally had a lower mass per unit length, i.e. lower condition factor, throughout the trial, but this did not seem to reflect any consistent dietary or ploidy effects on either dietary digestibility or the growth of the fish. By the end of the trial fish in all treatment groups had achieved a weight of 50+ g, and had completed the parr-smolt transformation. The triploids had slightly, but significantly, fewer vertebrae (Triploids STD 58.74 ± 0.10; HFM 58.68 ± 0.05) than the diploids (Diploids STD 58.97 ± 0.14; HFM 58.89 ± 0.01), and the incidence of skeletal (vertebral) abnormalities was higher in triploids (Triploids STD 31 ± 0.90%; HFM 15 ± 1.44%) than in diploids (Diploids STD 4 ± 0.80%; HFM 4 ± 0.83%). The HFM diet gave a significant reduction in the numbers of triploid salmon with vertebral anomalies in comparison with the triploids fed the STD diet possibly as a result of differences in phosphorus bioavailability between the two diets. Overall, the incidence of skeletal deformities was lower than reported in previous studies (Diploids 20+%, Triploids 40+%), possibly as a result of the combination of rearing at low-temperature and phosphorus-rich diets being used in the present study.