Heart rate as an indicator of stress during the critical swimming speed test of farmed Atlantic salmon (Salmo salar L.).

A swim tunnel is to fish as a treadmill is to humans, and is a device used for indirect measuring of the metabolic rate. This study aims to explore the fish stress (if any) during the critical swimming test routines (fish handling, confinement, and swimming) using heart rate (fH , heartbeat per minute) bio-loggers in farmed Atlantic salmon (Salmo salar L.). In addition, the recovery dynamics of exercised fish using fH were explored for 48 h post swim tests. Continuous fH data were acquired following the surgical implantation and throughout the trials, such as during fish handling, swim tests (critical swimming speed, Ucrit ), and 48 h post swim tests. After 3 weeks of surgical recovery, fH stabilized at 46.20 ± 1.26 beats min-1 , equalizing a ~38% reduction in fH recorded post-surgical tachycardia (74.13 ± 1.44 beats min-1 ). Interestingly, fH was elevated by ~200% compared to baseline levels not only due to the Ucrit (92.04 ± 0.23 beats min-1 ) but also due to fish handling and confinement in the swim tunnel, which was 66% above the baseline levels (77.48 ± 0.34 beats min-1 ), suggesting fish stress. Moreover, significantly higher plasma cortisol levels (199.56 ± 77.17 ng mL-1 ) corresponding to a ~300% increase compared to baseline levels (47.92 ± 27.70 ng mL-1 ) were identified after Ucrit , predicting post-swim test stress (physiological exhaustion). These findings reinforce the importance of fish acclimation in the swim tunnel prior to the swimming tests. However, fH dropped over the course of the 48-h post-swim test, but remained comparatively higher than the basal levels, suggesting fish should be given at least 48 h to recover from handling stress for better fish welfare. This study further explored the influence of fish tagging on Ucrit , which resulted in reduced swimming capabilities of tagged fish (1.95 ± 0.37 body lengths s-1 ) compared to untagged fish (2.54 ± 0.42 body length s-1 ), although this was not significant (p = 0.06), and therefore future tagging studies are warranted.

Bone without minerals and its secondary mineralization in Atlantic salmon (Salmo salar): the recovery from phosphorus deficiency.

Calcium and phosphorus (P) are the main bone minerals, and P deficiency can cause hypomineralized bones (osteomalacia) and malformations. This study used a P-deficient salmon model to falsify three hypotheses. First, an extended period of dietary P deficiency does not cause pathologies other than osteomalacia. Second, secondary mineralization of non-mineralized bone is possible. Third, secondary mineralization can restore the bones' mineral composition and mechanical properties. For 7 weeks, post-smolt Atlantic salmon (Salmo salar) received diets with regular P content (RP) or with a 50% lowered P content (LP). For additional 9 weeks, RP animals continued on the regular diet (RP-RP). LP animals continued on the LP diet (LP-LP), on a regular P diet (LP-RP) or on a high P diet (LP-HP). After 16 weeks, animals in all groups maintained a non-deformed vertebral column. LP-LP animals continued bone formation albeit without mineralization. Nine weeks of RP diet largely restored the mineral content and mechanical properties of vertebral bodies. Mineralization resumed deep inside the bone and away from osteoblasts. The history of P deficiency was traceable in LP-RP and LP-HP animals as a ring of low-mineralized bone in the vertebral body endplates, but no tissue alterations occurred that foreshadow vertebral body compression or fusion. Large quantities of non-mineralized salmon bone have the capacity to re-mineralize. If 16 weeks of P deficiency as a single factor is not causal for typical vertebral body malformations, other factors remain to be identified. This example of functional bone without minerals may explain why some teleost species can afford to have an extremely low mineralized skeleton.

Effect of temperature and diet on wound healing in Atlantic salmon (Salmo salar L.).

Compromised skin integrity of farmed Atlantic salmon, commonly occurring under low temperature and stressful conditions, has major impacts on animal welfare and economic productivity. Even fish with minimal scale loss and minor wounds can suffer from secondary infections, causing downgrading and mortalities. Wound healing is a complex process, where water temperature and nutrition play key roles. In this study, Atlantic salmon (260 g) were held at different water temperatures (4 or 12 °C) and fed three different diets for 10 weeks, before artificial wounds were inflicted and the wound healing process monitored for 2 weeks. The fish were fed either a control diet, a diet supplemented with zinc (Zn) or a diet containing a combination of functional ingredients in addition to Zn. The effect of diet was assessed through subjective and quantitative skin histology and the transcription of skin-associated chemokines. Histology confirmed that wound healing was faster at 12 °C. The epidermis was more organised, and image analyses of digitised skin slides showed that fish fed diets with added Zn had a significantly larger area of the epidermis covered by mucous cells in the deeper layers after 2 weeks, representing more advanced healing progression. Constitutive levels of the newly described chemokines, herein named CK 11A, B and C, confirmed their preferential expression in skin compared to other tissues. Contrasting modulation profiles at 4 and 12 °C were seen for all three chemokines during the wound healing time course, while the Zn-supplemented diets significantly increased the expression of CK 11A and B during the first 24 h of the healing phase.

Functional genomics reveals increases in cholesterol biosynthetic genes and highly unsaturated fatty acid biosynthesis after dietary substitution of fish oil with vegetable oils in Atlantic salmon (Salmo salar).

BACKGROUND: There is an increasing drive to replace fish oil (FO) in finfish aquaculture diets with vegetable oils (VO), driven by the short supply of FO derived from wild fish stocks. However, little is known of the consequences for fish health after such substitution. The effect of dietary VO on hepatic gene expression, lipid composition and growth was determined in Atlantic salmon (Salmo salar), using a combination of cDNA microarray, lipid, and biochemical analysis. FO was replaced with VO, added to diets as rapeseed (RO), soybean (SO) or linseed (LO) oils. RESULTS: Dietary VO had no major effect on growth of the fish, but increased the whole fish protein contents and tended to decrease whole fish lipid content, thus increasing the protein:lipid ratio. Expression levels of genes of the highly unsaturated fatty acid (HUFA) and cholesterol biosynthetic pathways were increased in all vegetable oil diets as was SREBP2, a master transcriptional regulator of these pathways. Other genes whose expression was increased by feeding VO included those of NADPH generation, lipid transport, peroxisomal fatty acid oxidation, a marker of intracellular lipid accumulation, and protein and RNA processing. Consistent with these results, HUFA biosynthesis, hepatic beta-oxidation activity and enzymic NADPH production were changed by VO, and there was a trend for increased hepatic lipid in LO and SO diets. Tissue cholesterol levels in VO fed fish were the same as animals fed FO, whereas fatty acid composition of the tissues largely reflected those of the diets and was marked by enrichment of 18 carbon fatty acids and reductions in 20 and 22 carbon HUFA. CONCLUSION: This combined gene expression, compositional and metabolic study demonstrates that major lipid metabolic effects occur after replacing FO with VO in salmon diets. These effects are most likely mediated by SREBP2, which responds to reductions in dietary cholesterol. These changes are sufficient to maintain whole body cholesterol levels but not HUFA levels.

Conjugated linoleic acid affects lipid composition, metabolism, and gene expression in gilthead sea bream (Sparus aurata L).

To maximize growth, farmed fish are fed high-fat diets, which can lead to high tissue lipid concentrations that have an impact on quality. The intake of conjugated linoleic acid (CLA) reduces body fat in mammals and this study was undertaken to determine the effects of dietary CLA on growth, composition, and postprandial metabolic variables in sea bream. Fish were fed 3 diets containing 48 g/100 g protein and 24 g/100 g fat, including fish oil supplemented with 0 (control), 2, or 4% CLA for 12 wk. Feed intake, specific growth rate, total body fat, and circulating somatolactin concentration were lower in fish fed CLA than in controls. Feed efficiency was greater in fish fed 2% CLA than in controls. Liver triglyceride concentrations were higher in fish fed 4% CLA and muscle triglyceride concentrations were lower in fish fed both CLA diets than in controls. Hepatic fatty acyl desaturase and elongase mRNA levels in fish fed CLA were lower than in controls. Metabolic differences between controls and CLA-fed fish were observed at 6 h but not at 24 h after the last meal, including lower postprandial circulating triglyceride concentrations, higher hepatic acyl-CoA-oxidase, and lower L-3-hydroxyacyl-CoA dehydrogenase activities in CLA-fed fish than in controls. Dietary CLA did not affect enzymes involved in lipogenesis including hepatic fatty acid synthase and malic enzyme, but it decreased glucose 6-phosphate dehydrogenase activity at 24 h, but not at 6 h after feeding. The data suggest that CLA intake in sea bream has little effect on hepatic lipogenesis, channels dietary lipid from adipose tissue to the liver, and switches hepatic mitochondrial to peroxisomal beta-oxidation.

Effect of dietary conjugated linoleic acid (CLA) on lipid composition, metabolism and gene expression in Atlantic salmon (Salmo salar) tissues.

Dietary conjugated linoleic acid (CLA) affects fat deposition and lipid metabolism in mammals, including livestock. To determine CLA effects in Atlantic salmon (Salmo salar), a major farmed fish species, fish were fed for 12 weeks on diets containing fish oil or fish oil with 2% and 4% CLA supplementation. Fatty acid composition of the tissues showed deposition of CLA with accumulation being 2 to 3 fold higher in muscle than in liver. CLA had no effect on feed conversion efficiency or growth of the fish but there was a decreased lipid content and increased protein content after 4% CLA feeding. Thus, the protein:lipid ratio in whole fish was increased in fish fed 4% CLA and triacylglycerol in liver was decreased. Liver beta-oxidation was increased whilst both red muscle beta-oxidation capacity and CPT1 activity was decreased by dietary CLA. Liver highly unsaturated fatty acid (HUFA) biosynthetic capacity was increased and the relative proportion of liver HUFA was marginally increased in salmon fed CLA. CLA had no effect on fatty acid Delta6 desaturase mRNA expression, but fatty acid elongase mRNA was increased in liver and intestine. In addition, the relative compositions of unsaturated and monounsaturated fatty acids changed after CLA feeding. CLA had no effect on PPARalpha or PPARgamma expression in liver or intestine, although PPARbeta2A expression was reduced in liver at 4% CLA feeding. CLA did not affect hepatic malic enzyme activity. Thus, overall, the effect of dietary CLA was to increase beta-oxidation in liver, to reduce levels of total body lipid and liver triacylglycerol, and to affect liver fatty acid composition, with increased elongase expression and HUFA biosynthetic capacity.

Compressed vertebrae in Atlantic salmon Salmo salar: evidence for metaplastic chondrogenesis as a skeletogenic response late in ontogeny.

Anterior/posterior (a/p) compression of the vertebral column, referred to as 'short tails', is a recurring event in farmed Atlantic salmon. Like other skeletal deformities, the problem usually becomes evident in a late life phase, too late for preventive measures, making it difficult to understand the aetiology of the disease. We use structural, radiological, histological, and mineral analyses to study 'short tail' adult salmon and to demonstrate that the study of adult fish can provide important insights into earlier developmental processes. 'Short tails' display a/p compressed vertebrae throughout the spine, except for the first post-cranial vertebrae. The vertebral number is unaltered, but the intervertebral space is reduced and the vertebrae are shorter. Compressed vertebrae are characterized by an unchanged central part, altered vertebral end plates (straight instead of funnel-shaped), an atypical inward bending of the vertebral edges, and structural alterations in the intervertebral tissue. The spongiosa is unaffected. The growth zones of adjacent vertebrae fuse and blend towards the intervertebral space into chondrogenic tissue. This tissue produces different types of cartilage, replacing the notochord. The correspondence in location of intervertebral cartilage and deformed vertebral end plates, and the clearly delimited, unaltered, central vertebral parts suggest that the a/p compression of vertebral bodies is a late developmental disorder that may be related to a metaplastic shift of osteogenic tissue into chondrogenic tissue in the vertebral growth zone. Given the lack of evidence for infections, metabolic disorders and/or genetic disorders, we propose that an altered mechanical load could have caused the transformation of the bone growth zones and the concomitant replacement of the intervertebral (notochord) tissue by cartilaginous tissues in the 'short tails' studied here. This hypothesis is supported by the role that notochord cells are known to play in spine development and in maintaining the structure of the intervertebral disk.

Formation of short-lived multinucleated giant cells (MGCS) from cultured gilthead seabream macrophages.

Monocytes/macrophages obtained from the head kidney and peritoneal cavity of gilthead seabream (Sparus aurata) were cultured using plates from three different manufacturers, and were maintained under different conditions. The effects on the morphology and fusion of monocytes/macrophages of initial cell loading, removal of non-adherent cells at different times after plating, and addition of serum and antibiotics were evaluated by light microscopy, and transmission (TEM) and scanning (SEM) electron microscopy. Despite variations in adherence, the behaviour and the morphological changes in kidney monocytes/macrophages were similar in all three types of plates. When foetal calf serum (FCS) was added to the incubation medium, most of the cells resembling monocytes/macrophages were connected by cytoplasmic extensions that formed bridges after 24 hr in culture. After 30 hr, the monocytes/macrophages started to fuse, forming multinucleated giant cells (MGCs) which gradually increased in size until the culture was 4-5 days old. After 5 days the MGCs started to die, and after a week most had disappeared from the cultures. Cells incubated with medium without serum showed changes similar to those fed with FCS, but some cells survived for 3 weeks. The addition of fish serum to the medium appeared to accelerate all processes: the monocytes/macrophages and MGCs died after 3 days in culture. Antibiotics had no apparent effect on the cultures. Removal of non-adherent cells at different times after plating did not appear to affect cell fusion. Coating the wells with extracellular matrix proteins reduced adherence but did not inhibit cell fusion. Curiously, not all macrophages fused with MGCs, and, unlike MGCs, these macrophages phagocytosed sheep red blood cells (SRBCs). Peritoneal macrophages also fused and formed MGCs in culture, similarly to kidney cells.