Disease resistance, stress response and effects of triploidy in growth hormone transgenic coho salmon

Diploid and triploid coho salmon Oncorhynchus kisutch transgenic for growth hormone (GH) and control coho salmon were compared for differences in disease resistance and stress response. Resistance to the bacterial pathogen Vibrio anguillarum was not affected in transgenic fish relative to their non‐transgenic counterparts when they were infected at the fry stage, but was lower in transgenic fish when infected near smolting. Vaccination against vibriosis provided equal protection to both transgenic and non‐transgenic fish. Triploid fish showed a lower resistance to vibriosis than their diploid counterparts. Diploid transgenic fish and non‐transgenic fish appeared to show similar physiological and cellular stress responses to a heat shock. These studies provide information useful for both performance and ecological risk assessments of growth‐accelerated coho salmon.     

Oxygen uptake of growth hormone transgenic coho salmon during starvation and feeding

Oxygen uptake of growth hormone transgenic coho salmon Oncorhynchus kisutch was measured in individual fish with a closed-system respirometer and was compared with that of similar-sized non-transgenic control coho salmon during starvation and when fed a fixed ration or to satiation. Transgenic and control fish did not differ in their standard oxygen uptake after 4 days of starvation, although control fish had a higher routine oxygen uptake, scope for spontaneous activity and initial acclimation oxygen uptake. During feeding, transgenic fish ate significantly more than control fish, and had an overall oxygen uptake that was 1·7 times greater than control fish. When fish that had eaten the same per cent body mass were compared, transgenic fish had an oxygen uptake that was 1·4 times greater than control fish. Differences in oxygen uptake in growth hormone transgenic coho salmon and non-transgenic fish appear to be due to the effects of feeding, acclimation and activity level, and not to a difference in basal metabolism.     

The glutathione antioxidant system is enhanced in growth hormone transgenic coho salmon (<Emphasis Type="Italic">Oncorhynchus kisutch</Emphasis>)

Insertion of a growth hormone (GH) transgene in coho salmon results in accelerated growth, and increased feeding and metabolic rates. Whether other physiological systems within the fish are adjusted to this accelerated growth has not been well explored. We examined the effects of a GH transgene and feeding level on the antioxidant glutathione and its associated enzymes in various tissues of coho salmon. When transgenic and control salmon were fed to satiation, transgenic fish had increased tissue glutathione, increased hepatic glutathione reductase activity, decreased hepatic activity of the glutathione synthesis enzyme γ-glutamylcysteine synthetase, and increased intestinal activity of the glutathione catabolic enzyme γ-glutamyltranspeptidase. However, these differences were mostly abolished by ration restriction and fasting, indicating that upregulation of the glutathione antioxidant system was due to accelerated growth, and not to intrinsic effects of the transgene. Increased food intake and ability to digest potential dietary glutathione, and not increased activity of glutathione synthesis enzymes, likely contributed to the higher levels of glutathione in transgenic fish. Components of the glutathione antioxidant system are likely upregulated to combat potentially higher reactive oxygen species production from increased metabolic rates in GH transgenic salmon.     

Disease resistance and health parameters of growth-hormone transgenic and wild-type coho salmon,Oncorhynchus kisutch

To extend previous findings regarding fish health and disease susceptibility of growth-enhanced fish, hematological and immunological parameters have been compared between growth hormone (GH) transgenic and wild-type non-transgenic coho salmon (Oncorhynchus kisutch). Compared to non-transgenic coho salmon, transgenic fish had significantly higher hematocrit (Hct), hemoglobin (Hb), mean cellular hemoglobin (MCH), mean cellular volume (MCV), and erythrocyte numbers, and lower white cell numbers. In addition, resistance to the bacterial pathogen Aeromonas salmonicida (causal agent of furunculosis) has been assessed between the strains. Higher susceptibility of transgenic fish to this disease challenge was observed in two separate year classes of fish. The present findings provide fundamental knowledge of the disease resistance on GH enhanced transgenic coho salmon, which is of importance for assessing the fitness of transgenic strains for environmental risk assessments, and for improving our understanding effects of growth modification on basic immune functions.     

Fasting modulates metabolic responses to cortisol, GH and IGF‐I in Arctic charr hepatocytes

Hepatocytes in primary culture from fed and 2 month fasted Arctic charr Salvelinus alpinus were exposed to physiological doses of either cortisol, salmon growth hormone (GH), salmon insulin‐like growth factor‐I (IGF‐I) or a combination of salmon GH and salmon IGF‐I. Fasting significantly lowered medium glucose levels compared to the fed fish, but had no significant effects on hepatocyte glycogen content or on the activities of enzymes involved in the intermediary metabolism. Cortisol treatment had no effect on hepatocyte glycogen content or on the enzyme activities investigated, but resulted in a significant increase in medium glucose concentration in hepatocytes isolated from fasted, but not fed fish. GH and IGF‐I treatments, both singly and in combination, significantly increased the glycogen content of hepatocytes isolated from fed fish, with less pronounced effects on hepatocytes isolated from fasted fish. The combination of GH and IGF‐I significantly increased lactate dehydrogenase activity regardless of the feeding state and significantly reduced the phosphenolpyruvate carboxykinase activity and medium glucose concentration in hepatocytes isolated from fed fish. Further, GH and IGF‐I significantly increased the activities of alanine aminotransferase and aspartate aminotransferase in hepatocytes isolated from fasted fish, but not fed fish. There were no effects of GH, IGF‐I, or their combination, on glucose 6‐phosphate dehydrogenase or 3‐hydroxyacyl‐CoA dehydrogenase activities. The results demonstrated that nutritional status of the animal modulates hepatocyte responsiveness to metabolic hormones, and suggested a role for GH and IGF‐I in hepatic glycogen conservation.     

Embryo survival and smolt to adult survival in second‐generation outbred coho salmon

Outbreeding depression was not detectable in observations of embryonic survival and of survival of smolts to adulthood relative to controls in coho salmon Oncorhynchus kisutch experimentally outbred over two generations by crossing fish from three widely separated populations. Survival in outbred coho salmon was not detectably less than that in second‐generation hybrid controls or second‐generation parental controls. Variation of embryonic survival was affected by the maternal source population and by individual females but not by the paternal source population or by individual males. Survival of smolts to adulthood at sea was greater in one second‐generation control group than in two others, but survival in second‐generation outbred groups was no less than in parental controls or hybrid controls.     

Gut morphology in growth hormone transgenic Atlantic salmon

Growth hormone transgenic Atlantic salmon Salmo salar reared at 12–13°C were F₂ generation derived, using eggs from a transgenic F₁ female and fertilized with milt from a non-transgenic male. At the time of tissue sampling the transgenic salmon were growing 1·6 times faster than control salmon. Transgenic salmon tended to have more intestinal folds that were longer than those of control salmon. Consequently, the transgenic salmon had a larger digestive surface area both in the anterior intestine (surface area 1·5 times control) and in the pyloric caeca (surface area 1·2 times control). Most morphological features of the intestine and of the pyloric caeca of transgenic salmon were larger than those of control salmon; in particular, the surface area of the anterior intestine was concordant with the growth rate difference.     

Interaction of growth hormone overexpression and nutritional status on pituitary gland clock gene expression in coho salmon,Oncorhynchus kisutch

Clock genes are involved in generating a circadian rhythm that is integrated with the metabolic state of an organism and information from the environment. Growth hormone (GH) transgenic coho salmon, Oncorhynchus kisutch, show a large increase in growth rate, but also attenuated seasonal growth modulations, modified timing of physiological transformations (e.g. smoltification) and disruptions in pituitary gene expression compared with wild-type salmon. In several fishes, circadian rhythm gene expression has been found to oscillate in the suprachiasmatic nucleus of the hypothalamus, as well as in multiple peripheral tissues, but this control system has not been examined in the pituitary gland nor has the effect of transgenic growth modification been examined. Thus, the daily expression of 10 core clock genes has been examined in pituitary glands of GH transgenic (T) and wild-type coho salmon (NT) entrained on a regular photocycle (12L: 12D) and provided either with scheduled feeding or had food withheld for 60?h. Most clock genes in both genotypes showed oscillating patterns of mRNA levels with light and dark cycles. However, T showed different amplitudes and patterns of expression compared with wild salmon, both in fed and starved conditions. The results from this study indicate that constitutive expression of GH is associated with changes in clock gene regulation, which may play a role in the disrupted behavioural and physiological phenotypes observed in growth-modified transgenic strains.     

Regulation of feeding behavior and food intake by appetite-regulating peptides in wild-type and growth hormone-transgenic coho salmon

Survival, competition, growth and reproductive success in fishes are highly dependent on food intake, food availability and feeding behavior and are all influenced by a complex set of metabolic and neuroendocrine mechanisms. Overexpression of growth hormone (GH) in transgenic fish can result in greatly enhanced growth rates, feed conversion, feeding motivation and food intake. The objectives of this study were to compare seasonal feeding behavior of non-transgenic wild-type (NT) and GH-transgenic (T) coho salmon (Oncorhynchus kisutch), and to examine the effects of intraperitoneal injections of the appetite-regulating peptides cholecystokinin (CCK-8), bombesin (BBS), glucagon-like peptide-1 (GLP-1), and alpha-melanocyte-stimulating hormone (α-MSH) on feeding behavior. T salmon fed consistently across all seasons, whereas NT dramatically reduced their food intake in winter, indicating the seasonal regulation of appetite can be altered by overexpression of GH in T fish. Intraperitoneal injections of CCK-8 and BBS caused a significant and rapid decrease in food intake for both genotypes. Treatment with either GLP-1 or α-MSH resulted in a significant suppression of food intake for NT but had no effect in T coho salmon. The differential response of T and NT fish to α-MSH is consistent with the melanocortin-4 receptor system being a significant pathway by which GH acts to stimulate appetite. Taken together, these results suggest that chronically increased levels of GH alter feeding regulatory pathways to different extents for individual peptides, and that altered feeding behavior in transgenic coho salmon may arise, in part, from changes in sensitivity to peripheral appetite-regulating signals.     

Gill Morphometry in Growth Hormone Transgenic Pacific Coho Salmon,Onchorhynchus kisutch,Differs Markedly from that in GH Transgenic Atlantic Salmon

In a previous study we showed that many of the morphological features of the respiratory system of GH (growth hormone) transgenic Atlantic salmon are greater than similarly sized control salmon. Here we show that the manifestation of GH transgene is similar in two different lines of GH transgenic Pacific coho salmon, but that it is very different from that in the GH transgenic Atlantic salmon. The GH transgenic Pacific coho salmon do not have a larger gill surface area than similarly sized control fish.     

Estimation of protein digestibility—IV. Digestive proteinases from the pyloric caeca of coho salmon (Oncorhynchus kisutch) fed diets containing soybean meal

The goal of this study was to better understand why dietary soybean products are poorly utilized by salmonids. The influence of dietary intake on coho salmon fingerling weight gain and specific properties of pyloric caeca enzymes was investigated. Fingerlings were fed diets containing heated or unheated soybean meal (SBM) or Promoveal™, as 15–25% herring meal replacer, for 8–12 weeks. Fish fed to apparent satiation with diets containing heated SBM replacer gained more weight than those fed unheated SBM at the same level. Fish increased in body weight at the same rate when fed restricted rations containing either 15% SBM replacer that was variously heated up to 20 min, 15% Promoveal™ replacer or the herring meal basal diet. After the experimental diets were fed, digestive proteinases were isolated from the pyloric caeca. Yield of pyloric caeca enzymes (PCE), recovery of trypsin in PCE, soybean trypsin inhibitor (SBTI) sensitivity of PCE trypsin, specific activity of PCE trypsin and in vitro casein digestibility by PCE were determined for each dietary group. Weight gain vs in vitro casein digestibility by PCE was linear for animals fed unheated SBM to apparent satiation (r² = 0.71, P < 0.1) but not for animals fed either heated SBM to apparent satiation or variously heated SBM as 15% replacer at restricted levels. Trypsin from fish fed diets with heated or unheated SBM, but not Promoveal™ replacer, was less sensitive to SBTI than fish fed no SBM. For fish fed diets with variously heated SBM as 15% replacer, the SBTI activity of the SBM and SBTI inhibition of PCE trypsin were inversely related (r² = 0.88, P < 0.05). The yield of PCE was higher for fish fed 25% of heated SBM replacer than it was for diet groups fed less SBM. The yield of PCE trypsin was higher from animals fed 25% heated SBM replacer than those fed diets with a lower percentage of heated SBM replacer. Feeding coho fingerlings rations with SBM replacer appears to promote physiological compensation of PCE. Heat stable and/or heat-activated factor(s) and SBTI appear to cause the compensation of salmon digestive proteinases from coho salmon fed diets with SBM.     

Hormonal control of sulfate uptake by branchial cartilage of coho salmon: role of IGF-I.

The direct hormonal control of sulfate uptake by cartilage matrix of coho salmon was examined by exposing branchial cartilage to 1 microCi.ml-1 35SO4 for 48 hours at 15 degrees C in a defined medium. Sulfate uptake occurred primarily in cartilage (rather than bone) and the amount of specific uptake was similar in epibranchial and ceratobranchial cartilages. Intact and hypophysectomized coho salmon starved for 22 days had equivalent in vitro sulfate uptake, which in both cases were 30% of the uptake seen in branchial cartilage of fed, intact controls. In branchial cartilage from starved coho salmon, in vitro exposure to recombinant bovine insulin-like growth factor I (rbIGF-I) at 1, 10, 100, and 1,000 ng.ml-1 caused a dose-dependent increase in sulfate uptake, with a maximum 3-fold increase over control at 1,000 ng.ml-1 rbIGF-I. Coho salmon insulin (1, 10, 100, and 1,000 ng.ml-1) resulted in a maximum 30% increase in sulfate uptake at the highest dose. Growth hormone and triiodo-L-thyronine had no direct effect on in vitro sulfate uptake. The results indicate that IGF-I has direct effects on coho salmon cartilage and may be an important regulator of growth in salmon and other teleosts.     

Genetic evidence for lacustrine spawning of the non‐anadromous Atlantic salmon population of Little Gull Lake, Newfoundland

Allozyme markers were used to investigate the spatial boundaries of the non‐anadromous Atlantic salmon population of Little Gull Lake, Eastern Canada. Mixed year class samples of juvenile salmon were analysed from inlet streams, upstream lakes, the outlet river and sites downstream. No evidence was found that individuals from the Lake's non‐anadromous population were present in these samples, strongly suggesting that the non‐anadromous population is confined to the lake and has a lacustrine spawning habit.     

Molecular cloning and sequencing of coho salmon growth hormone cDNA

A cDNA library was constructed using mRNA isolated from coho salmon pituitaries. By employing rainbow trout growth hormone cDNA as a probe, the coho salmon cDNA was isolated and the complete nucleotide (nt) sequence determined. The coding region contains 630 nt while the 5'- and 3'-untranslated regions are 64 and 489 nt in length, respectively. Comparison of the noncoding regions of coho and chum salmon cDNAs reveal identity at the 5' end but significant variation in the 3' end. Chum salmon and rainbow trout have identical amino acid (aa) sequences, but coho salmon growth hormone has a sequence that differs by 6 of the 188 predicted aa. Since salmonids are tetraploid, this difference may be the result of either divergence of the same growth hormone locus or of variation between different loci. Comparisons of the cDNA restriction maps of these three fish species suggest the former possibility.     

Determination of body shape variation in Irish hatchery‐reared and wild Atlantic salmon

Discriminant function analysis was used to distinguish morphologically between samples of parr, smolts and adult Atlantic salmon Salmo salar from several hatchery and river systems in Ireland. The effect of habitat shift was investigated in Atlantic salmon parr. Parr grown from the eyed‐egg stage with a non‐sibling group in a hatchery environment, came to resemble the mean body shape of their host hatchery Atlantic salmon stock more closely than that of a full sibling group grown at their natal hatchery. Wild Atlantic salmon smolts differed in shape from hatchery‐reared smolts. This difference was less pronounced, but still statistically significant when wild adults were compared with hatchery‐reared adults captured in the coastal drift‐net fishery after a year spent at sea. Rearing conditions had a significant impact on the production and growth of fish body shape. This in turn may have affected adaptability and survivorship of ranched Atlantic salmon in the marine environment.     

Effect of dietary vitamin E level on growth, tissue lipid peroxidation, and erythrocyte fragility of transgenic coho salmon, Oncorhynchus kisutch

This study was conducted to investigate the effect of dietary vitamin E concentration on growth performance, iron-catalyzed lipid peroxidation in liver and muscle tissue, and erythrocyte fragility of transgenic growth hormone coho salmon (Oncorhynchus kisutch). Fish were fed one of four isoenergetic and isonitrogenous experimental diets that contained either 11, 29, 50, or 105 IU of vitamin E/kg. Following the 10-week feeding trial, no significant (P>0.05) diet-related differences were detected in growth, whole body proximate composition or erythrocyte fragility. The vitamin E contents of liver and muscle, however, were affected by the dietary treatment. Fish fed diets containing > or =50 IU of vitamin E/kg had significantly increased vitamin E concentrations in their tissues. Iron-catalyzed lipid peroxidation of liver and muscle tissue of fish fed elevated dietary vitamin E (> or =50 IU vitamin E/kg diet) was significantly lower (P<0.05) than that noted for fish fed the diet containing no supplemental vitamin E. The results indicated that changes in tissue lipid peroxidation measurements precede clinical signs of sub-optimal vitamin E intake.     

Rearing in Seawater Mesocosms Improves the Spawning Performance of Growth Hormone Transgenic and Wild-Type Coho Salmon

Growth hormone (GH) transgenes can significantly accelerate growth rates in fish and cause associated alterations to their physiology and behaviour. Concern exists regarding potential environmental risks of GH transgenic fish, should they enter natural ecosystems. In particular, whether they can reproduce and generate viable offspring under natural conditions is poorly understood. In previous studies, GH transgenic salmon grown under contained culture conditions had lower spawning behaviour and reproductive success relative to wild-type fish reared in nature. However, wild-type salmon cultured in equal conditions also had limited reproductive success. As such, whether decreased reproductive success of GH transgenic salmon is due to the action of the transgene or to secondary effects of culture (or a combination) has not been fully ascertained. Hence, salmon were reared in large (350,000 L), semi-natural, seawater tanks (termed mesocosms) designed to minimize effects of standard laboratory culture conditions, and the reproductive success of wild-type and GH transgenic coho salmon from mesocosms were compared with that of wild-type fish from nature. Mesocosm rearing partially restored spawning behaviour and success of wild-type fish relative to culture rearing, but remained lower overall than those reared in nature. GH transgenic salmon reared in the mesocosm had similar spawning behaviour and success as wild-type fish reared in the mesocosm when in full competition and without competition, but had lower success in male-only competition experiments. There was evidence of genotype×environmental interactions on spawning success, so that spawning success of transgenic fish, should they escape to natural systems in early life, cannot be predicted with low uncertainty. Under the present conditions, we found no evidence to support enhanced mating capabilities of GH transgenic coho salmon compared to wild-type salmon. However, it is clear that GH transgenic salmon are capable of successful spawning, and can reproduce with wild-type fish from natural systems.     

Differential lifetime success and performance of native and non‐native Atlantic salmon examined under communal natural conditions

The lifetime success and performance characteristics of communally reared offspring of wild native Burrishoole (native), ranched native (ranched) and non‐native (non‐native) Atlantic salmon Salmo salar from the adjacent Owenmore River were compared. Non‐native 0+ year parr showed a substantial downstream migration, which was not shown by native and ranched parr. This appears to have been an active migration rather than competitive displacement and may reflect an adaptation to environmental or physiographic conditions within the Owenmore River catchment where the main nursery habitat is downstream of the spawning area. There were no differences between native and ranched in smolt output or adult return. Both of these measures, however, were significantly lower for the non‐native group. A greater proportion of the non‐native Atlantic salmon was taken in the coastal drift nets compared to the return to the Burrishoole system, probably as a result of the greater size of the non‐native fish. The overall lifetime success of the non‐native group, from fertilized egg to returning adult, was some 35% of native and ranched. The ranched group showed a significantly greater male parr maturity, a greater proportion of 1+ year smolts, and differences in sex ratio and timing of freshwater entry of returning adults compared to native, which may have fitness implications under specific conditions.     

Intestinal morphology in growth hormone transgenic coho salmon

In two GH transgenic coho salmon Oncorhynchus kisutch , the surface area of the intestine was 2·2 times that of control salmon and the growth rate was about twice that of controls. It seems likely that the enhanced intestinal surface area is a compensatory feature that is manifested in fast growing salmonids.     

Upper thermal tolerance of wild‐type,domesticated and growth hormone‐transgenic coho salmon Oncorhynchus kisutch

In coho salmon Oncorhynchus kisutch, no significant differences in critical thermal maximum (c. 26·9° C, C_Tmax) were observed among size‐matched wild‐type, domesticated, growth hormone (GH)‐transgenic fish fed to satiation, and GH‐transgenic fish on a ration‐restricted diet. Instead, GH‐transgenic fish fed to satiation had significantly higher maximum heart rate and Arrhenius breakpoint temperature (mean ± s.e. = 17·3 ± 0·1° C, T_AB). These results provide insight into effects of modified growth rate on temperature tolerance in salmonids, and can be used to assess the potential ecological consequences of GH‐transgenic fishes should they enter natural environments with temperatures near their thermal tolerance limits.