Effects of dietary arachidonic acid on the reproductive physiology of female Atlantic cod (Gadus morhua L.).

The present study was designed to investigate potential effects of arachidonic acid (ARA) on the reproductive physiology of female Atlantic cod (Gadus morhua L.). Two-year old Atlantic cod of both sexes were equally distributed into eight sea cages after completion of their first spawning in May 2005. Four experimental groups were established and fed diets with different levels of ARA corresponding to 0.5, 1, 2 and 4% of total fatty acid. Ovarian growth and development was documented every month. Fatty acid composition was analysed in ovaries, liver and plasma at the beginning of the experiment, one month prior to spawning, and in spent fish, one month after spawning was completed. Plasma concentrations of estradiol-17ß, testosterone and vitellogenin, and ovarian gene transcript levels of steroidogenic acute regulatory protein (star), P450aromatase (cyp19a1a) and 20ß-hydroxy steroid dehydrogenase (20bhsd/cbr1) were monitored every month in fish fed the experimental diets and related to oocyte stage. Potential fecundity was calculated based on ovarian samples taken one month before onset of spawning. Ovarian and plasma ARA levels were highly correlated to dietary ARA levels. There was a net accumulation of ARA compared to other essential fatty acids in ovarian tissue that was reflected in a decrease in EPA:ARA ratio. Plasma concentrations of vitellogenin, estradiol-17ß and testosterone and key gene transcript levels were affected by dietary ARA and stage of maturation. The results show that ARA has a significant influence on the reproductive physiology of female Atlantic cod.

Ocean warming and acidification modulate energy budget and gill ion regulatory mechanisms in Atlantic cod (Gadus morhua).

Ocean warming and acidification are threatening marine ecosystems. In marine animals, acidification is thought to enhance ion regulatory costs and thereby baseline energy demand, while elevated temperature also increases baseline metabolic rate. Here we investigated standard metabolic rates (SMR) and plasma parameters of Atlantic cod (Gadus morhua) after 3-4 weeks of exposure to ambient and future PCO2 levels (550, 1200 and 2200 µatm) and at two temperatures (10, 18 °C). In vivo branchial ion regulatory costs were studied in isolated, perfused gill preparations. Animals reared at 18 °C responded to increasing CO2 by elevating SMR, in contrast to specimens at 10 °C. Isolated gills at 10 °C and elevated PCO2 (≥1200 µatm) displayed increased soft tissue mass, in parallel to increased gill oxygen demand, indicating an increased fraction of gill in whole animal energy budget. Altered gill size was not found at 18 °C, where a shift in the use of ion regulation mechanisms occurred towards enhanced Na(+)/H(+)-exchange and HCO3 (-) transport at high PCO2 (2200 µatm), paralleled by higher Na(+)/K(+)-ATPase activities. This shift did not affect total gill energy consumption leaving whole animal energy budget unaffected. Higher Na(+)/K(+)-ATPase activities in the warmth might have compensated for enhanced branchial permeability and led to reduced plasma Na(+) and/or Cl(-) concentrations and slightly lowered osmolalities seen at 18 °C and 550 or 2200 µatm PCO2 in vivo. Overall, the gill as a key ion regulation organ seems to be highly effective in supporting the resilience of cod to effects of ocean warming and acidification.

Pre-anaesthetic metomidate sedation delays the stress response after caudal artery cannulation in Atlantic cod (Gadus morhua).

Recovery from caudal artery cannulation with and without pre-anaesthesia metomidate sedation was assessed in Atlantic cod (Gadus morhua). The levels of plasma cortisol, glucose, electrolytes and acid-base parameters were compared between sedated and unsedated cod and to those in uncannulated individuals, where the samples were obtained by sacrificial sampling (reference level). Metomidate sedation delayed the stress response, causing sedated cod plasma cortisol to return to the reference level more slowly [day 4 post surgery (PS)] than in unsedated cod (day 2 PS). Plasma glucose was elevated in both sedated and unsedated cod up to and including day 5 PS. Plasma K(+) was lower and pH was higher in cannulated cod than in the reference from 24 h PS until the end of experimentation, indicating a stress effect of sacrificial sampling on plasma K(+) and pH that was likely caused by an acute stress response. Metomidate sedation delayed the stress response following CA cannulation and should therefore not be used as a pre-anaesthetic sedation in Atlantic cod. The caudal artery cannulation can be a useful tool in obtaining repeated blood samples from Atlantic cod given an adequate recovery time, which was determined to be 6 days irrespective of pre-anaesthesia sedation status.

An estrogen-responsive plasma protein expression signature in Atlantic cod (Gadus morhua) revealed by SELDI-TOF MS.

Compound-specific protein expression signatures (PESs) can be revealed by proteomic techniques. The SELDI-TOF MS approach is advantageous due to its simplicity and high-throughput capacity, however, there are concerns regarding the reproducibility of this method. The aim of this study was to define an estrogen-responsive PES in plasma of Atlantic cod (Gadus morhua) using the SELDI-TOF MS technique. Protein expression analysis of male cod exposed to 17ß-estradiol (E2) showed that 27 plasma peaks were differentially expressed following exposure. The reproducibility of this result was evaluated by reanalyzing the samples six months later, and a significant change in expression was confirmed for 13 of the 27 peaks detected in the first analysis. The performance of the reproducible E2-responsive PES, constituting these 13 peaks, was then tested on samples from juvenile cod exposed to 4-nonylphenol, North Sea oil, or North Sea oil spiked with alkylphenols. Principal component analysis revealed that nonylphenol-exposed cod could be separated from unexposed cod based on the E2-responsive PES, indicating that the PES can be used to assess estrogenic exposure of both juvenile and adult specimens of cod. A targeted antibody-assisted SELDI-TOF MS approach was carried out in an attempt to identify the E2-responsive peaks. Results indicated that 2 peaks were fragments of the well-known biomarkers VTG and/or ZRP. In this study, the SELDI-TOF MS technology has shown its potential for defining compound-specific PESs in fish. Nevertheless, thorough validation of reproducibility, specificity and sensitivity of a PES is required before it can be applied in environmental monitoring.

Endocrine modulation in Atlantic cod (Gadus morhua L.) exposed to alkylphenols, polyaromatic hydrocarbons, produced water, and dispersed oil.

Effluent from oil production activities contains chemicals that are suspected of inducing endocrine disruption in fish. In this study, Atlantic cod (Gadus morhua L.) were exposed to mixtures of low- and medium-molecular-weight alkylphenols (AP) (methyl- to heptylphenol), polycyclic aromatic hydrocarbons (PAH), diluted produced water, and dispersed oil for 15 d in a flow-through exposure system. Condition index (CI), hepatosomatic index (HSI), gonadosomatic index (GSI), concentration of the estrogenic biomarker vitellogenin (Vtg), and modulation of the total sex steroid-binding capacity in plasma were determined to assess whether these mixtures were capable of interfering with endocrine-regulated physiological processes in Atlantic cod. No marked differences in plasma Vtg levels were found between control and exposed groups of either males or females, possibly due to high intergroup variances and low sample numbers. An apparent numerical increase in the number of male and female fish with high plasma Vtg levels was, however, observed in some exposure groups compared to control. This purported weak estrogenic effect was several orders of magnitude lower than that observed for potent estrogens and suggested that the levels of estrogen receptor (ER) agonists were low. Exposure of female fish to a mixture of dispersed oil and a mixture of AP, PAH, and dispersed oil led to upregulation of the plasma total sex steroid-binding capacity, indicating interference with the normal blood steroid transport. No significant effects were seen for CI, HSI, and GSI, suggesting that the endocrine-disrupting potential was not sufficient to elicit effects on general physiological conditions and gonad development during this short exposure period.

Water column monitoring of the biological effects of produced water from the Ekofisk offshore oil installation from 2006 to 2009.

The Norwegian water column monitoring program investigates the biological effects of offshore oil and gas activities in Norwegian waters. In three separate surveys in 2006, 2008, and 2009, bioaccumulation and biomarker responses were measured in mussels (Mytilus edulis) and Atlantic cod (Gadus morhua) held in cages at known distances from the produced water (PW) discharge at the Ekofisk oil field. Identical monitoring studies performed in all three years have allowed the biological effects and bioaccumulation data to be compared, and in addition, enabled the potential environmental benefits of a PW treatment system (CTour), implemented in 2008, to be evaluated. The results of the 2009 survey showed that caged animals were exposed to low levels of PW components, with highest tissue concentrations in mussels located closest to the PW discharge. Mussels located approximately 1-2 km away demonstrated only background concentrations of target compounds. Concentrations of polycyclic aromatic hydrocarbons (PAH) and alkyl phenol (AP) metabolites in the bile of caged cod were elevated at stations 200-250 m from the discharge. There was also a signal of exposure relative to discharge for the biomarkers CYP1A in fish and micronuclei in mussels. All other fish and mussel biomarkers showed no significant exposure effects in 2009. The mussel bioaccumulation data in 2009 indicated a lower exposure to the PW effluent than seen previously in 2008 and 2006, resulting in an associated general improvement in the health of the caged mussels. This was due to the reduction in overall discharge of PW components (measured as oil in water) into the area in 2009 compared to previous years as a result of the improved PW treatment system.

Candidate biomarker discovery in plasma of juvenile cod (Gadus morhua) exposed to crude North Sea oil, alkyl phenols and polycyclic aromatic hydrocarbons (PAHs).

In this study we have investigated protein changes in plasma of juvenile Atlantic cod (Gadus morhua) induced by crude North Sea oil and North Sea oil spiked with alkyl phenols and polycyclic aromatic hydrocarbons, a surrogate produced water composition. Using a proteomic approach, we identified 137 differentially expressed proteins at different levels of crude oil exposure. Many of the induced protein changes occurred at low levels of exposure. The results obtained with protein expression profiles after exposure to oil and surrogate produced water indicate effects on fibrinolysis and the complement cascade, the immune system, fertility-linked proteins, bone resorption, fatty acid metabolism as well as increased oxidative stress, impaired cell mobility and increased levels of proteins associated with apoptosis. Although the number of individuals and samples in this study is limited within each treatment group, the protein changes observed in this study represent a first screening for potential biomarker candidates in cod plasma reflecting potential effects of crude oil and produced water exposure on fish.

Photoperiod-modulated testis maturation in Atlantic cod (Gadus morhua, L.).

Precocious male puberty is a significant problem in Atlantic cod aquaculture. While photoperiod manipulation can inhibit testis growth, a detailed analysis of effects on spermatogenesis is missing. Starting July 1, 2004, prepubertal fish were exposed to different photoperiod regimens in indoor tanks for 17 mo. Testis histology, germ cell dynamics (proliferation and apoptosis), and plasma androgen levels were analyzed. In the natural light (NL) group, testis growth started in September 2004 and was completed in February 2005, when a 2-mo spawning period started. In the constant light (LL) group, none or very few spermatogenic cysts were recruited into spermatogenesis, and apoptotic germ cell loss was high. A change of photoperiod from NL to LL at winter solstice (December 21, 2004) resulted in premature (2 mo) completion of the reproductive cycle, while changing from LL to NL at winter solstice triggered faster than normal testis development. Plasma testosterone levels increased in the NL group from spermatogonial proliferation toward meiosis, while those of 11-ketotestosterone increased toward spermiogenesis and spermiation. Plasma androgen levels did not rise under LL conditions. Comparing fish with developing testes from all groups indicated that low androgen levels were associated with a high incidence of spermatogonial apoptosis; we also found that androgen receptor mRNA expression was most prominent in Sertoli cells in contact with growing spermatogonial clones. Our data show that an inhibitory photoperiod (LL) reduced or blocked differentiation of spermatogonia, increased apoptosis (particularly among proliferating spermatogonia), and was associated with reduced androgen levels, a situation possibly reflecting insufficient gonadotropic stimulation.

Investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil.

The induction of micronuclei and other nuclear abnormalities (nuclear buds, bi-nucleated and fragmented-apoptotic cells) was analyzed in the erythrocytes of peripheral blood and cephalic kidney of turbot (Scophthalmus maximus) and Atlantic cod (Gadus morua), treated with crude oil (Statfjord B, Norway) and with nonylphenol. Significant increase in MN was observed in turbot kidney and blood after exposure to 30 ppb of nonylphenol, 0.5 ppm of oil, and after co-exposure to 0.5 ppm of oil spiked with additional mixture of alkylphenols and PAHs (P varied between 0.0054 and <0.0001). The induction of micronuclei was observed only in cod kidney after exposure to spiked oil (P=0.0317). Significant inter-specific differences after the exposure to 0.5 ppm of oil (P=0.0385) and after treatment with spiked oil (P=0.0067) were observed. In turbot cephalic kidney, the elevated levels of bi-nucleated cells were observed in all treatment groups (P values varied in a range from 0.05 to 0.0025) while the increase in cells with nuclear buds was noted after the exposure to 0.5 ppm of oil (P=0.05). The fragmented-apoptotic cells appeared after the exposure to nonylphenol (P=0.0039) and to spiked oil (P<0.0001). In turbot blood, only the significant induction in nuclear buds was detected. Statistically significant inter-tissue differences were found only in the induction of fragmented-apoptotic cells after the exposure to nonylphenol and to spiked oil.

Vitellogenin in the blood plasma of male cod (Gadus morhua): a sign of oestrogenic endocrine disruption in the open sea?

An ELISA for cod vitellogenin (VTG) has been set up using cod lipovitellin for plate coating and standardisation. The assay has been applied to plasma samples collected from male and female cod caught in three distinct areas around the UK, three areas off the Norwegian coast and also to cod reared initially at an aquaculture site and subsequently maintained at a research station. The aim of the study was to determine whether there were any signs of oestrogenic endocrine disruption in a fish species living offshore. VTG induction was found in male cod caught in the North Sea, the Shetland Box area, in Oslofjord and also in cultivated fish. There was a strong relationship between concentrations of VTG and fish size. There was no evidence that the presence of VTG in the plasma of males is a natural part of their life cycle. On the other hand, the size of fish at which these elevated VTG concentrations appear (ca. 5 kg) is about the size that cod change from feeding primarily on benthic invertebrates to mainly other fish, both benthic and pelagic. The possibility is suggested that large cod pick up oestrogenic endocrine disrupters through the food chain.