Validation of the HemoCue Hb 801 portable haemoglobin analyser for fish blood.

Haemoglobin concentration ([Hb]) assessment in fish blood has become a routine parameter to measure the health and welfare status of the animals. The original method (haemoglobincyanide method, best known as the Drabkin method) for measuring Hb in human and animals is not well suited for work outside of a laboratory setting. It is relatively time consuming, contains hazardous cyanide elements, and requires specific laboratory material. As an alternative to the Drabkin method, portable analysers have been developed for human blood, but they need to be first validated for fish blood before being used in experiments. In this study, the performance of the new HemoCue Hb 801 portable haemoglobin analyser was compared to the validated Drabkin method to determine [Hb] in three fish species. Hb readings between the two methods were not different for any of the species tested (rainbow trout, Onchorynchus mykiss, Atlantic wolffish, Anarhichas lupus, and Nile tilapia, Oreochromis niloticus). Therefore, this new portable device can be readily used to measure Hb in fish blood. Unlike the previous model from HemoCue, the Hb 201+, this device does not need an incubation time or a correction factor, representing a major gain of time and precision.

Local variation in stress response of juvenile anadromous brown trout, Salmo trutta.

Habitat fragmentation may cut off anadromous salmonids from parts of their potential native habitat and separate previously connected populations. Understanding the consequences of this is vital for fish management and prioritization of restoration activities. Here, we show that there is a significant difference in the body morphology, physiological stress response, and aspects contributing to aerobic capacity between juvenile anadromous brown trout, Salmo trutta, collected at a downstream site and an upstream site, separated by 2 km and several challenging stream sections, in a small unfragmented stream system in western Sweden. Following a standardized stress test, there were significant differences between fish from the upstream and downstream sites (plasma cortisol concentration, plasma osmolality, hematocrit, hemoglobin concentration, and mean corpuscular hemoglobin concentration). Plasma glucose concentration did not significantly differ between fish from the two sites. Fish from the upstream site had larger spleen mass, although there was no evidence of differences in ventricle mass or proportion of compact ventricular myocardium. These physiological differences indicate local variation in stress response and highlight the importance of considering local trait variation in river management. If a section of the river becomes fragmented or degraded, and there are differences in the juveniles in different parts of the river, the consequence for the population might be larger than the proportional loss of habitat.

High protein requirements of juvenile Atlantic wolffish, Anarhichas lupus: Effects of dietary protein levels on growth, health, and welfare.

The objective of the present study was to investigate the optimal dietary protein requirement and the effect of varying protein levels on the growth and health of juvenile, wild-caught Atlantic wolffish, Anarhichas lupus, a promising candidate for cold-water aquaculture diversification. Six iso-energetic (ca. 18.3 MJ kg-1), fish meal-based experimental diets were formulated with crude protein levels ranging from 35% to 60%, with graded increments of 5% in a 12-week feeding trial in a recirculating aquaculture system (RAS). Weight gain, specific growth rate (SGR), and condition factor (K) were evaluated in response to dietary protein levels. Liver, muscle, and blood parameters were assessed for possible changes in protein and lipid metabolism and welfare. Overall growth was highly variable throughout the experiment on all diets, as expected for a wild population. The feed with highest in protein (60%) inclusion resulted in the highest growth rates, with an average weight gain of 37.4% ± 33.8% and an SGR of 0.31% ± 0.2% day-1. This was closely followed by feeds with 55% and 50% protein inclusion with an average weight gain of 22.9% ± 34.8% and 28.5% ± 38.3%, respectively, and an SGR of 0.18% ± 0.3% day-1 and 0.22% ± 0.3% day-1, respectively. Fish fed the high protein diets generally had increased hepatic lipid deposition (17%-18%) and reduced free fatty acid levels (3.1-6.8 µmol L-1) in the plasma relative to fish that were fed the lower protein diets (35%-45%). No effects of diet were found on plasma protein levels or muscle protein content. Furthermore, stress parameters such as plasma cortisol and glucose levels were unaffected by diet, as were plasma ghrelin levels. Overall, these results suggest that a high protein inclusion in the diet for Atlantic wolffish is required to sustain growth with a minimum protein level of 50%.

Evaluation of starvation status in the early developmental stages of black rockfish (Sebastes schlegelii) based on morphological and histological characteristics.

Assessing the nutritional status and identifying major causes of mortality in larvae experiencing varying degrees of starvation are crucial for establishing appropriate feeding protocols and enhancing the welfare of hatchery-reared fish. The black rockfish Sebastes schlegelii is an important species in aquaculture and stock enhancement efforts in China, Japan, and Korea. This study aimed to identify optimal diagnostic morphometric indicators of starvation in newly hatched (0-6 days post-hatch, DPH) and postlarval stages (27-37 DPH) of this valuable fish species through histological analyses. Our findings revealed that certain morphometric parameters, including body length, the ratios of eye diameter to head height, body height to body length, and abdomen height to body height, exhibit sensitivity to starvation during both larval and postlarval stages. Particularly, the ratios of body height to body length and abdomen height to body height emerged as the most sensitive morphometric indicators of starvation. Histological examinations of the digestive system revealed rapid alterations in the morphology of hepatic parenchymal cells, accompanied by a significant decrease in the number of lipid cells in the liver during episodes of food deprivation. Starvation induced cellular degeneration in the digestive organs, manifested by reduced heights of epithelial cells and mucosal layers in the intestine, oesophagus, and stomach, along with degeneration and separation of muscle fibers. Among these variables, the height of the intestinal submucosa and muscle layer emerged as the most sensitive indicators reflecting nutritional conditions in newly hatched larvae. In contrast, the height of intestinal striated borders and mucosal folds proved to be the most sensitive indicators in the postlarval stage. Furthermore, the height of intestinal epithelial cells and the number of lipid vacuoles in enterocytes exhibited high sensitivity to food deprivation in both newly hatched larvae and postlarvae. These findings underscore the varying resilience of fish to starvation during different developmental phases and highlight the utility of morphological sensitivity characteristics as reliable diagnostic indices for assessing nutritional status in relation to starvation or suboptimal feeding during the early developmental stages of black rockfish in hatchery-reared processes.

Fish processing side streams are promising ingredients in diets for rainbow trout (Oncorhynchus mykiss) - Effects on growth physiology, appetite, and intestinal health.

Due to the growth of aquaculture and the finite supply of fishmeal and oil, alternative marine protein and lipid sources are highly sought after. Particularly promising is the use of side streams from the fish processing industry, allowing for the recovery and retention of otherwise lost nutrients in the food production chain. The aim of the present study was to evaluate the potential of three fish processing side streams as fish feed ingredients. The side streams originated from different stages of the production chain, were used without further processing, and included sprat trimmings (heads, frames, viscera), marinated herring (fillets) and mackerel in tomato sauce (fillets and sauce). The three side streams contained moderate levels of protein (28-32% dry matter) and high levels of lipid (34-43%). The sprat trimmings included ca. 29% ash and 1.5% phosphorous which may add value due to the high level of essential minerals but needs to be considered in feed formulations. Three diets were formulated to include 50% of each side stream replacing all fishmeal and ca. 80% of the fish oil of the control diet, which contained 35% fishmeal and 10% fish oil. The diets were evaluated in a 12-week feeding trial using rainbow trout (Oncorhynchus mykiss). Fish fed the sprat diet displayed the highest feed intake and growth, and showed no negative effects on the intestinal health. The mackerel side stream displayed a good digestibility but resulted in lower growth rates compared to the sprat trimmings. Fish fed the herring diet, displayed the lowest performance regarding growth, feed intake and digestibility. They further exhibited a reduction in nutrient uptake in both proximal and distal intestine, likely contributing to the observed lower digestibility and growth, and a reduction in plasma ghrelin levels. As part of a circular approach to increase marine lipid and protein production for fish feed, the tested sprat and mackerel side streams are promising raw materials however additional studies using more commercial-like feed formulations are encouraged.

Two-phase microalgae cultivation for RAS water remediation and high-value biomass production.

The overall goal of this study was to provide solutions to innovative microalgae-based technology for wastewater remediation in a cold-water recirculating marine aquaculture system (RAS). This is based on the novel concept of integrated aquaculture systems in which fish nutrient-rich rearing water will be used for microalgae cultivation. The produced biomass can be used as fish feed, while the cleaned water can be reused, to create a highly eco-sustainable circular economy. Here, we tested three microalgae species Nannochloropis granulata (Ng), Phaeodactylum tricornutum (Pt), and Chlorella sp (Csp) for their ability to remove nitrogen and phosphate from the RAS wastewater and simultaneously produce high-value biomass, i.e., containing amino acids (AA), carotenoids, and polyunsaturated fatty acids (PUFAs). A high yield and value of biomass were achieved for all species in a two-phase cultivation strategy: i) a first phase using a medium optimized for best growth (f/2 14x, control); ii) a second "stress" phase using the RAS wastewater to enhance the production of high-value metabolites. Ng and Pt performed best in terms of biomass yield (i.e., 5-6 g of dry weight, DW.L-1) and efficient cleaning of the RAS wastewater from nitrite, nitrate, and phosphate (i.e., 100% removal). Csp produced about 3 g L-1 of DW and reduced efficiently only nitrate, and phosphate (i.e., about 76% and 100% removal, respectively). The biomass of all strains was rich in protein (30-40 % of DW) containing all the essential AA except Methionine. The biomass of all three species was also rich in PUFAs. Finally, all tested species are excellent sources of antioxidant carotenoids, including fucoxanthin (Pt), lutein (Ng and Csp) and ß-carotene (Csp). All tested species in our novel two-phase cultivation strategy thus showed great potential to treat marine RAS wastewater and provide sustainable alternatives to animal and plant proteins with extra added values.

Invader at the edge - Genomic origins and physiological differences of round gobies across a steep urban salinity gradient.

Species invasions are a global problem of increasing concern, especially in highly connected aquatic environments. Despite this, salinity conditions can pose physiological barriers to their spread, and understanding them is important for management. In Scandinavia's largest cargo port, the invasive round goby (Neogobius melanostomus) is established across a steep salinity gradient. We used 12,937 SNPs to identify the genetic origin and diversity of three sites along the salinity gradient and round goby from western, central and northern Baltic Sea, as well as north European rivers. Fish from two sites from the extreme ends of the gradient were also acclimated to freshwater and seawater, and tested for respiratory and osmoregulatory physiology. Fish from the high-salinity environment in the outer port showed higher genetic diversity, and closer relatedness to the other regions, compared to fish from lower salinity upstream the river. Fish from the high-salinity site also had higher maximum metabolic rate, fewer blood cells and lower blood Ca2+. Despite these genotypic and phenotypic differences, salinity acclimation affected fish from both sites in the same way: seawater increased the blood osmolality and Na+ levels, and freshwater increased the levels of the stress hormone cortisol. Our results show genotypic and phenotypic differences over short spatial scales across this steep salinity gradient. These patterns of the physiologically robust round goby are likely driven by multiple introductions into the high-salinity site, and a process of sorting, likely based on behaviour or selection, along the gradient. This euryhaline fish risks spreading from this area, and seascape genomics and phenotypic characterization can inform management strategies even within an area as small as a coastal harbour inlet.

Pain Recognition in Fish.

Empirical evidence has demonstrated that fish experience pain, and so to ensure their good welfare, it is vital that we can recognize and assess pain. A range of general, behavioral, and physiologic indicators can be used when assessing pain in fish. Many of these can be used at the tank side and are termed operational welfare indicators, whereas some require further computer or laboratory analysis. Behavioral indicators are valid and have been shown to profoundly differ between nonpainful and painful treatments in fish. However, these are not universal, and species-specific differences exist in behavioral responses to pain.

Low Holding Densities Increase Stress Response and Aggression in Zebrafish.

With laboratory zebrafish (Danio rerio) being an established and popular research model, there is a need for universal, research-based husbandry guidelines for this species, since guidelines can help promote good welfare through providing appropriate care. Despite the widespread use of zebrafish in research, it remains unclear how holding densities affect their welfare. Previous studies have mainly evaluated the effects of holding densities on a single parameter, such as growth, reproductive output, or social interactions, rather than looking at multiple welfare parameters simultaneously. Here we investigated how chronic (nine weeks) exposure to five different holding densities (1, 4, 8, 12, and 16 fish/L) affected multiple welfare indicators. We found that fish in the 1 fish/L density treatment had higher free water cortisol concentrations per fish, increased vertical distribution, and displayed aggressive behaviour more frequently than fish held at higher densities. On the other hand, density treatments had no effect on anxiety behaviour, whole-brain neurotransmitter levels, egg volume, or the proportion of fertilised eggs. Our results demonstrate that zebrafish can be held at densities between 4 and 16 fish/L without compromising their welfare. However, housing zebrafish in the density of 1 fish/L increased their stress level and aggressive behaviour.

Salinity Variation in a Mangrove Ecosystem: A Physiological Investigation to Assess Potential Consequences of Salinity Disturbances on Mangrove Crabs.

Dimitri Theuerkauff, Georgina A. Rivera-Ingraham, Jonathan A.C. Roques, Laurence Azzopardi, Marine Bertini, Mathilde Lejeune, Emilie Farcy, Jehan-Hervé Lignot, and Elliott Sucré (2018) Salinity is one of the main environmental factors determining coastal species distribution. However, in the specific case of mangrove crabs, salinity selection cannot be understood through ecological approaches alone. Yet understanding this issue is crucial in the context of mangrove conservation, since this ecosystem is often used as biofilter of (low- salinity) wastewater. Crabs are keystone species in this mangrove ecosystem and are differentially affected by salinity. We hypothesize that crab salinity selection may be partly explained by specific salinity-induced physiological constraints associated with osmoregulation, energy and redox homeostasis. To test this, the response to salinity variation was analysed in two landward mangrove crabs: the fiddler crab Tubuca urvillei, which inhabits low-salinity areas of the mangrove, and the red mangrove crab Neosarmatium meinerti, which lives in areas with higher salinity. Results confirm that both species are strong hypo-/hyper-osmoregulators that deal easily with large salinity variations. Such shifts in salinity do not induce changes in energy expenditure (measured as oxygen consumption) or in the production of reactive oxygen species. However, T. urvillei is physiologically suited to habitats with brackish water, since it presents i) high hemolymph osmolalities over a wider range of salinities and lower osmoregulatory capacity in seawater, ii) high Na+/K+-ATPase (NKA) activity in the posterior osmoregulatory gills and iii) a thicker osmoregulatory epithelium along the posterior gill lamellae. Therefore, while environmental salinity alone cannot directly explain fiddler and red mangrove crab distributions, our data suggest that salinity selection is indeed influenced by specific physiological adjustments.

Stress in African catfish (Clarias gariepinus) following overland transportation.

Of the many stressors in aquaculture, transportation of fish has remained poorly studied. The objective of this study was therefore to assess the effects of a (simulated) commercial transportation on stress physiology of market-size African catfish (Clarias gariepinus). Catfish weighing approximately 1.25 kg were returned to the farm after 3 h of truck-transportation, and stress-related parameters were measured for up to 72 h following return. Recovery from transportation was assessed through blood samples measuring plasma cortisol, glucose and non-esterified fatty acids (NEFA) and gill histology. Also, the number of skin lesions was compared before and after transport. Pre-transport handling and sorting elevated plasma cortisol levels compared to unhandled animals (before fasting). Plasma cortisol levels were further increased due to transportation. In control fish, plasma cortisol levels returned to baseline values within 6 h, whereas it took 48 h to reach baseline values in transported catfish. Plasma glucose and NEFA levels remained stable and were similar across all groups. Transported catfish did not, on average, have more skin lesions than the handling group, but the number of skin lesions had increased compared to unhandled animals. The macroscopic condition of the gills was similar in control, transported and unhandled catfish; however, light microscopy and immunohistochemistry revealed atypical morphology and chloride cell migration normally associated with adverse water conditions. From our data, we conclude that transportation may be considered a strong stressor to catfish that may add to other stressors and thus inflict upon the welfare of the fish.

Forced sustained swimming exercise at optimal speed enhances growth of juvenile yellowtail kingfish (Seriola lalandi).

Swimming exercise at optimal speed may optimize growth performance of yellowtail kingfish in a recirculating aquaculture system. Therefore, optimal swimming speeds (U opt in m s(-1) or body lengths s(-1), BL s(-1)) were assessed and then applied to determine the effects of long-term forced and sustained swimming at U opt on growth performance of juvenile yellowtail kingfish. U opt was quantified in Blazka-type swim-tunnels for 145, 206, and 311 mm juveniles resulting in values of: (1) 0.70 m s(-1) or 4.83 BL s(-1), (2) 0.82 m s(-1) or 3.25 BL s(-1), and (3) 0.85 m s(-1) or 2.73 BL s(-1). Combined with literature data from larger fish, a relation of U opt (BL s(-1)) = 234.07(BL)(-0.779) (R (2) = 0.9909) was established for this species. Yellowtail kingfish, either forced to perform sustained swimming exercise at an optimal speed of 2.46 BL s(-1) ("swimmers") or allowed to perform spontaneous activity at low water flow ("resters") in a newly designed 3600 L oval flume (with flow created by an impeller driven by an electric motor), were then compared. At the start of the experiment, ten fish were sampled representing the initial condition. After 18 days, swimmers (n = 23) showed a 92% greater increase in BL and 46% greater increase in BW as compared to resters (n = 23). As both groups were fed equal rations, feed conversion ratio (FCR) for swimmers was 1.21 vs. 1.74 for resters. Doppler ultrasound imaging showed a statistically significant higher blood flow (31%) in the ventral aorta of swimmers vs. resters (44 ± 3 vs. 34 ± 3 mL min(-1), respectively, under anesthesia). Thus, growth performance can be rapidly improved by optimal swimming, without larger feed investments.

Physiological and behavioral responses to an electrical stimulus in Mozambique tilapia (Oreochromis mossambicus).

Consumer awareness of the need to improve fish welfare is increasing. Electrostunning is a clean and potentially efficient procedure more and more used to provoke loss of consciousness prior to killing or slaughtering (reviewed by Van de Vis et al. in Aquac Res 34:211-220, 2003). Little is known how (powerful) electrical stimuli, which do not stun immediately, are perceived by fish. We investigated responses of hand-held Mozambique tilapia (Oreochromis mossambicus) to a standardized electric shock applied to the tailfin. The handling with the resulting unavoidable acute stress response was carefully controlled for. Fish responses were analyzed up to 24 h following the shock. Electric shock resulted in slightly higher levels in plasma cortisol, lactate, ionic levels, and osmolality, than handling alone. Plasma glucose had significantly increased 6 h after shock compared to handling, indicative of enhanced adrenergic activity. Mucus release from the gills, branchial Na⁺/K⁺ ATPase activity, and chloride cell migration and proliferation, parameters that will change with strong adrenergic activation, were not affected. Decreased swimming activity and delay in resumption of chafing behavior indicated a stronger and differential response toward the electric shock. Responses to handling lasted shorter compared to those to an electric shock. The differential and stronger responses to the electric shock suggest that fish perceived the shock potentially as painful.

Tailfin clipping, a painful procedure: Studies on Nile tilapia and common carp.

The fish welfare debate is intensifying. Consequently, more research is carried out to further our knowledge on fish welfare in aquaculture. We define here a series of key parameters to substantiate an acute response to a supposedly painful stimulus: a standardized tailfin clip. Ultrastructural analysis of common carp (Cyprinus carpio) tailfin indicates the presence of A-δ and C-type axons, which are typical for transmitting nociceptive signals in (higher) vertebrates. In Nile tilapia (Oreochromis niloticus), responses to a tailfin clip were studied and the unavoidable acute stress associated with the handling required for this procedure. A series of key parameters for further studies was defined. The responses seen in 'classical' stress parameters (e.g., changes in plasma cortisol, glucose and lactate levels) did not allow discrimination between the clipping procedure and the handling stress. However, three parameters indicated a differential, stronger response to the clip stimulus itself: first, swimming activity increased more and clipped fish spent more time in the light (in a tank where half the volume is covered by dark material); second, the gill's mucus cells released their content as observed 1h after the clip, and this response is transient (no longer observed at 6h post clipping). Third, branchial Na(+)/K(+)-ATPase activity assayed in vitro was not affected by the procedures, but a remarkable migration of Na(+)/K(+)-ATPase immunoreactive (chloride) cells into the lamellar epithelium was observed as of 6h post clipping. We conclude that the differential response to clipping supports that this is a painful procedure that evokes a transient specific physiological status.