Fatty acid composition of black soldier fly larvae (Hermetia illucens) - Possibilities and limitations for modification through diet.

Black soldier fly larvae (Hermetia illucens; BSFL) can convert organic wastes into a nutrient-rich biomass suitable in animal feed, which could be a way to achieve more sustainable production of food. However, little is known about how the diet fed to BSFL affects their nutritional value, especially their fatty acid composition. In this study, BSFL were fed 11 diets based on four different organic waste sources (mussels, bread, fish and food waste). Fatty acid and proximate composition (dry matter, crude fat, crude protein and ash) were analysed in the diets, in two-week-old larvae and substrate residues. Larval weight, survival and feed conversion were also recorded. The diet was found to affect all parameters investigated. Irrespective of diet, the larval fat consisted mainly of lauric acid and other saturated fatty acids and these were found to be synthesised by the larvae. However, both the fatty acid composition of the substrate, and the larval weight were found to affect the fatty acid profile of the larvae. In general, larvae with a higher weight contained a higher percentage of saturated fatty acids and a lower percentage of unsaturated fatty acids, such as eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). It was concluded that the possibilities to tailor the fatty acid composition of the BSFL through the diet are limited; thus, the BSFL fat may not be suitable to replace fish oil, but has potential of inclusion in other food, feed and fuel products.

Remote physiological monitoring provides unique insights on the cardiovascular performance and stress responses of freely swimming rainbow trout in aquaculture.

Investigating the mechanisms that fish employ to maintain homeostasis in their everyday life requires measurements of physiological and behavioural responses in the field. With multivariate bio-loggers, we continuously measured gastrointestinal blood flow (GBF), heart rate, activity and body temperature in rainbow trout (Oncorhynchus mykiss) swimming freely amongst ~5000 conspecifics in a sea cage. Our findings clearly demonstrate that while both acute aquaculture-related stress and spontaneous activity resulted in transient reductions in GBF (i.e. reductions of up to 65%), recovery from stressful handling practices subsequently involved a substantial and prolonged gastrointestinal hyperemia far beyond the level observed prior to the stressor. The gastrointestinal hyperemia may be necessary to repair the damage to the gastrointestinal tract caused by acute stress. Furthermore, heart rate responses to acute stress or voluntary activity differed depending on the individual's physiological state. Stressed fish (i.e. mean heart rates >70 beats min-1) exhibited a bradycardic response to acute stress or activity, whereas fish with mean heart rates <60 beats min-1 instead demonstrated strong tachycardic responses. Remote monitoring of physiological and behavioural variables using bio-loggers can provide unique insights into 'real-life' responses of animals, which can largely differ from the responses observed in confined laboratory settings.

Haematological and intestinal health parameters of rainbow trout are influenced by dietary live yeast and increased water temperature.

Live yeast may be a sustainable protein source in salmonid diets while exhibiting a probiotic effect to counteract environmental stressors, such as increased water temperature that is being exacerbated by climate change. The objective of this study was to evaluate the effects of feeding a high dietary inclusion of live yeast and increased water temperature on growth, haematological and intestinal physiology of rainbow trout. For six weeks, 129 g fish in 16 tanks (n = 4) were fed either a diet based on fishmeal or based on live yeast (214 g kg-1 of diet or 7.6 log CFU g-1 of Saccharomyces cerevisiae) that replaced 40% of fishmeal protein while fish were reared in water temperatures of either 11 °C (cold) or 18 °C (warm). Fish weights, caudal blood and proximal and distal intestines were collected and analysed. Fish fed live yeast resulted in reduced growth (SGR and WG) and higher FCR, while growth in cold and warm water was similar despite differences in TGC. However, increased mortality, plasma cortisol, and intestinal oedema and villous damage indicated fish reared in warm water were subjected to chronic stress. Temperature had a significant effect on haematocrit and red blood cell counts that resulted in significantly higher haemoglobin levels in fish kept in warm water attributed to an elevated oxygen demand. In the proximal intestine, increased temperature resulted in reduced expression of pro-inflammatory cytokines, e.g. TNFα and IL8, that were further reduced in fish fed live yeast. In addition, feeding live yeast reduced gene expression of CLD6 involved in gut barrier function, which suggests that the level of yeast was too high and masked any beneficial effects on fish health. In conclusion, feeding a high inclusion of live yeast reduced fish growth and expression of intestinal genes, while increasing the temperature from 11 to 18 °C subjected fish to chronic stress that restricted growth, suppressed innate immunity and induced intestinal damage. Replacing 40% of fishmeal protein with live yeast did not counteract negative effects caused by increased temperature, thus alternative strategies need to be explored and implemented to protect the growth and health of rainbow trout from seasonal and long-term rises in water temperature.

Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

Effects of dietary yeast inclusion and acute stress on post-prandial whole blood profiles of dorsal aorta-cannulated rainbow trout.

Yeast is a potential alternative to fish meal in diets for farmed fish, yet replacing more than 50 % of fish meal results in reduced fish growth. In a 4-week experiment, 15 rainbow trout (Oncorhynchus mykiss) were cannulated and fed three diets each week: 30 % fish meal as a control (FM); 60 % replacement of fish meal protein, on a digestible basis, with Saccharomyces cerevisiae (SC); and 60 % replacement with Wickerhamomyces anomalus and S. cerevisiae mix (WA). Blood was collected at 0, 3, 6, 12 and 24 h after feeding. In the final week, fish were exposed to a 1-min netting stressor to evaluate possible diet-stress interactions. Significant increases in pH, TCO2, HCO3 and base excess were found after fish were fed the SC and WA diets compared with FM, which elevated blood alkaline tides. Yeast ingredients had lower buffering capacity and ash content than fish meal, which explained the increase in alkaline tides. In addition, fish fed the WA diet had significantly reduced erythrocyte area and fish fed SC and WA diets had increased mean corpuscular haemoglobin levels, indicating haemolytic anaemia. Higher levels of nucleic acid in yeast-based diets and potentially higher production of reactive oxygen species were suspected of damaging haemoglobin, which require replacement by smaller immature erythrocytes. Acute stress caused the expected rise in cortisol and glucose levels, but no interaction with diet was found. These results show that replacing 60 % of fish meal protein with yeasts can induce haemolytic anaemia in rainbow trout, which may limit yeast inclusion in diets for farmed fish.

Genetic improvement of feed conversion ratio via indirect selection against lipid deposition in farmed rainbow trout (Oncorhynchus mykiss Walbaum).

In farmed fish, selective breeding for feed conversion ratio (FCR) may be possible via indirectly selecting for easily-measured indicator traits correlated with FCR. We tested the hypothesis that rainbow trout with low lipid% have genetically better FCR, and that lipid% may be genetically related to retention efficiency of macronutrients, making lipid% a useful indicator trait. A quantitative genetic analysis was used to quantify the benefit of replacing feed intake in a selection index with one of three lipid traits: body lipid%, muscle lipid% or viscera% weight of total body weight (reflecting visceral lipid). The index theory calculations showed that simultaneous selection for weight gain and against feed intake (direct selection to improve FCR) increased the expected genetic response in FCR by 1·50-fold compared with the sole selection for growth. Replacing feed intake in the selection index with body lipid%, muscle lipid% or viscera% increased genetic response in FCR by 1·29-, 1·49- and 1·02-fold, respectively, compared with the sole selection for growth. Consequently, indirect selection for weight gain and against muscle lipid% was almost as effective as direct selection for FCR. Fish with genetically low body and muscle lipid% were more efficient in turning ingested protein into protein weight gain. Both physiological and genetic mechanisms promote the hypothesis that low-lipid% fish are more efficient. These results highlight that in breeding programmes of rainbow trout, control of lipid deposition improves not only FCR but also protein-retention efficiency. This improves resource efficiency of aquaculture and reduces nutrient load to the environment.

Evaluation of chitinolytic activities and membrane integrity in gut tissues of Arctic charr (Salvelinus alpinus) fed fish meal and zygomycete biomass.

Chitinolytic activity, nutrient uptake and intestinal barrier functions were investigated in gut tissues of Arctic charr (Salvelinus alpinus) fed iso-nitrogenous diets based on fish meal, with or without inclusion of zygomycetes (Rhizopus oryzae). We found that gut tissue of Arctic charr had significant chitinase activity, of both endo- and exo-chitinase iso-forms. Moreover, the distribution pattern along the GI tract of Arctic charr differed between endo-chitinase and exo-chitinase. The endo-chitinase activity in stomach tissue and in the distal intestine was several hundred-fold higher than the exo-chitinase activity in stomach tissue. The greatest exo-chitinase activity was found in the distal intestine. The zygomycete-based diet resulted in higher chitinolytic activity in gut tissue compared to the fish meal-based diet. Disturbed intestinal integrity and increased uptake rate of the amino acid lysine were observed in the distal, but not proximal, intestine of fish fed the zygomycete-based feed.

Post-surgical analgesia in rainbow trout: is reduced cardioventilatory activity a sign of improved animal welfare or the adverse effects of an opioid drug?

The use of fish models in biomedical research is increasing. Since behavioural and physiological consequences of surgical procedures may affect experimental results, these effects should be defined and, if possible, ameliorated. Thus, the use of post-surgical analgesia should be considered after invasive procedures also in fish, but presently, little information exists on the effects of analgesics in fish. This study assessed the effects of an opioid drug, buprenorphine (0.05 mg/kg IM), on resting ventilation and heart rates during 7 days of postsurgical recovery in rainbow trout (Oncorhynchus mykiss) at 10°C by non-invasively recording bioelectric potentials from the fish via electrodes in the water. Baseline ventilation and heart rates were considerably lower compared to previously reported values for rainbow trout at 10°C, possibly due to the non-invasive recording technique. Buprenorphine significantly decreased both ventilation and heart rates further, and the effects were most pronounced at 4-7 days after anaesthesia, surgical procedures and administration of the drug. Somewhat surprisingly, the same effects of buprenorphine were seen in the two control groups that had not been subject to surgery. These results indicate that the reductions in ventilation and heart rates are not caused by an analgesic effect of the drug, but may instead reflect a general sedative effect acting on both behaviour as well as e.g. central control of ventilation in fishes. This resembles what has previously been demonstrated in mammals, although the duration of the drug effect is considerably longer in this ectothermic animal. Thus, before using buprenorphine for postoperative analgesic treatment in fish, these potentially adverse effects need further characterisation.

Strain- and temperature-dependent changes of fatty acid composition in Wickerhamomyces anomalus and Blastobotrys adeninivorans.

The fatty acid (FA) profiles of two strains of the yeasts Wickerhamomyces anomalus and Blastobotrys (Arxula) adeninivorans at cultivation temperatures from 15 to 30 °C were characterized. Besides the common even-numbered C16 and C18 FAs, substantial proportions of the uneven-numbered C17:1 were found in both species. C18:3(n-3) (alpha linolenic acid) made up to 3% of the total FAs in all strains. Considerable strain differences occurred, with regard to both the presence of single FAs and parameters like the double binding index (DBI) and C16:C18 ratio. W. anomalus J121 formed C18:1(n-5) (up to 10.9% of the total FAs) but no C18:1(n-7), whereas in W. anomalus VKM160, no C18:1(n-5) was found but up to 14.6% C18:1(n-7). Similarly, B. adeninivorans CBS 8244 formed exclusively C18:1(n-7) (maximum 9%) and CBS 7377 C18:1(n-5) (maximum 12.6%). W. anomalus J121 had the lowest DBI (0.72) at 15 °C and the highest (0.92) at 20 °C, at which point the values decreased with increasing temperatures. In W. anomalus VKM160 and both B. adeninivorans strains, DBI was highest at 15 °C and decreased with increasing temperature. In J121, the C16:C18 ratio was highest at 15 °C, decreasing at higher temperatures, whereas in the other strains, the opposite trend was observed.

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.

Anaesthesia of farmed fish: implications for welfare.

During their life cycle as farmed animals, there are several situations in which fish are subjected to handling and confinement. Netting, weighing, sorting, vaccination, transport and, at the end, slaughter are frequent events under farming conditions. As research subjects, fish may also undergo surgical procedures that range from tagging, sampling and small incisions to invasive procedures. In these situations, treatment with anaesthetic agents may be necessary in order to ensure the welfare of the fish. The main objective of this paper is to review our knowledge of the effects of anaesthetic agents in farmed fish and their possible implications for welfare. As wide variations in response to anaesthesia have been observed both between and within species, special attention has been paid to the importance of secondary factors such as body weight, water temperature and acute stress. In this review, we have limited ourselves to the anaesthetic agents such as benzocaine, metacaine (MS-222), metomidate hydrochloride, isoeugenol, 2-phenoxyethanol and quinaldine. Anaesthetic protocols of fish usually refer to one single agent, whereas protocols of human and veterinary medicine cover combinations of several drugs, each contributing to the effects needed in the anaesthesia. As stress prior to anaesthesia may result in abnormal reactions, pre-anaesthetic sedation is regularly used in order to reduce or avoid stress and is an integral part of the veterinary protocols of higher vertebrates. Furthermore, the anaesthetic agents that are used in order to obtain general anaesthesia are combined with analgesic agents that target nociception. The increased use of such combinations in fish is therefore included as a special section. Anaesthetic agents are widely used to avoid stress during various farming procedures. While several studies report that anaesthetics are effective in reducing the stress associated with confinement and handling, there are indications that anaesthesia may in itself induce a stress response, measured by elevated levels of cortisol. MS-222 has been reported to elicit high cortisol release rates immediately following exposure, while benzocaine causes a bimodal response. Metomidate has an inhibitory effect on cortisol in fish and seems to induce the lowest release of cortisol of the agents reported in the literature. Compared to what is observed following severe stressors such as handling and confinement, the amount of cortisol released in response to anaesthesia appears to be low but may represent an extra load under otherwise stressful circumstances. Furthermore, anaesthetics may cause secondary adverse reactions such as acidosis and osmotic stress due to respiratory arrest and insufficient exchange of gas and ions between the blood and the water. All in all, anaesthetics may reduce stress and thereby improve welfare but can also have unwanted side effects that reduce the welfare of the fish and should therefore always be used with caution. Finally, on the basis of the data reported in the literature and our own experience, we recommend that anaesthetic protocols should always be tested on a few fish under prevailing conditions in order to ensure an adequate depth of anaesthesia. This recommendation applies whether a single agent or a combination of agents is used, although it appears that protocols comprising combinations of agents provide wider safety margins. The analgesic effects of currently used agents, in spite of their proven local effects, are currently being debated as the agents are administrated to fish via inhalation rather than locally at the target site. We therefore recommend that all protocols of procedures requiring general anaesthesia should be complemented by administration of agents with analgesic effect at the site of tissue trauma.

Changes in arterial PO2, physiological blood parameters and intracellular antioxidants in free-swimming Atlantic cod (Gadus morhua) exposed to varying levels of hyperoxia.

Free-swimming Atlantic cod (Gadus morhua) were exposed to water oxygen pressures (P(w)O2) ranging from 18.1 to 41.5 kPa and sampled for blood using an indwelling caudal artery cannula. Arterial blood oxygen pressure (P(a)O2) increased with increasing P(w)O2, from 12.0 kPa in normoxia (18.1 kPa) to 34.2 kPa in the highest hyperoxic level tested (41.5 kPa). Blood CO2 pressure and plasma bicarbonate concentration increased with P(w)O2, indicating reduced ventilation with increased P(w)O2. Plasma glucose, sodium and potassium were not affected by water oxygen level. Blood oxidative stress biomarkers, reduced glutathione, oxidized glutathione and the oxidative stress index (ratio between oxidized and total glutathione) differed intermittently between normoxia and hyperoxia. The oxidative stress index was higher in the blood of exposed compared to unexposed control cod. Together with elevated P(a)O2, these findings suggest increased production of reactive oxygen species and increased oxidative stress in Atlantic cod exposed to hyperoxia.

Anesthesia induces stress in Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus).

Stress in response to anesthesia with benzocaine, MS-222, metomidate and isoeugenol was studied in Atlantic salmon (Salmo salar), Atlantic halibut (Hippoglossus hippoglossus), and Atlantic cod (Gadus morhua) with no concomitant stress from handling or confinement in association with anesthesia or sampling. All of the anesthetics tested induced a stress response in all species, displayed by a release of cortisol to the water. MS-222 anesthesia elicited the highest cortisol release rates, reaching maximum levels 0.5 h post-exposure and returning to basal levels after 3-4 h. Benzocaine anesthesia caused a bimodal response where the initial peak in cortisol release rate was followed by a second increase lasting towards the end of the trial (6 h). This bimodality was more profound in Atlantic salmon than in Atlantic halibut and Atlantic cod. Metomidate anesthesia induced the lowest release of cortisol of the agents tested in both Atlantic halibut and Atlantic cod, but resulted in a bimodal response in Atlantic salmon where the initial increase in cortisol release was followed by a larger increase peaking at 2-2.5 h post exposure before returning to basal levels after 5 h. The stress induced in Atlantic salmon by isoeugenol anesthesia resembled that of MS-222, but did not reach the same elevated level. Overall, the cortisol release was most profound in Atlantic salmon followed by Atlantic halibut and Atlantic cod.

Postprandial changes in plasma free amino acid levels obtained simultaneously from the hepatic portal vein and the dorsal aorta in rainbow trout (Oncorhynchus mykiss).

For the first time, changes in plasma concentrations of free amino acid (AA) and their metabolites were followed simultaneously in pre- and post-hepatic blood following a single meal in non-anaesthetized and free-swimming fish. Rainbow trout (Oncorhynchus mykiss), kept in 10 degrees C water and fitted with cannulae in the hepatic portal vein (HPV) and the dorsal aorta (DA), were force-fed 1% of their body mass and blood samples were taken from both cannulae at 0, 3, 6, 12, 24 and 48 h postprandially to follow the free AA profile. Almost all free AAs increased rapidly within the first 3 h and only a few free AAs did not change significantly over time. By 6 h, the total free AA concentration had peaked in blood taken from both the DA (7107+/-369 nmol ml(-1)) and HPV (9999+/-572 nmol ml(-1)). However, individual free AAs showed three main profiles beyond this time: for type I, a peak concentration occurred only at 6 h; for type II, there was a more gradual rise in concentration to a peak at 24 h; and for type III there were two peaks, at 6 h and 24 h. All free AAs returned to or were lower than baseline levels within 48 h, with the exception of threonine and proline. The total free AA concentrations were consistently higher (P<0.05) in the HPV than in the DA at 3 h, 6 h, 12 h and 24 h. Our data provide clear evidence that, during the first pass through the liver, hepatic modification altered individual free AA concentrations as indicated by variable ratios among the simultaneous blood samples. Furthermore, the elevation of ammonium and urea in the HPV indicates intestinal catabolism of ingested free AA before release into the HPV. In conclusion, the dual HPV and DA cannulation shows promise as a useful technique for qualitative and quantitative investigations of absorption and turnover of nutrients, especially if the measurements can be combined with reliable estimates of blood flow and labelled substances.

Effects of isoeugenol on in vitro neuromuscular blockade of rat phrenic nerve-diaphragm preparations.

OBJECTIVE: To investigate in vitro effects of isoeugenol on neuromuscular transmission in tissues obtained from rats. SAMPLE POPULATION: Tissues (phrenic nerve and diaphragm) obtained from 15 male Sprague-Dawley rats. PROCEDURE: Rats were euthanatized, and tissues (phrenic nerves and diaphragm) were obtained. Phrenic nerve-diaphragm preparations were examined in vitro. The phrenic nerve was stimulated with weak electrical impulses. Muscle-twitch responses were recorded before and after the addition of drugs (pancuronium, neostigmine, isoeugenol, and benzocaine). RESULTS: Pancuronium and isoeugenol in low concentrations (10 to 206 microM) caused a distinct decrease in twitch response, which could be reversed by the addition of neostigmine. The decrease in twitch response caused by benzocaine or high concentrations of isoeugenol could not be reversed by the addition of neostigmine. CONCLUSIONS AND CLINICAL RELEVANCE: Isoeugenol caused a competitive blockade of neuromuscular transmission. Neostigmine restored this transmission by inhibiting acetylcholinesterase, which led to increased concentrations of acetylcholine. Because isoeugenol is used as an anesthetic in fish, further investigations are necessary to determine whether fish exposed to isoeugenol are sedated and unconscious or whether they are only paralyzed but have intact perception in afferent sensory nerves.