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[EN] Protocols for the cryopreservation of fish gametes have been developed for many different fish species, in special, freshwater salmonids and cyprinids. Methods for sperm freezing have progressed during the last decades due to the increasing number of potential applications: aquaculture (genetic improvement programs, broodstock management, helping with species having reproductive problems), biotechnology studies using model fish species (preservation of transgenic or mutant lines), cryobanking of genetic resources from endangered species, etc. This mini-review tries to give an overview of the present situation of this area of research, identifying the main challenges and perspectives, redirecting the reader to more in-depth reviews and papers., Partially funded by the European Training Network IMPRESS (Marie Sklodowska Curie Actions; Grant agreement nº: 642893), COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE), the Research Centre of Excellence- 9878-3/2016/FEKUT, KMR_12-1-2012-0436, NKFIH (OTKA) 109847, KLING 31-03-05-FEP-73, CRIOBIV 31-03-05-FEP-59, REPLING 31-03-05-FEP-69 all from PROMAR programme.

Individual differences in physiological and behavioural responses to stressors are increasingly recognised as adaptive variation and thus raw material for evolution and fish farming improvements including selective breeding. Such individual variation has been evolutionarily conserved and is present in all vertebrate taxa including fish. In farmed animals, the interest in consistent trait associations, that is coping styles, has increased dramatically over the last years because many studies have demonstrated links to performance traits, health and disease susceptibility and welfare. This study will review (i) the main behavioural, neuroendocrine, cognitive and emotional differences between reactive and proactive coping styles in farmed fish; (ii) the methodological approaches used to identify coping styles in farmed fish, including individual (group) mass-screening tests; and (iii) how knowledge on coping styles may contribute to improved sustainability of the aquaculture industry, including welfare and performance of farmed fish. Moreover, we will suggest areas for future research, where genetic basis (heritability/epigene tic) of coping styles, and the neuroendocrine mechanisms behind consistent as well as flexible behavioural patterns are pinpointed as central themes. In addition, the ontogeny of coping styles and the influence of age, social context and environmental change in coping styles will also be discussed. European Commission under the 7th Framework Programme FP7-KBBE [265957]

Fish hatcheries must adapt larval feeding protocols to feeding behavior and metabolism patterns to obtain more efficient feed utilization. Fish larvae exhibit daily ingesting rhythms rather than ingesting food continuously throughout the day. The aim of this study was to determine the daily patterns of feed intake, protein digestibility, protein retention and catabolism in Senegalese sole post-larvae (Solea senegalensis; 33 days post-hatching) using 14C-labeled Artemia protein and incubation in metabolic chambers. Sole post-larvae were fed at 09:00, 15:00, 21:00, 03:00 and 09:00+1 day; and those fed at 09:00, 21:00, 03:00 and 09:00+1 day showed significantly higher feed intake than post-larvae fed at 15:00 h (P=0.000). Digestibility and evacuation rate of ingested protein did not change during the whole cycle (P=0.114); however, post-larvae fed at 21:00 and 03:00 h showed the significantly highest protein retention efficiency and lowest catabolism (P=0.002). Therefore, results confirm the existence of daily rhythmicity in feeding activity and in the utilization of the ingested nutrients in Senegalese sole post-larvae., This work received national funds from Fundação para a Ciência e a Tecnologia (FCT; Portugal) through projects CCMAR/Multi/04326/2013 and SOLEAWIN (310305/FEP/71). It was partially supported by the PROMAR Program with funds from Fundo Regional para a Ciência e Tecnologia (FEDER), and also through the Ministerio de Economía y Competitividad (MINECO; Spain) by project EFISHDIGEST (AGL2014-52888-R) with FEDER/European Region Development Fund (ERDF) contribution granted to M.Y. (Spain). Thanks are also due to European Cooperation in Science and Technology (Cost Action) FA0801 (Larvanet). C.N.-G. was supported by a doctoral fellowship (BES-2012-051956) from Ministerio de Economía y Competitividad (Spain). S.E. acknowledges a FCT investigator grant IF/00482/2014/CP1217/CT0005 funded by Fundação para a Ciência e a Tecnologia (FCT; Portugal) and the European Social Fund under the Operational Programme for the Enhancement of Human Potential.

The aim of this study was to assess electrical stunning of Atlantic cod and turbot in seawater to develop a protocol for the process of stunning and killing. An induced general epileptiform insult (unconscious) had a duration of 40 ± 27 s (n =14) in cod (2.6 ± 0.5 kg) and 34 ± 18 s (n = 19) in turbot (520 ± 65 g). Seven cod and 3 turbot displayed a physical reaction, and 11 turbot registered an electroencephalogram (EEG) response to pain stimuli administered 30 s post-stun. The heart rate was 32 ± 6 beats/min in cod and 25 ± 7 beats/min in turbot prior to stunning. Post-stunning, the electrocardiogram (ECG) revealed fibrillation and reduced activity post-stun. EEG, ECG recordings, and behavioral observations indicate that when a bipolar square wave current was applied with a frequency of 133 Hz and 43% duty cycle side to side (turbot) and at 170 Hz and 33% duty cycle (cod) head to tail, both species were stunned in seawater at current densities of 3.2 A/dm2 and 2.5 A/dm2, respectively. For turbot, a 5 s exposure to electricity followed by chilling in ice water for 15 min is sufficient to prevent recovery. For cod, a killing method needs to be established.

Animals should be rendered unconscious before slaughter in order to avoid suffering or pain. The objective of this study was to evaluate an electrical stunning after dewatering to induce instantaneous unconsciousness and insensibility in yellowtail kingfish (Seriola lalandi), common sole (Solea solea) and pike-perch (Stizostedion lucioperca). To kill without recovery, the current was applied for 5 s, followed by chilling in ice water for 10 min. Loss of consciousness and sensibility were assessed by neural (EEG), physiological (ECG) and behavioural parameters. An epileptiform insult was observed in all yellowtail kingfish (n = 27), common sole (n = 25) and pike-perch (n = 25) after administering a current of 0.54 +/- 0.12 A(rms) (124 V dc and 11 V-rms ac; 100 Hz), 0.65 +/- 0.23 A(rms) (98 V dc and 8.4 V-rms ac; 100 Hz) and 0.75 +/- 0.24 A(rms) (144 V dc and 13 V-rms ac; 100 Hz) during 1 s through the head of individual fish, respectively. When yellowtail kingfish (n = 11) was submitted to a 5 s electrical stun followed by chilling in ice water, this resulted in passing 0.72 +/- 0.13 A(rms) for 5 s and no recovery during chilling. In the case of common sole (n = 10) and pike-perch (n = 12), passing 1.4 +/- 0.64 and 0.75 +/- 0.24 A(rms) during 5 s followed by chilling in ice water for 10 min resulted in an irrecoverable stun in 9 out 10 fish and 11 out 12 fish, respectively. We conclude that for yellowtail kingfish the investigated combination of electrical stunning and chilling is suitable for humane slaughter, whereas for common sole and pike-perch this procedure needs to be optimised. (C) 2012 Elsevier B.V. All rights reserved.

It has been suggested that fish cultured in recirculating aquaculture systems (RAS) grow less as compared with fish cultured in flow-through systems due to the accumulation of substances. In the Netherlands, the commercial culture of Nile tilapia Oreochromis niloticus in 300 and 600 MT's systems is done exclusively in RAS operated at water exchange rates as low as 30 L/kg feed/day due to nitrate control by single-sludge denitrification reactors. The use of such nearly closed RAS raises the question whether growth retardation (GR) is present in Nile tilapia. This study is the first to investigate the existence of growth retardation in Nile tilapia by comparing the growth, feeding behaviour and stress response of Nile tilapia cultured in RAS with different levels of substances accumulated. Three RAS, operated at 30 L/kg feed/day (HIGH accumulation), 70 L/kg feed/day (MIDDLE accumulation) and 1500 L/kg feed/day (LOW accumulation) were used. Each RAS contained 24 glass aquaria with individually housed fish. To determine whether GR is size-dependent, per RAS 3 fish size categories were tested in the 57 day experimental period: large (288.7 ± 34.2 g; N = 8), medium (162.4 ± 23.4 g; N = 8) and small (81.4 ± 21.0 g; N = 8). Experimental fish were fed ad libitum , twice per day. Feeding behaviour was determined once per week and was measured as the time taken by each fish to eat the first pellet (latency, LAT) and the total time spent feeding (total feeding time, TFT). Temperature, pH, conductivity, alkalinity, dissolved oxygen, dissolved CO 2 , nitrogen compounds (TAN, NO 2 -N and NO 3 -N), chemical oxygen demand and orthophosphate-P were measured over time. At day 57 fish were weighed, blood sampled and returned to their tanks for an extra experimental period of 15 days, and subjected at day 72 to an acute stress followed by blood sampling. Blood was analysed for glucose and cortisol. Results showed that the water quality parameters measured in the 3 RAS (with the exception of alkalinity) were still within the optimum range for growth of Nile tilapia. Large individuals showed a tendency to grow more in the LOW treatment (2.66 ± 1.35 g/kg 0.8 /day) as compared with the MIDDLE treatment (0.93 ± 1.63 g/kg 0.8 /day). On the contrary, small individuals grew significantly less in the LOW treatment (3.60 ± 1.74 g/kg 0.8 /day) as compared with the HIGH (7.22 ± 1.58 g/kg 0.8 /day) and MIDDLE (6.82 ± 4.54 g/kg 0.8 /day) treatments. Small fish were more motivated to eat (lower latency) in the MIDDLE (4.63 ± 5.24 min) as compared with the LOW treatment (8.94 ± 6.41 min). In the HIGH accumulation treatment higher glucose levels were observed in the small fish, before and after acute stress, as compared with the LOW accumulation treatment. In conclusion, this study showed that the extent to which the accumulation of substances in RAS affects growth depends on fish size: large individuals show a trend towards growth retardation in the highest accumulation RAS while small individuals, on the contrary, seem to grow better in such systems.

Behavioural, neural and physiological parameters were scored after electro-stunning of Nile tilapia ( Oreochromis niloticus ) in three positions to an electric field in fresh water. An overall current density, 1.0 A rms /dm 2 , 50 Hz sinusoidal, applied for 1 s top-to-bottom in water (700 μS/cm), induced a general epileptiform insult on the EEG for 26 ± 10 s ( n = 24). The ECG revealed fibrillation for 9 ± 4 s in 15 out of 24 fish. To kill without recovery, the current was applied for 5 s, followed by chilling in ice water for 15 min. Killing of the stunned fish by gill-cutting instead of chilling is not recommended, as 2 out 4 tilapias responded 10 min post stun. When electricity was applied head-to-tail on tilapia ( n = 8), the overall current density could be reduced to 0.4 A rms /dm 2 , 50 Hz sinusoidal, to induce a general epileptiform insult, which lasted 27 ± 10 s. A pulsed square wave alternating current (overall density of 0.6 A rms /dm 2 , 133 Hz and 43% duty cycle) was applied for 1 s side-to-side on tilapia in water (650 μS/cm). This induced a general epileptiform insult for 51 ± 37 s ( n = 14). Fibrillation occurred for 16 ± 7 s ( n = 10). Free-swimming fish righted between 68 and 95 s ( n = 5) and between 438 and 1139 s ( n = 5) after 1 and 5 s electro-stunning, respectively. Our results show that protection of welfare of tilapia at slaughter can be obtained with electro-stunning followed by killing the stunned fish in ice water.

The objective was to assess neural, behavioural responses and product quality in farmed sea bass (Dicentrarchus labrax) upon electrical stunning in seawater. The electrical sinusoidal 50 Hz or pulse square wave alternating 133 Hz current induced a general epileptiform insult with a current of 3.3±0.2 or 3 A dm¿2, respectively, for 1 s head to tail in seawater. The total duration of the insult was 48±34 and 23±11 s. After stunning, the electro-cardiogram revealed fibrillation and ceased or showed malfunction. Product quality was assessed in a group electrically stunned, followed by chilling in ice water and the controls were only chilled in ice water. The pH of the fillets was 0.1¿0.2 lower (P

International audience; Feed efficiency is a major goal for aquaculture sustainability, and selecting fish to genetically enhance this trait would be highly valuable. However, no selective breeding program specifically targeted to feed efficiency exists for farmed fish, mostly because of the difficulty of measuring individual feed intake. However, a negative phenotypic correlation between feed efficiency and weight loss at fasting has been previously demonstrated in sea bass submitted to feed deprivation (FD). We mated sea bass parents selected for their high (FD+) or low (FD-) weight loss at fasting to produce FD+ and FD- progeny, which were reared in a single tank to avoid common environmental effects. At 8 months of age, 1200 of those fish were submitted to three alternating periods of fasting (3 weeks) and re-feeding (3 weeks). Individuals were weighed at the end of each feeding and fasting period. Their line of origin was identified by genotyping of 12 microsatellite markers, resulting in 1130 unambiguously assigned fish (484 FD-, 686 FD+). FD- offspring lost significantly less weight than FD+ offspring in this feed deprivation trial. After that the feed efficiency of eight groups of 50 FD+ fish and eight groups of 50 FD- fish was evaluated in four successive 20-day periods. At the end of the fourth period, 10 fish per tank were sacrificed to evaluate their carcass yield. The FD- fish had a better overall growth and were fatter, and FD+ fish had a better carcass yield. A better feed efficiency was expected for the FD- fish, but differences between the two groups for this trait, measured either with feed efficiency ratio or with residual feed intake, were not consistently significant Although the two lines were clearly divergent for several traits, demonstration of feed efficiency differences between the FD+ and the FD- lines was not consistently observed in sea bass. A second generation of selection may allow further divergence in the lines and reveal differences in feed efficiency. (C) 2013 Elsevier B.V. All rights reserved.

The overall objective of the study was to evaluate a percussive and an electrical stunning method under laboratory conditions in Atlantic salmon. Evidence of unconsciousness and insensibility of the salmon was provided on the electroencephalogram (EEG) by the appearance of slow waves and spikes, followed by a strong depression in electrical activity. This phenomenon was observed in 17 salmon after percussive stunning using an air pressure of 8.1 to 10 bars, whilst 8 fish were considered conscious at pressures below 8.1 bars although some were seemingly unconscious on behaviour. Consequences were a haemorrhage in the brain cavity in 15 out of 17 fish, broken upper or lower jaws in 9 fish and eye burst in 8 fish. A general epileptiform insult (unconscious and insensible) was obtained by delivering a voltage, consisting of a direct current (DC) coupled with 100 Hz alternating current (AC) with a peak value of ≈112 volt (V), head to body, for ≈ 0.5 s. The total duration of the insult was 62 ± 44 s (mean ± SD; n = 25) which was followed by minimal brain activity in 19 fish. The heart rate was 20 ± 7 beats/min prior to stunning. After stunning the electrocardiogram (ECG) revealed fibrillation for 22 ± 15 s and became irregular and showed extra systolae (ventrical contraction) afterwards. Exposing the salmon for 5 s with electricity followed by a gill cut resulted that 1 out of 3 fish recovered temporary after 3 min. Haemorrhages were not observed in the fillets. Average current for head to body electrical dry stunning was 668 milliampere (mA) root mean square (RMS) with an average stunning voltage of 107.9 V rms . Electrical head to body stunning can be recommended when using coupled AC and DC current of 668 mA rms and ≈ 107 V rms . The salmon can be stunned in ≈ 0.5 s. However, a correct bleeding procedure should be developed. For percussive stunning we conclude that if sufficient force is used the fish will be rendered unconscious insensible which result in damage of the carcass, whereas a combined AC and DC can be recommended source for dry electrical head to body stunning.

The objective was to assess neural, behavioural responses and product quality in farmed sea bass (Dicentrarchus labrax) upon electrical stunning in seawater. The electrical sinusoidal 50 Hz or pulse square wave alternating 133 Hz current induced a general epileptiform insult with a current of 3.3±0.2 or 3 A dm¿2, respectively, for 1 s head to tail in seawater. The total duration of the insult was 48±34 and 23±11 s. After stunning, the electro-cardiogram revealed fibrillation and ceased or showed malfunction. Product quality was assessed in a group electrically stunned, followed by chilling in ice water and the controls were only chilled in ice water. The pH of the fillets was 0.1¿0.2 lower (P

The overall objective of the study was to evaluate electrical and percussive stunning methods under laboratory conditions in common carps. The electrical current needed to induce a general epileptiform insult was assessed in 13 carps. The insult was obtained by delivering a current of 0.24 ± 0.03 A (163 ± 1 V; 50 Hz, a.c.) to the head, via scissor-model stunning tongs for approximately 1 s. The duration of the tonic, the clonic and the exhaustion phases were 11 ± 4 s, 5 ± 3 s and 20 ± 14 s on the EEG, respectively. An actual clonic phase was observed in two fish. The total duration of the insult was 31 ± 14 s. The heart rate was 22 ± 12 beats/min prior to stunning while after stunning, the ECG revealed fibrillation for 18 ± 7 s and was irregular. The behaviour of 10 individual carp, which were allowed to move freely in water, was recorded following head-only stunning (164 V, 0.23 ± 0.6 A). The duration of the tonic phase was approximately 10 s and the fish started fin movements after 48 ± 8 s and resumed swimming after 121 ± 83 s. A general epileptiform insult could be induced in carps (n = 25) by passing an electrical current through fresh water using 0.14 ± 0.03 A/dm2 (113 ± 17 V; electrode distance 16 cm) for 1 s at a water conductivity of 200 ¿S/cm. Twenty three carps were rendered unconscious and insensible during electrical stunning in fresh water, using 0.73 ± 0.05 A/dm2 (411 ± 2 V; electrode distance 16 cm) for 5 s at a water conductivity of 330 ¿S/cm, followed by chilling in ice water. As a second stunning procedure, carps were stunned mechanically using a percussion pistol at a maximal velocity of 10.99 ± 0.88 J. Evidence of unconsciousness and insensibility of the carps was provided by the appearance of theta, delta waves and spikes on the EEG, which were proceeded by no brain activity. However, two carp responded after 0.5 min to administered pain stimuli and one carp after 3 min. It may be concluded from this study that common carp are effectively stunned in fresh water. The application of an electrical current of 0.73 A/dm2 (26 V/cm, 50 Hz, a.c., 330 ¿S/cm) to individual carps in a tank in combination with chilling in ice water is an effective procedure for slaughter. The mechanical method of percussion stunning did not provide assurance of effective stunning as not all carp were unconscious and insensible after its administration.