Long-term body tactile stimulation reduces aggression and improves productive performance in Nile tilapia groups.

One concern of the Anthropocene is the effects of human activities on animal welfare, revealing the urgency to mitigate impacts of rearing environments. Body tactile stimulation (TS), like massage therapy, has emerged as an enrichment method to counteract stress and anxiety in vertebrates. In the current study, we evaluated the effects of long-term TS on four-member groups of male Nile tilapia, a worldwide reared species whose socially aggressive behavior is an essential source of stress. We placed a rectangular PVC frame fitted with vertical plastic sticks sided with silicone bristles in the center of aquarium to enable the fish to receive body TS when passing through the bristles. A similar apparatus without bristles was used as the control. Fish subjected to TS for 21 days showed a gradual lowering of overt fights over time, but with no reduction in cortisol or androgen levels. Nevertheless, TS improved the specific growth rate, maintained balanced length/weight gain, and increased feed efficiency, probably owing to the lowered energy expenditure during fights. Thus, we show for the first time that long-term TS provided by a simple device can be used as a tool to improve the welfare and productive performance of territorial fish.

Cannabidiol improves Nile tilapia cichlid fish welfare.

Cannabidiol (CBD) is a substance derived from Cannabis sativa, widely studied in medicine for controlling neural diseases in humans. Besides the positive effects on humans, it also presents anxiolytic proprieties and decreases aggressiveness and stress in mammals. Therefore, CBD has the potential to increase welfare in reared animals, as it seems to reduce negative states commonly experienced in artificial environments. Here, we tested the effect of different CBD doses (0, 1, 10 and 20 mg/kg) on aggressiveness, stress and reproductive development of the Nile tilapia (Oreochromis niloticus) a fish reared worldwide for farming and research purposes. CBD mixed with fish food was offered to isolated fish for 5 weeks. The 10 mg/kg dose decreased fish's aggressiveness over time, whereas 20 mg/kg attenuated non-social stress. Both doses decreased the baseline cortisol level of fish and increased the gonadosomatic index. However, CBD 1 and 10 mg/kg doses decreased the spermatozoa number. No CBD dose affected feeding ingestion and growth variables, showing that it is not harmful to meat production amount. Despite the effect on spermatozoa, CBD supplementation exhibits high potential to benefit animals' lives in artificial environments. Therefore, we showed for the first time that CBD could be used as a tool to increase non-mammal welfare, presenting a great potential to be explored in other husbandry and captivity species.

Preference and Motivation Tests for Body Tactile Stimulation in Fish.

We tested whether territorial fish (Nile tilapia) perceive body tactile stimulation as a positive or negative resource. Individual male fish were placed for eight days in an aquarium containing a rectangular PVC frame, which was filled with vertical plastic sticks sided with silicone bristles in the middle of the tank. Fish passing this device received a tactile stimulus. The fish then underwent a preference test by choosing between areas half-with and half-without tactile bristles. Then, fish were submitted to a motivation test where they had to pass an aversive stimulus (bright light) to access the device. Fish were, then, paired to settle social rank, which occurs by way of fights (social stressor), and were assigned again to preference and motivation tests. A group without social stress was used as a control. Contrary to our expectations, fish preferred the area without tactile bristles, although subordinate fish reached tactile stimulation more than the dominant one. Social stress did not affect the preference and motivation, suggesting that fish do not perceive tactile stimulation as a stressor reliever. However, as fish did not avoid the stimulation, reached the device spontaneously, and faced an aversive stimulus to access it, we conclude that tactile stimulation is not a negative condition and, therefore, can be used in further studies regarding fish welfare.

Understanding behaviour to improve the welfare of an ornamental fish.

Some common practices in aquaculture, ornamental trade and fish facilities may disturb the behavioural repertoire of fish and its natural adaptive value, reducing welfare and impairing fish production. Hence, it is necessary to understand fish behaviour, as well as the factors affecting it, to improve the quality of fish's life under artificial environment. Here, we reviewed the behaviour of the angelfish Pterophyllum scalare, an Amazonian cichlid used worldwide both as an ornamental fish and as a fish model in scientific research. We characterized social, reproductive and feeding behaviour, as well as the amazing cognitive ability of the angelfish. In addition, we reviewed the effects of environmental enrichment and suggested some important variables that need to be considered for rearing P. scalare. In this review, we show for the first time a synthesis on behaviour and a best practice overview to improve the welfare of angelfish as a target species. Nonetheless, most topics reviewed fit a broader set of fish species, particularly ornamental ones. This synthesis can therefore open a path for further behavioural research applied to the welfare of angelfish and bring insights to other fish species.

Male and female cichlid fish show cognitive inhibitory control ability.

Inhibitory control is a way to infer cognitive flexibility in animals by inhibiting a behavioral propensity to obtain a reward. Here we tested whether there are differences in inhibitory control between females and males of the fish Nile tilapia owing to their distinct reproductive roles. Individuals were tested under a detour-reaching paradigm, consisting of training fish to feed behind an opaque barrier and, thereafter, testing them with a transparent one. Fish is expected to avoid trying to cross through the transparent barrier to achieve food (reward), thus showing inhibitory control by recovering the learned detour with the opaque apparatus. Both males and females learned to detour the transparent barrier with similar scores of correct responses, whereas females reached the food faster. This result is probably associated to their different sex roles in reproduction: females care for the eggs and fry inside their mouth (thus requiring a high inhibitory control not to swallow them), whereas males have to stay inside the territory defending it against intruder males, which also demands some inhibitory ability not to leave the spawning site and take the risk of losing it. Furthermore, this evidence of cognitive flexibility can enable social fish to deal with unpredictable interactions.

The Bias of combining variables on fish's aggressive behavior studies.

Quantifying animal aggressive behavior by behavioral units, either displays or attacks, is a common practice in animal behavior studies. However, this practice can generate a bias in data analysis, especially when the variables have different temporal patterns. This study aims to use Bayesian Hierarchical Linear Models (B-HLMs) to analyze the feasibility of pooling the aggressive behavior variables of four cichlids species. Additionally, this paper discusses the feasibility of combining variables by examining the usage of different sample sizes and family distributions to aggressive behaviour variables. The subject species were: the angelfish (Pterophyllum scalare), the tiger oscar (Astronotus ocellatus), the Cichlasoma paranaense and the Nile tilapia (Oreochromis niloticus). For each species, 15 groups of 3 individuals were assigned to daily observations (10-min recordings) for 5 days. Aggressive behavior data was labeled according to its aggressive intensity. The variables chase (C), tail beating (TB), push (P), lateral attack (LA) and bite (B) were classified as high intensity. The variables undulation (U), lateral threat (LT) and frontal displays (FD) were classified as low intensity. These behaviors, however, were not present in all species. Model parameters were estimated by Monte Carlo Markov chains using non-informative priors. B-HLMs were performed to assess the impact probability of each variable in the analysis. Results revealed that when combining variables, the resulting distribution is strongly influenced by only one variable in each category. Moreover, in some cases the aggregate values altered the results, which changed the probabilities of the main variables. Species with low aggressive behavior frequencies, such as A. ocellatus, are more sensitive to this bias. LT was the main low intensity variable for all species, while B was the main high intensity variable for the P. scalare and the O. niloticus. LA was the high intensity category variable that was the most relevant for the C. paranaense and A. ocellatus. Moreover, combining the variables did not impact the feasibility of reducing the sample size when compared to using the most quantitative variable. For all species a sample size of 12 did not change the study conclusions. With respect to family distribution, based on DIC values the Gaussian model is more suitable for most of the studied species. However, caution should be taken, because the Gaussian posterior probability distribution overlapped 0 in some cases, which is biologically impossible in aggressive behaviors. The only exception is the A. ocellatus, which, based on DIC values, was the only species better modeled by a Poisson distribution. Bayesian analysis can be therefore considered a strong tool for analyzing aggressive behavior.

Tactile stimulation reduces aggressiveness but does not lower stress in a territorial fish.

Body tactile stimulation has a positive effect upon highly social animals, such as mammals and cleaner-client coral-reef fish, by relieving stress and improving health. Conversely, some tactile contacts are naturally detrimental, such as those resulted from aggressive interactions. To study whether positive responses from tactile stimulation are generalized among vertebrates, we tested its effect on stress response and aggressive behavior in a territorial fish species, Nile tilapia. We developed an apparatus made of a row of sticks bordered by silicone bristles that was positioned in the middle of the aquarium, and through which fish had to pass to access food, thus receiving tactile stimulation. Isolated fish experienced tactile stimulation for 7 days, and  were assigned to 2 types of stressors: non-social (confinement) or social (aggressive interaction). Each of them had a corresponding control treatment without tactile stimulation. Although fish spontaneously crossed the apparatus, we did not observe a decrease in plasma cortisol levels immediately after stressor application as a response to the use of the apparatus, either for social or non-social treatment. However, tactile stimulation reduced aggressive interaction in the social treatment, showing a positive effect on a territorial fish species, and pointing to a way to improve welfare.

Unusual effect of chemical communication on social aggression in juvenile cichlid fish Cichlasoma paranaense (Cichliformes: Cichlidae)

Some fish species are socially organized and show a social rank order which is achieved through aggressive interactions. After hierarchy is settled, such species communicate their ranks through several sensorial cues; this communication is adaptive because it reduces detrimental effects from physical contests. Cichlid fish are socially organized and signal their social ranks through visual, acoustic and chemical communication. The response to signaling may vary according to the species and environment; the knowledge of different species is fundamental to understand the evolutionary forces upon their social communication. We tested the effect of chemical signaling on social groups of juvenile cichlid Cichlasoma paranaense by renewing the water in the aquarium, a procedure that washes away chemical information and increases aggressive interactions in other cichlid species. Two treatments were designed: 50% and 0% water renewal. Aggressive interactions were video-recorded immediately before water renewal, 1min, 1h, 2h, and 24h after water renewal. The treatment with the water renewal did not increase aggressive interactions within the group. The 50% water renewal apparently reduced aggressive interactions in this species, indicating an interspecific difference on the aggressive response to chemical variation in the social environment.(AU)

Algumas espécies de peixes são organizadas socialmente e apresentam uma ordem de rank social que é alcançada por meio de interações agressivas. Após o estabelecimento da hierarquia, essas espécies comunicam seu rank por diversas pistas sensoriais, essa comunicação é adaptativa, pois reduz os efeitos prejudiciais das lutas físicas. Peixes ciclídeos são socialmente organizados e sinalizam o rank social por comunicação visual, acústica e química. A resposta à sinalização pode variar de acordo com a espécie e o ambiente; o conhecimento sobre diferentes espécies é necessário para entender as forças evolutivas sobre sua comunicação social. Nós testamos o efeito da sinalização química em grupos sociais de juvenis do ciclídeo Cichlasoma paranaense por meio da renovação de água do aquário, um procedimento que dilui informações químicas e aumenta a interação agressiva em outras espécies de ciclídeos. Dois tratamentos foram realizados: 50% e 0% de renovação da água. A interação agressiva foi registrada imediatamente antes da renovação da água, 1min, 1h, 2h e 24h após a renovação da água. O tratamento com renovação não aumenta as interações agressivas dentro do grupo. A renovação de 50% da água do aquário aparentemente diminui as interações agressivas nessa espécie, indicando uma diferença interespecífica na resposta agressiva a variação química no ambiente social.(AU)

Water temperature affects aggressive interactions in a Neotropical cichlid fish

Changes in water temperature may affect the aggressive behavior of aquatic organisms, such as fish, either by changing some physiological mechanisms or by increasing the probability of encounters between individuals as a result of variation in their swimming activity. In our study, we evaluated the influence of increasing and decreasing temperature on the aggressive behavior of the Neotropical cichlid fish Cichlasoma paranaense. Firstly, we tested the critical thermal maximum (CTMax) tolerated by this species. Then, we tested the effect of decreasing or increasing the water temperature in 6o C (starting at 27° C) on the aggressive interactions of fish under isolation or housed in groups. We found a CTMax value of 39° C for C. paranaense. We also observe that a 6° C decrease in water temperature lowers swimming activity and aggressive interactions in both isolated and group-housed fish, as expected. On the other hand, the increase in temperature had no effect on the fish's aggressive behavior, neither for isolated nor for grouped fish. We concluded that C. paranaense shows high tolerance to elevated temperatures and, in turn, it does not affect aggressive behavior. Nevertheless, we cannot dismiss possible effects of elevated temperatures on aggressive interactions over longer periods.(AU)

Mudanças na temperatura da água podem afetar o comportamento agressivo de organismos aquáticos, como peixes, seja alterando alguns mecanismos fisiológicos ou aumentando a probabilidade de encontros entre os indivíduos como um resultado da variação na sua atividade natatória. Em nosso estudo, nós avaliamos a influência do aumento e da diminuição da temperatura sobre o comportamento agressivo do peixe ciclídeo neotropical Cichlasoma paranaense. Primeiramente, nós testamos a temperatura crítica máxima (CTMax) tolerada por essa espécie. Depois disso, nós testamos o efeito de reduzir ou aumentar a temperatura da água em 6° C (a partir de 27° C) sobre as interações agressivas de peixes sob isolamento ou alojados em grupos. Nós encontramos um valor de CTMax de 39° C para C. paranaense. Nós também observamos que uma redução de 6° C na temperatura da água diminui a atividade natatória e as interações agressivas tanto em peixes isolados quanto agrupados, como esperado. Por outro lado, o aumento na temperatura não teve efeito no comportamento agressivo dos peixes nem isolados, nem agrupados. Nós concluímos que C. paranaense apresenta alta tolerância a temperaturas elevadas, e que isso, por sua vez, não afeta o comportamento agressivo. No entanto, nós não podemos descartar possíveis efeitos de temperaturas elevadas sobre interações agressivas durante longos períodos.(AU)

Bayesian analysis improves experimental studies about temporal patterning of aggression in fish.

This study aims to describe a Bayesian Hierarchical Linear Model (HLM) approach for longitudinal designs in fish's experimental aggressive behavior studies as an alternative to classical methods In particular, we discuss the advantages of Bayesian analysis in dealing with combined variables, non-statistically significant results and required sample size using an experiment of angelfish (Pterophyllum scalare) species as case study. Groups of 3 individuals were subjected to daily observations recorded for 10min during 5days. The frequencies of attacks, displays and the total attacks (attacks+displays) of each record were modeled using Monte Carlo Markov chains. In addition, a Bayesian HLM was performed for measuring the rate of increase/decrease of the aggressive behavior during the time and to assess the probability of difference among days. Results highlighted that using the combined variable of total attacks could lead to biased conclusions as displays and attacks showed an opposite pattern in the experiment. Moreover, depending of the study, this difference in pattern can happen more clearly or more subtly. Subtle changes cannot be detected when p-values are implemented. On the contrary, Bayesian methods provide a clear description of the changes even when patterns are subtle. Additionally, results showed that the number of replicates (15 or 11) invariant the study conclusions as well that using a small sample size could be more evident within the overlapping days, that includes the social rank stability. Therefore, Bayesian analysis seems to be a richer and an adequate statistical approach for fish's aggressive behavior longitudinal designs.

Fish adjust aggressive behavior to audience size with limited information on bystanders' fighting ability.

In a social environment, individual behavior is modulated by surrounding observers (a phenomenon known as the audience effect). Here, we used mirrors to test the effect of two audience sizes (one virtual bystander vs. three virtual bystanders) on the aggressive behavior of a focal fish when bystander's fighting ability was not clear (i.e., information about the ability of virtual conspecifics limited by their mirror images). We found that the Nile tilapia, a cichlid fish, responds to its image as an audience by reducing overt aggression in the presence of larger audience.

Bile acids as potential pheromones in pintado catfish Pseudoplatystoma corruscans (Spix & Agassiz, 1829): eletrophysiological and behavioral studies

Bile acids are potent olfactory and gustatory stimulants for fish. Electro-olfactogram recording was used to test whether the olfactory epithelium of pintado catfish Pseudoplatystoma corruscans is specifically sensitive to bile acids, some of which have been hypothesized to function as pheromones. Five out of 30 bile acids that had been pre-screened for olfactory activity in fish were selected. Cross-adaptation experiments demonstrated that sensitivity to bile acids is attributable to at least 3 independent classes of olfactory receptor sites. The taurocholic acid (TCA) and taurochenodeoxycholic acid (TCD) were the most potent compounds. By using avoidance/preference tests, we found that P. corruscans prefers water containing TCA. Bile acids are discriminated by olfactory epithelium of pintado, supporting that these compounds could function as pheromones.

Os ácidos biliares são potentes estimulantes olfatórios e gustatórios em peixes. Registros em eletro-olfactograma foram usados para testar se o epitélio olfatório de Pseudoplatystoma corruscans, pintado, é sensível aos ácidos biliares, alguns dos quais têm sido propostos como feromônios. Foram selecionados cinco de uma lista de trinta ácidos biliares previamente testados em atividade olfatória em peixes. Testes de adaptação cruzada demonstraram que a sensibilidade aos ácidos biliares se dá por 3 classes independentes de sites de receptores olfatórios. O ácido taurocólico (TCA) e o ácido tauroquenodesoxicólico (TCD) foram os compostos mais potentes. Em testes de evasão/preferência, P. corruscans prefere água contendo o ácido TCA. Os ácidos biliares são discriminadas por epitélio olfatório de pintado, evidenciando que estes compostos podem funcionar como feromônios.

Photoperiod modulation of aggressive behavior is independent of androgens in a tropical cichlid fish.

Photoperiod is a major environmental cue that signals breeding conditions in animals living in temperate climates. Therefore, the activity of the reproductive (i.e. hypothalamic-pituitary-gonadal, HPG) axis and of the expression of reproductive behaviors, including territoriality, is responsive to changes in day length. However, at low latitudes the seasonal variation in day length decreases dramatically and photoperiod becomes less reliable as a breeding entraining cue in tropical species. In spite of this, some tropical mammals and birds have been found to still respond to small amplitude changes in photoperiod (e.g. 17min). Here we tested the effect of 2 photoperiod regimes, referred to as long-day (LD: 16L:08D) and short-day (SD: 08L:16D), on the activity of the HPG axis, on aggressive behavior and in the androgen response to social challenges in males of the tropical cichlid fish Tilapia rendalli. For each treatment, fish were transferred from a pre-treatment photoperiod of 12L:12D to their treatment photoperiod (either LD or SD) in which they were kept for 20days on stock tanks. Afterwards, males were isolated for 4days in glass aquaria in order to establish territories and initial androgen levels (testosterone, T; 11-ketotestosterone, KT) were assessed. On the 4th day, territorial intrusions were promoted such that 1/3 of the isolated males acted as residents and another 1/3 as intruders. Territorial intrusions lasted for 1h to test the effects of a social challenge under different photoperiod regimes. Photoperiod treatment (either SD or LD) failed to induce significant changes in the HPG activity, as measured by androgen levels and gonadosomatic index. However, SD increased the intensity of aggressive behaviors and shortened the time to settle a dominance hierarchy in an androgen-independent manner. The androgen responsiveness to the simulated territorial intrusion was only present in KT but not for T. The percent change in KT levels in response to the social challenge was different between treatments (SD>LD) and between male types (resident>intruder). The higher androgen response to a social challenge in residents under SD may be explained by the time course of the androgen response that due to the long time it takes to fight resolution under LD, might have been delayed. This result illustrates the importance of incorporating time response data in social endocrinology studies.

Social instability promotes hormone-behavior associated patterns in a cichlid fish.

Androgens are known to respond to social challenges and to control the expression of social behavior and reproductive traits, such as gonadal maturation and sperm production, expression of secondary sex characters and reproductive behaviors. According to the challenge hypothesis variation in androgen levels above a breeding baseline should be explained by the regime of social challenges faced by the individual considering the trade-offs of androgens with other traits (e.g. parental care). One prediction that can be derived from the challenge hypothesis is that androgen levels should increase in response to social instability. Moreover, considering that a tighter association of relevant traits is expected in periods of environmental instability, we also predict that in unstable environments the degree of correlations among different behaviors should increase and hormones and behavior should be associated. These predictions were tested in a polygamous cichlid fish (Mozambique tilapia, Oreochromis mossambicus) with exclusive maternal care. Social instability was produced by swapping dominant males among groups. Stable treatment consisted in removing and placing back dominant males in the same group, in order to control for handling stress. Cortisol levels were also measured to monitor stress levels involved in the procedure and their relation to the androgen patterns and behavior. As predicted androgen levels increased in males in response to the establishment of a social hierarchy and presence of receptive females. However, there were no further differential increases in androgen levels over the social manipulation phase between social stable and social unstable groups. As predicted behaviors were significantly more correlated among themselves in the unstable than in the stable treatment and an associated hormone-behavior pattern was only observed in the unstable treatment.

Insights into the concept of fish welfare.

Fish welfare issues are predicated on understanding whether fish are sentient beings. Therefore, we analyzed the logic of the methodologies used for studying this attribute. We conclude that empirical science is unable to prove or to disprove that fish are sentient beings. Thus, we propose a combined ethical-scientific approach for considering fish as sentient beings. The most difficult ongoing question is to determine which conditions fish prefer. Approaches to assess fish preferences should be rigorously and cautiously employed. In light of these considerations, attempts to establish physiological standards for fish welfare are discouraged, and a preference-based definition of fish welfare is proposed.