RESSALVA 

Atendendo solicitação do 
autor, o texto completo 
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disponibilizado somente 
a partir de 30/04/2021.



Guilherme Delgado Martins 

Preference for reaching tactile stimulation in a territorial fish is not affected by social 

stress 

São José do Rio Preto, SP, Brasil. 

2019 



 

Guilherme Delgado Martins 

 

 

 

 

Preference for reaching tactile stimulation in a territorial fish is not affected by social stress 

 

 

 

 

 

 

 

Dissertação apresentada como parte dos requisitos para 

obtenção do título de Mestre em Biologia Animal, junto ao 

Programa de Pós-Graduação em Biologia Animal, do 

Instituto de Biociências, Letras e Ciências Exatas da 

Universidade Estadual Paulista “Júlio de Mesquita Filho”, 

Campus de São José do Rio Preto.  

 

Orientadora: Profª. Drª. Eliane Gonçalves de Freitas 

 

Financiador: CNPq, proc.: 131338/2017-0 

 

 

 

 

 

 

 

 

 

 

 

São José do Rio Preto, SP, Brasil. 

2019 



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 

Guilherme Delgado Martins 

 

 

 

 

 

 

 

 

 

 Preference for reaching tactile stimulation in a territorial fish is not 

affected by social stress 

 

 

Dissertação apresentada como parte dos requisitos 

para obtenção do título de Mestre em Biologia 

Animal, junto ao Programa de Pós-Graduação em 

Biologia Animal, do Instituto de Biociências, Letras e 

Ciências Exatas da Universidade Estadual Paulista 

“Júlio de Mesquita Filho”, Câmpus de São José do Rio 

Preto. 

 

Financiadora: CNPq, proc.: 131338/2017-0  
 

 
 

Comissão Examinadora 

 

Profª. Drª. Eliane Gonçalves de Freitas 

UNESP – Câmpus de São José do Rio Preto 

Orientador 

 

Prof. Dr. João Luis Saraiva 

Centro de Ciências do Mar (CCMAR), Universidade do Algarve, Portugal. 

 

Dra. Caroline Marques Maia 

Instituto Gilson Volpato de Educação Científica (IGVEC) 

 

 

 

São José do Rio Preto 

30 de abril de 2019 



 

AGRADECIMENTOS 

Agradeço aos membros do laboratório pelas discussões sobre o assunto e por todo o 

suporte para a pesquisa, em especial ao Kawan Carvalho Martins e à Marcela Cesar Bolognesi 

pela ajuda com a confecção dos estimuladores tácteis e dos aparatos dos testes, e à professora 

Eliane Gonçalves-de-Freitas pela oportunidade, confiança e apoio.  

Agradeço à minha namorada Francielly por todo o amor, companheirismo, ajuda 

compreensão e carinho durante toda essa jornada, sentimentos que serão presentes para o 

resto de nossas vidas. 

Agradeço a meus pais pelo cuidado e amor dedicados desde o início de minha vida 

passando por todo meu período dentro da universidade. 

Esse trabalho foi financiado por bolsa de mestrado concedida pelo CNPq – Conselho 

Nacional de Desenvolvimento Científico e Tecnológico (auxílio concedido a GDM, 

#131338/2017-0; EGF #428296/2016-5). 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

RESUMO 

O bem-estar animal é avaliado por indicadores fisiológicos e comportamentais, como nível de 

estresse, taxa de crescimento, comportamentos estereotipados e desempenho cognitivo. Uma 

abordagem recente utiliza a percepção do próprio animal como uma forma de saber se o 

mesmo se encontra em bom estado de bem-estar, por meio de testes de preferência e 

motivação, considerando que o animal está bem saúde quando está em condições por ele 

preferidas. Neste estudo, usamos essa abordagem para testar se peixes territoriais percebem a 

estimulação táctil corporal como um recurso positivo, uma vez que essa condição tem sido 

considerada uma maneira de melhorar o bem-estar em peixes. Assim, testamos se a tilápia-do-

nilo prefere e está motivada a acessar a estimulação tátil; e se a preferência aumenta após um 

estímulo negativo, como o estresse oriundo de interações agressivas (estresse social). Machos 

adultos foram isolados por oito dias em aquários contendo um estimulador táctil central 

formado por hastes plásticas verticais com cerdas de silicone, pelas quais os peixes passam 

recebendo estimulação táctil corporal. Posteriormente, os animais foram submetidos a um 

teste de preferência no qual o peixe tinha a opção de atravessar uma área com estimulação 

táctil ou uma área sem estimulação. Em seguida, os peixes passaram por um teste de 

motivação, no qual deviam superar estímulos aversivos (alta iluminação) para acessar a 

estimulação táctil. Depois disso, os animais foram pareados para estabelecer a hierarquia de 

social por meio de interações agressivas, e novamente submetidos a testes de preferência e 

motivação. Um grupo controle foi submetido aos mesmos procedimentos, mas sem o estressor 

social. Observamos que o número de atravessamentos foi maior pela área sem estimulação 

táctil, tanto nos testes de preferência quanto nos de motivação, embora algumas diferenças 

individuais tenham sido detectadas. No entanto, os peixes não evitaram a área com 

estimulação, mesmo com a opção de permanecer em áreas neutras; os animais também 

superaram a rota aversiva de alta iluminação para obter o acesso ao estimulador táctil. O 

estresse social não afetou o comportamento de preferência e motivação. Além disso, 

observamos que os peixes que atravessaram mais o estimulador se tornaram submissos, de 

modo que a estimulação táctil atuou modulando a hierarquia social reduzindo a agressividade. 

No geral, concluímos que os peixes não percebem a estimulação tátil como um alívio de 

estresse social. No entanto, como os animais não evitaram a estimulação e enfrentaram um 

estímulo aversivo para obter acesso a ela, sugerimos que a estimulação táctil pode representar 

uma condição positiva para peixes territoriais. 

Palavras-chave: bem-estar de peixes, escolha, motivação, comportamento social. 



 

ABSTRACT 

Animal welfare is evaluated by several physiological and behavioral indicators such as stress 

level, growth rate, stereotyped behavior and cognitive performance. A recent approach uses 

the perception of the animal itself as a way to evaluate if this animal is in a good welfare state, 

by preference and motivation tests, considering that the animal is in good welfare when it is in 

conditions that it choses itself. Here, we used this approach to test whether territorial fish 

perceive body tactile stimulation as a positive resource, since this condition has been 

considered a way to improve fish welfare. Thus, we tested whether the fish Nile tilapia prefers 

and are motivated to access tactile stimulation; and if the preference increases after a negative 

stimulus, such as the stress from aggressive interaction (social stress). Adult males were 

isolated for eight days in aquaria containing a central tactile stimulator device formed by 

vertical plastic sticks with silicone bristles, whose fish pass throughout, receiving body tactile 

stimulation. Afterwards, they were assigned to a preference test in which the fish had the 

option of crossing between an area with tactile stimulation and an area without stimulation. 

Then, fish went to a motivation test, in which it must overcome aversive stimuli (bright light) 

to access the tactile stimulation. Thereafter, they were paired to establish social rank by 

aggressive interactions, and subjected again to preference and motivation tests. A control 

group underwent the same procedures but without the social stressor. We observed that the 

number of crossings was higher in the area without tactile stimulation either in preference or 

motivation tests, although some individual differences were detected.  Nevertheless, the fish 

did not avoid the area with stimulation, even with the option of remaining in neutral areas; 

and also overcame the aversive route to obtain the access to the tactile stimulator. Social 

stress did not affect the preference and motivation behavior. Additionally, we observed that 

fish that crossed more through the apparatus became subordinate, thus that tactile stimulation 

modulates social rank hierarchy by reducing aggressiveness. Overall, we conclude that fish 

does not perceive tactile stimulation as a social stressor relieve. However, as fish did not 

avoid the stimulation and faced an aversive stimulus to gain access to it, we suggest that 

tactile stimulation may represent a positive condition for territorial fish.  

Keywords: fish welfare, choice, motivation, social behavior. 

 

 

 



 

Sumário 

1. INTRODUCTION ............................................................................................................. 7 

2. METHODS ........................................................................................................................ 9 

2.1. Fish housing .................................................................................................................... 9 

2.2. Strategy of study........................................................................................................... 10 

2.3. Experiencing tactile stimulation ................................................................................. 10 

2.4. Experimental Procedures ............................................................................................ 12 

2.4.1. Preference Test .......................................................................................................... 13 

2.4.2. Motivation Test ......................................................................................................... 13 

2.4.3. Social stress ................................................................................................................ 14 

2.5. Data analysis ................................................................................................................. 15 

2.5.1. Preference Index ....................................................................................................... 16 

3. ETHICAL NOTE ............................................................................................................ 17 

4. RESULTS ........................................................................................................................ 17 

4.1. Number of crossings from day 1 to 8.......................................................................... 17 

4.2. Preference test .............................................................................................................. 18 

4.3. Preference Index .......................................................................................................... 18 

4.4. Motivation test .............................................................................................................. 23 

4.5. Social rank .................................................................................................................... 25 

5. DISCUSSION .................................................................................................................. 29 

6. CONCLUSION................................................................................................................ 32 

REFERENCES .................................................................................................................... 33 

 

 

 

 

 

 

 

 



7 
 

 

1. INTRODUCTION 

Attention to animal welfare has been a frequent concern of society in recent years
 

(BROOM, 2011). Studies on fish welfare and aquaculture conditions have been increasing 

considerably
 
(ASHLEY, 2007) as long as fish are considered sentient beings

 
(VOLPATO et 

al., 2007)⁠, capable of presenting consciousness of feelings and sensations
 
(GALHARDO; 

OLIVEIRA, 2006). The most traditional animal welfare research relies on indicators of 

animal state as physiological responses, such as catecholamine and corticosteroid levels in 

blood plasma, which suggest stress levels variation
 
(HUNTINGFORD, 2006); behavioral 

responses such as the presence of abnormal behaviors
 
(HUNTINGFROD, 2006), stereotyped 

behaviors
 
(ALMAZÁN-RUEDA et al., 2004), changes in aggressive behavior

 
(BOSCOLO et 

al., 2011) and cognitive performance
 
(BRANDÃO et al., 2015). A more recent approach 

considers that a welfare condition can be inferred by the perception of animal needs and from 

the own animal perspective
 
(DUNCAN, 2006; KIRKDEN; PAJOR, 2006; NICOL et al., 

2009; GALHARDO; OLIVEIRA, 2011; FRANKS, 2019). 

An efficient way to investigate whether a particular condition confers welfare by 

animal perception is to test the choice and preference of these animals for such condition
 

(DUNCAN, 1992; VOLPATO et al., 2007; MAIA; VOLPATO, 2016). These tests are 

designed to analyze how the animal feels by functional and behavioral traits
 
(DUNCAN, 

2006) and the animal's ability to choose the absence of negative states
 
(DAWKINS, 206; 

BOISSY et al., 2007) and the presence of positive ones
 
(DUNCAN, 2002). Thus, if the 

animal presents this positive perception regarding some environment’s condition, it is 

expected that it will choose to access or remain in that condition
 
(VOLPATO et al., 2007; 

DUNCAN, 2002), for example the preference for structured environment in zebrafish (Danio 

rerio) and checker barbs (Puntius oligolepis)
 
(KISTLER et al., 2011) and the preference for 

substrate in Mozambique tilapia
 
(GALHARDO; OLIVEIRA, 2011). It is important to 

emphasize that choice and preference may represent different concepts depending on the 

context
 
(MAIA; VOLPATO, 2016). The preference-based approach requires consistency 

between the choices over time
 
(VOLPATO et al., 2007; MAIA; VOLPATO, 2016).  

As a complement to the preference tests, it is important to understand how animals are 

motivated to achieve a particular condition
 
(DUNCAN, 1992; DAWKINS, 2006; MILLOT et 

al., 2014; MAIA et al., 2017; FRANKS, 2019) according to the idea that the more important a 

condition is to the animal, the higher is the "price it is willing to pay" to reach it
 
(KIRKDEN; 



8 
 

PAJOR, 2006; GALHARDO; OLIVEIRA, 2011). The study of motivation is crucial to 

understand how animals make their decisions
 
(BERRIDGE, 2004; JENSEN; PEDERSEN, 

2008). It corresponds to the balance between the external stimuli and the internal state of the 

animal in the previous moment to a decision making
 
(KIRKDEN; PAJOR, 2006), for 

example, the choice to access a particular resource. Motivation tests can, then, indicate how 

valuable a resource is to the animals
 
(MAIA; VOLPATO, 2016), establishing a connection 

across perception, needing and preference in the analysis of welfare
 
(JENSEN; PEDERSEN, 

2008). In this context, one of the most suitable experimental designs to study motivation is the 

use of aversive obstacles or stimuli, which the animal must overcome in order to reach a 

particular resource
 
(DUNCAN, 1992; MAIA et al., 2017). The way an individual cope with 

these obstacles and stimuli indicates the importance of the condition or resource to be 

achieved by the animal
 
(GALHARDO; OLIVEIRA, 2011). Physical obstacles like push-door 

operant devices are commonly used
 
(OLSSON et al., 2002). In this model, increased costs are 

imposed by adding weights to a door and the animal has to push these heavy doors to access a 

certain resource. Psychological effort tests are also efficient in demonstrating animal 

motivation
 
(MAIA et al., 2017), for example pathways with aversive stimulus that animals 

have to cross to reach the resource. Theses stimuli can be a high light route
 
(MAIA et al., 

2017) or the presence of artificial water currents for fish
 
(SULLIVAN et al., 2016). Thus, 

different resources or attributes in the environment can be tested by preference and 

motivation. 

A recent way thought to improve welfare and increase positive state perception in 

vertebrates is the body tactile stimulation
 
(GONÇALVES-DE-FREITAS et al., 2019). In 

mammals, for example, physical interactions as body massage or touches are able to reduce 

stress
 
(FIELD et al., 2005. HERNANDEZ-REIF et al., 2007; PROBST et al., 2012); to 

relieve pain
 
(FIELD, 1998; JANE et al., 2009); to elevate serotonin levels

 
(FIELD et al., 

2005) to minimize behavioral traits of anxiety and depression
 
(FREITAS et al., 2015; 

ANTONIAZZI et al., 2017) and to prevent learning deficit
 
(DE LOS ANGELES et al., 2016). 

Furthermore, teleost fishes have been tested for this effect as well. Soares et al. (2009) were 

the first to show that tactile stimulation reduces stress in a coral reef fish which perform a 

client-cleaner interaction (Ctenochaetus striatus and Labroides dimidiatus, respectively). 

These authors observed that the clients seek for tactile stimulus promoted by the cleaners, and 

that such stimulation reduces cortisol levels of the client fish after confinement stress. 

Another study demonstrated that tactile stimulation reduces aggressive interaction in pairs of 

male Nile tilapia, a territorial species
 
(BOLOGNESI et al., 2019), although did not reduce 



9 
 

cortisol levels. In Soares et al.
 
(2009) study, tactile stimulation is part of the species’ natural 

behavior. However, Nile tilapia is a territorial fish; therefore it is not clear yet if tactile 

stimulation is perceived as a positive condition, in order to improve their welfare. In this 

context, a preference-based approach is adequate to answer this question.  

Thus, in this study we tested whether Nile tilapia freely choose tactile stimulation and 

also their motivation to access this resource. We also tested if such preference and motivation 

increase after a negative stimulus, such as the stress from aggressive interactions (social 

stress). If the perception of tactile stimulation is generalized and positive in vertebrates, we 

expected that territorial fish such as Nile tilapia would freely choose to access this resource 

and that they would be more motivated to access tactile stimulation after experiencing a 

stressful situation. Nile tilapia, Oreochromis niloticus (L.) is a very important species in 

aquaculture
 
(BARCELLOS et al., 1999. GONÇALVES-DE-FREITAS et al., 2019) and the 

knowledge of tools that promote welfare of these animals is highly relevant
 
(VOLPATO et 

al., 2009; GONÇALVES-DE-FREITAS et al., 2019). In social system of the Nile tilapia, the 

males establish a dominance hierarchy by aggressive confrontations
 

(ALVARENGA; 

VOLPATO, 1995, CARVALHO et al., 2008; BARRETO et al., 2011). The occurrence of 

these confrontations between conspecifics causes social stress, one of the main causes of 

chronic stress in Nile tilapia rearing
 
(BARCELLOS et al., 1999; BOSCOLO et al., 2011). The 

social stress arising from the establishment of the dominance hierarchy can generate serious 

damages to these animals, thus becoming an important factor of poor welfare
 
(GONÇALVES-

DE-FREITAS et al., 2019). Therefore, it is important to investigate conditions that reduce 

detrimental effects from social interaction in Nile tilapia, such as tactile stimulation.  

2. METHODS 

2.1. Fish housing 

Adult males of GIFT Nile tilapia from a commercial supplier (Ribeirão Preto, SP, 

Brazil), were kept in outdoor ponds at the IBILCE, UNESP, São José do Rio Preto, SP, 

Brazil. The fish were collected for the study and taken to the laboratory where they all were 

acclimated together for 15 days in a polyethylene water tank (ca. 500 L, 1 fish/10 L) with 

water at 28ºC and 12L:12D light regime (7:00 a.m. to 7:00 p.m.). The fish were fed with 

ration for tropical fish (28% CP, apparent satiety) once a day (10:00 a.m.). The water quality 

was maintained by biological filters with filtration of 400 L/h, and constant aeration. 

 



32 
 

accessed more frequently the stimulation to become subordinate. In fact, tactile stimulation 

reduces the aggressiveness of Nile tilapia
 
(BOLOGNESI et al., 2019), therefore individuals 

who experienced more tactile stimulation should become less aggressive and lose the contest, 

as aggressive ability define the winner and the loser individual
 
(BOSCOLO et al., 2011). 

Another interesting fact regarding subordinate fish is that in the preference test, they equally 

accessed the areas with and without stimulation, unlike the dominant ones. This result is in 

agreement with the pattern of crossings in the initial 8 days of the fish that would become 

subordinate. However, after social stressor, subordinate and dominant showed the same 

number of crossings by the stimulator. It is known that the establishment social hierarchy in 

Nile tilapia could be stressful in a similar way either for subordinate or dominant animals
 

(CORREA et al., 2003). Therefore, after pairing, the stress levels of dominant and 

subordinate individuals could be equal, allowing a similar pattern of access to tactile 

stimulation for both. Despite this, some studies show that the effects of agonistic interactions 

can be higher in subordinate individuals in a longer period
 
(EJICK; SCHRECK, 1980) what 

could explain the decrease in crossings through the area with tactile simulation and through 

the area without tactile stimulation after the pairing for aggressive interactions in subordinate 

fish. 

In this study, we could observe that the pattern of preference and motivation responses 

by tactile stimulation is very variable for Nile tilapia. We had the purpose of testing whether 

or not the fish prefers tactile stimulation and, therefore, it was limited to two options of 

choice. In conditions in which few choices are offered to animals, we cannot conclude 

assertively that the one chosen more frequently will always be preferred, especially if it is a 

variable environment (MAIA; VOLPATO, 2019)
 
and where the options of choice can have 

gradual effects on the animal, as it is the case of tactile stimulation. In some studies, such as 

this one, preference and motivation responses are analyzed together
 
(SULLIVAN et al., 

2016). The conclusions of these analyzes can sometimes extrapolate the individual views of 

each test, showing that even if an option of choice is not accessed more often, animals may 

rather be motivated to access this option and consequently, this condition may represent a 

positive aspect from the perspective of the animal itself. 

6. CONCLUSION 

We conclude that tactile stimulation is not aversive to Nile tilapia, since animals do 

not avoid contact with the stimulator; social stress has no effect on preference and motivation, 

since fish do not increase the search for tactile stimulation after this situation; tactile 



33 
 

stimulation can be determinant in the modulation of dominance hierarchy by reducing 

aggressiveness and making individuals become subordinate; animals are motivated to access 

tactile stimulation as they face an aversive stimulus to gain such access, suggesting that 

stimulation may represent a positive condition for these animals. 

 

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