heart rate in dominant and subordinate rainbow trout ...fish. these conflicts yield dominance and...

Ted Slight

Degree project for Bachelor of Science inBiology

BIO602 Biology: Degree project 15 hecSpring 2015

Department of Biological and Environmental SciencesUniversity of Gothenburg

Examiner: Catharina OlssonDepartment of Biological and Environmental Sciences

University of Gothenburg

Supervisors: Johan Höjesjö and Michael AxelssonDepartment of Biological and Environmental Sciences

University of Gothenburg

Heart rate in dominant and subordinaterainbow trout, Oncorhynchus mykiss

- a pilot study on physiological and behavioural responses, using a bio-logging system

http://en.wikipedia.org/wiki/Trout#/media/File:Rainbow_Trout.jpg

Ted Slight Heart rate in interacting rainbow trout June 2015

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Contents Abstract ....................................................................................................................................................4

Introduction .............................................................................................................................................5

Materials and Methods ...........................................................................................................................6

Experimental animals ...........................................................................................................................6

Experimental procedures .....................................................................................................................6

Day 1 - Scoring of social status ......................................................................................................6

Day 2 & 3 - Surgery and recovery ..................................................................................................7

Day 4 - Heart rate measurements and scoring ............................................................................8

Data Analysis ........................................................................................................................................8

Results ......................................................................................................................................................8

Utility of procedures .............................................................................................................................8

Heart rate and behavioural responses .................................................................................................9

Discussion.............................................................................................................................................. 12

Utility of procedures .......................................................................................................................... 12

Heart rate and behavioural responses .............................................................................................. 12

Conclusions and future research ...................................................................................................... 14

Acknowledgements .............................................................................................................................. 15

References ............................................................................................................................................ 15

Appendix A – Sample of scoring protocol ............................................................................................ 17


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Abstract It is well known that hierarchic conflicts occur among various animal species, including many salmonid fish. These conflicts yield dominance and subordination, followed by different physiological consequences of varying costs for the animals involved. Although a number of previous studies have investigated physio-logical responses like endocrinology and metabolism, there is no study on how heart rate varies simulta-neously in two agonistically interacting individuals. Neither is it known how adequate a bio-logging sys-tem is in answering this question. In this pilot study the heart rate was recorded continuously from 30 minutes before to 9 hours after the start of such an interaction, using implantable heart rate loggers. Paired rainbow trout (Oncorhynchus mykiss), one year old (+1), were behaviourally scored to determine social status, before and after the implantation. The mean heart rate increased from before to after inter-action in 7 out of 8 individuals. In two out of four pairs the increase was greater for the subordinate indi-vidual. In the remaining two pairs the increase was greater for the dominant individual. The scoring re-sults attained before surgery strongly coincided with the scoring results after surgery, and no outstanding changes in behaviour were perceived. The results suggest that bio-logging has great potential in the inves-tigation of heart rate responses in interacting animals, and that social interactions cause heart rate to temporarily increase, but that the variability and small sample size hindered conclusions to be drawn regarding differences between subordinates and dominants. Bio-logging can play an important role in the understanding of interactions between physiological and behavioural responses, as well as in the under-standing of costs involved in social conflicts.

Sammanfattning Det är idag väl känt att hierarkiska konflikter förekommer inom oräkneliga djurarter, däribland många laxfiskar. Dessa konflikter resulterar i dominans och subordination, vilket medför olika fysiologiska kon-sekvenser med varierande kostnader för de berörda individerna. Även om ett flertal tidigare studier har undersökt fysiologiska responser såsom endokrinologi och metabolism, saknas det studier som visar hur hjärtfrekvensen varierar simultant för två antagonistiskt interagerande individer. Inte heller finns det forskning som visar hur brukbart ett implanterbart bio-logging-system skulle vara i besvarandet av denna fråga. I denna pilotstudie mättes hjärtfrekvens kontinuerligt från 30 minuter före till 9 timmar efter det att en sådan interaktion inletts, med hjälp av implanterbara hjärtfrekvensmätare. Beteendet hos interage-rande, ettåriga (1+) regnbågar (Oncorhynchus mykiss) observerades både före och efter operationen, för att fastställa social status. Den genomsnittliga hjärtfrekvensen ökade från innan till efter interaktionen hos 7 av 8 individer. I två av fyra par var ökningen större för den subordinata individen. I de återstående två paren var ökningen större för den dominanta individen. Resultatet av dominansbestämningen från före operationen överensstämde med det från efter operationen, och inga påtagliga beteendeförändringar kunde noteras. Resultaten visar att bio-loggers har en lovande potential i studerandet av hjärtfrekvens hos interagerande djur. Dessutom antyder resultaten att sociala interaktioner leder till tillfälligt förhöjd hjärtfrekvens, men variabiliteten och den begränsade stickprovsstorleken förhindrar ytterligare härled-ningar angående skillnaden mellan dominanta och subordinata individer. Implanterbara hjärtfrekvens-mätare kan spela en viktig roll i människans förståelse för interaktioner mellan fysiologiska och bete-endemässiga responser, men även gällande insikter kring kostnader involverade i sociala konflikter.


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Introduction Hierarchic systems are found in various taxa, throughout the animal kingdom, and hierarchic con-flicts result in both costs and benefits for the interact-ing animals (Parker, 1974). The study of animal con-flicts and the understanding of aggressive behaviour have been greatly influenced by biological theories implemented by Maynard Smith, Price and Parker (Enquist and Leimar, 1983). Through application of game theory and mathematic modelling, the evolu-tion of different conflict strategies can be explained by natural selection at an individual level. Although actual animal conflicts are more complex than these models, they show through pay-off analyses that both aggressive and non-aggressive behaviour can be ad-vantageous to individuals (Maynard Smith and Price, 1973; Maynard Smith, 1974). In a review by Koolhaas and colleagues (1999) behavioural and physiological characteristics of stress related coping styles are discussed. Certain sets of characteristics can be observed not only in wild animals but also in domesticated animals. Be-havioural characteristics are categorised into two types of responses to social stress. The proactive response is characterised by resource control and aggressive behaviour, whereas the reactive response is characterised by immobility and low aggression. Although these two categories are commonly used, the authors also point out that a number of studies, performed on various animal species, indicate that there is a level of individual variation in these coping styles that should be taken into consideration (reviewed by Koolhaas et al., 1999). Social stress is connected to dominance and subordination which result in hierarchic structures, and these structures are commonly formed by salmonid fish (Abbott and Dill, 1989; Nakano, 1995; Adams et al., 1998; Gilmour et al., 2005; Höjesjö et al., 2007). Interacting salmon-ids show different types of agonistic behaviour which were early described by Keenleyside and Yamamoto (1962) in a study on Atlantic salmon fry (Salmo salar). Here, interacting individuals were observed during territorial defence in laboratory as well as in natural habitat, which resulted in six categories of agonistic behaviour: “charging, nipping, chasing, frontal display, lateral display and fleeing” (Keenleyside and Yamamoto, 1962). More recent studies have used similar behavioural variables in dominance scoring of salmonids, especially aggres-sive behaviour and tank position, but also colouration

(Abbott and Dill, 1989; Nakano, 1995; Johnsson et al., 1996; O'Connor et al., 1999; Øverli et al., 1999; Sloman et al., 2000a; Höjesjö et al., 2007; Höjesjö et al., 2015). Agonistic interactions have various physiologi-cal short term and long term consequences for both dominant and subordinate salmonids (Øverli et al., 1999; reviewed by Sloman and Armstrong, 2002). Øverli and his colleagues detected that both individu-als attained increased levels of circulating plasma cortisol, within five minutes after a staged conflict. Interestingly, the cortisol levels of the dominants decreased within three hours to a normal level, whereas the level of cortisol continued to increase in subordinate individuals. In a different study the plasma cortisol after two weeks of confinement was significantly higher in the subordinates (Sloman et al., 2000a). Furthermore, lower resistance to bacterial infection (Pottinger and Pickering, 1992), lower growth rate (Abbott and Dill, 1989) and higher standard metabolic rate (Sloman et al., 2000b) has been reported in subordinate individuals of salmon-ids. In a more recent project, researchers measured heart rate responses to visual exposure of dominants, which resulted in a postponed increase of heart rate in the exposed individuals, suggestively preparing these individuals for “fight or flight” (Höjesjö et al., 2007). The heart rate is one of many stress related physiological responses interconnected with, and constantly responding to, the sympathetic (“fight or flight”) and parasympathetic nervous system, as well as to the external environment. Consequently, heart rate has been used as an indicator for stress in sever-al studies (Cabanac and Cabanac, 2000; Cooke et al., 2004a; Höjesjö et al., 2007; Boman, 2014). Many previous studies on heart rate responses in aquatic animals have been performed with either electrodes attached to the animal or by using a sub-merged electrode cage that detects cardiac bioelectri-cal potentials through the water (Goodman and Weinberger, 1971; Höjesjö et al., 1999; Altimiras and Larsen, 2000). These methods confine the free movement of the animal, but other types of physio-logical monitoring have been developed in the ad-vancing field of biotelemetry, allowing remote meas-urements to be made on unrestrained animals (Cooke et al., 2004b; Ponganis, 2007). More recent tech-niques involve fully implantable devices that success-fully measure physiological parameters like blood flow, blood pressure, ECG and temperature (Axelsson et al., 2007; Gräns et al., 2009). Compared to semi-


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implantable and externally attached devices, fully implantable devices bring about advantages like de-creased risk of infections (Schulz, 2003) and in-creased chances of the subjected animal to be nor-mally treated by conspecifics (Connors et al., 2002). Today, some of the implantable biotelemetry devices have microprocessors storing data which allows smaller and lighter devices (Ponganis, 2007), such as the DST milli-HRT heart rate and temperature logger from STAR-ODDI (fig. 1). These 40 mm bio-loggers allow researchers to, for the first time, examine sim-ultaneous heart rate responses, in two interacting and freely swimming animals. The aim of this pilot study was to investigate physiological consequences of social status in inter-acting individuals, both subordinate and dominant, and to evaluate the method of bio-logging in this con-text. This was done through observations and heart rate measurements collected from paired rainbow trout (Oncorhynchus mykiss), interacting in laborato-ry conditions for 9 hours. It was also examined if the interaction affected the heart rate at all, and if there would be a difference between dominant and subor-dinate individuals. One posed hypothesis was that the heart rate is higher in subordinates compared to dominants due to higher stress levels caused by the subordination. It was predicted that the subordinates would show a larger and positive change in average heart rate from before to after the interaction, than would the dominant.

Figure 1. The DST milli-HRT heart rate and temperature log-ger from STAR-ODDI. (www.star-oddi.com)

Materials and Methods Experimental animals In this study 10 young rainbow trout (Oncorhynchus mykiss), obtained from the hatchery Antens Fiskodling outside of Gothenburg, were used. The weight ranged from 0.55 kg to 0.78 kg, all individuals were one to two years old (+1) and of unknown sex. Before the experiment the animals were held in 200 litre tanks for at least two weeks to acclimatise to laboratory conditions. During the experiment the animals were held in a 1500 litre tank. All tanks were continuously supplied with aerated fresh water and the tempera-ture was kept at 10°C. The animals were fed artificial pellets three times a week before the experiment and were fasted during the trials which lasted four to five days. Two of the animals were at an early stage ex-cluded from the experiment after discovery of buoy-ancy issues for one of the individuals. The oxygen saturation of the water in the experiment tank was measured on day 2 after each surgical procedure and again after each termination of the experiment on day 4, yielding a minimum of 85 % saturation. No visual injuries were acquired to the animals during the ex-periment. All experiments were approved by the regional ethical committee (89-2013). Experimental procedure Prior to the experiment 10 individuals of similar size were placed in two tanks. From these tanks one pair, consisting of one individual from each tank, was net-ted and transported to the experimental tank where the subjected animals underwent the following pro-cedure: scoring of social status, surgery with recov-ery and finally interaction with heart rate measure-ments and repeated dominance scoring. After the first trial was ended the whole procedure was re-peated until cardiac and behavioural data had been collected from four pairs, each pair yielding one dom-inant and one subordinate individual. Day 1 - Scoring of social status Prior to the surgery each pair was observed and scored during interaction in order to determine their relative social rank. Before the fish were placed in the experiment tank they were anesthetised and tagged with Floy tags beneath the dorsal fin so that the indi-viduals could be easily distinguished from each other. The anaesthetic water solution contained MS-222 (100 mg/L) and sodium bicarbonate (200 mg/L). The fish were placed in the solution until they lost control


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of balance (side facing up), where after they were tagged immediately and placed in the experiment tank with a dividing wall preventing visual contact. Here they were allowed to recover for four hours, which also allowed some acclimation to the experi-ment tank. After the recovery period the wall was lifted, allowing interactions between the two individuals. During the first 20 minutes the interacting subjects where observed through spyholes in a plastic cover by two persons, scoring the behaviour of each fish separately under intervals of 30 seconds (30-1200 seconds after start). The number of initiated interac-tions, initiated attacks, completed attacks and the position (most central or not) of each individual, for each 30 second period, was recorded on a protocol sheet (see appendix A). After the first 20 minute round of scoring the counts were summarised and calculations were performed; this yielded frequencies distributed on the two individuals for each of the four behavioural variables. Forty minutes after the first scoring round ended a second 20 minute scoring round was started, although the wall was kept in uplifted position the whole time. This procedure was repeated until dominance was determined. The fol-lowing criteria were set up in advance to decide the social status: for a given individual to be labelled as “temporarily” dominant after a 20 minute scoring round, this individual was required to represent more than 80 % of all recorded cases in each of the four behaviours. For a given individual to be labelled as “certainly” dominant, this individual had to be labelled as temporarily dominant in two consecutive 20 minute scoring rounds, starting from round num-ber two. Due to the plausible disturbance from the wall elevation, the first round was beforehand decid-ed to be excluded from the assessment of social sta-tus. When social status was determined the wall was lowered to keep the two individuals separated. Day 2 and 3 - Surgery and recovery DST milli-HRT heart rate and temperature loggers from STAR-ODDI were implanted into the two indi-viduals. They were weighed while anesthetised by the same solution as above (100 mg/L MS-222 and 200 mg/L sodium bicarbonate), but this time for a longer time until surgery level of anaesthesia was reached. During the surgery, which lasted for a max-imum of one hour per animal, they were kept anes-thetised by oxygenated water containing MS-222 (50

mg/L) and sodium bicarbonate (100 mg/L) at a tem-perature of 8°C, constantly pumped over the gills. A 30 mm incision was made from 35 mm to 65 mm posterior to the pectoral fins, along the mid-ventral line (see fig. 2). The logger was placed hori-zontally and pointed forward in the abdominal cavity between the pectoral fins and the incision. There the logger was fixed with the first stitch using a 3-0 poly-glactin suture while the remaining stitches were done using a 3-0 polypropylen monofilament suture. The position of the logger ensured that the electrodes pointed outwards horizontally, a position shown to give the highest signal quality (Boman, 2014). After the surgery the subjects were again placed in the experiment tank for a minimum of 47.5 hours of re-covery, separated by the dividing wall. The DST milli-HRT logger is a 40 mm long, implantable, ceramic heart rate and temperature logger that through its integrated electrodes can re-ceive cardiac bio-potentials, calculate an instantane-ous mean heart rate and store this data down to a resolution of once every minute (www.star-oddi.com, 2015-05-20). With attached silicon tubes, used to facilitate the fixing of the loggers, they weighed 12.5 g which is 2.3 % of the weight of the smallest individual used in this experiment.

Figure 2. Position of incision and implant. The incision is marked with a dotted line. (Boman, 2014)


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Day 4 – Heart rate measurements and scoring On the fourth day the wall was elevated 09:00 allow-ing the individuals to interact in the 1500 litre tank (see fig. 3), now with implanted heart rate loggers. The loggers were programmed using STAR-ODDI’s software program Mercury 4.02, to measure the heart rate every second minute between 08:30 and 10:00 in order to get a high resolution of measurements before the interaction and closely after its beginning. Between 10:00 and 18:00 the loggers stored data every 15 minutes in order to primarily show long-term effects, with the wall in an elevated position the whole time, Starting at 09:00 the interacting individuals where scored with a procedure identical to the one performed on the first day, until the social rank was determined. This was done in order to attain social scoring data from both before and after the surgery, to aid the detection of possible surgical effects on behaviour. An AXIS P3344 camera was mounted above the tank, providing visual recordings, which were later used to score the interactions by video. All visual recordings were managed through the soft-ware program AXIS Camera Station, version 4.08.048. The video scoring was performed according to the same protocol as before except now looking at a vid-eo recorded from above, to test an alternative scoring method. The video recordings were also used to in-vestigate the connection between levels of activity and certain data points. Apart from enabling the vid-eo scoring and analysis, the camera also provided a web based live view which aided the surveillance of interacting fish. After 18:00 the experiment was terminated, the fish were euthanized using the same MS-222 solution (100 mg/L), and the data was received using the same software program as before. Data analysis The measured variable was the heart rate (bpm), and the factor used was social status with the two levels subordinate and dominant. The overall measure-ments over time, and differences between the two groups in change of average heart rate from before to after wall elevation, was analysed using descriptive statistics in Excel 2010. No Further statistical analysis was performed due to the small sample size (n=4) and the high variability of the collected data. Apart from the analysis of cardiac data, scoring data and video material was analysed. The scoring data was analysed to determine social status and the

video recordings were analysed for method evalua-tions.

Figure 3. The experiment tank viewed from above. The dotted line illustrates the position of the dividing wall, prior to interac-tion.

Results Utility of procedures Three separate tests were performed in order to evaluate the utility of certain procedures. Firstly, each pair was scored both before and after the surgery, to investigate whether the surgery and a data-logger implanted in the abdominal cavity would alter the social behaviour or not. The results show that surgi-cal implantation had no effect on relative rank for any of the 8 individuals. Also, there were no outstanding changes perceived in behaviour from before to after the surgery. Secondly, an analysis was done on low quality data points attained from the heart rate loggers. Each point in time lacking values from both individuals was located in the video recordings and observed. Out of the total 53 data points, 43 (81%) were posi-tioned in time within 30 seconds from a high activity event, such as chasing. Finally, the utility of an alternative scoring method was tested. When an equivalent scoring pro-cedure was performed observing the four pairs dur-ing the same time slots, but now in front of a comput-er screen, the same social statuses were determined in every single 20 minute scoring round. When scor-


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ing one individual and one behaviour, the largest difference between live scorings and video scorings, in frequency calculations, was 17 percentage points (table 1). The Floy tags, which were easily distin-guished standing beside the tank, were hard to per-ceive looking at the video recordings, but on the oth-er hand the most central position was easier to de-termine.

Table 1. The maximum difference between video scoring and live scoring, in percentage points (derived from relative fre-quencies between individuals of Oncorhynchus mykiss), are presented for each pair and each behaviour. Because the behav-iours “Initiated attack” and “Completed attack” resulted in either 100 % or 0 % in all cases, these were excluded from the table.

PAIR 1 PAIR 2 PAIR 3 PAIR 4

Difference in initiated interactions 8 7 1 0

Difference in most central position 11 1 17 5

Heart rate and behavioural responses The dominant versus subordinate individuals weighed 0.78 versus 0.55 kg in pair 1, 0.68 versus 0.68 kg in pair 2, 0.72 versus 0.59 kg in pair 3 and 0.63 versus 0.63 kg in pair 4, respectively. In all four cases the individuals interacted ac-tively and agonistically with dominance settled after three 20 minute scoring rounds, yielding a clear social status at 11:20 am. This resulted in one dominant and one subordinate individual per pair, seen as red and blue, respectively, in figure 4 below. The figure shows all received data points between 08:30 and 18:00 that

were totally free from the logger’s own quality re-marks. The logger software has an integrated signal quality assessment system, which resulted in varying amounts of data across the eight individuals. As seen in figure 4d, there were no data points of sufficient quality received after 13:00 from the subordinate individual in pair 4. The overall trend is that subordinate individu-als had a higher heart rate than dominant individuals, once social rank was settled. However, pair 1 (fig. 4a) demonstrates the reverse with a slightly higher domi-nant heart rate.


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Figure 4. Heart rate measurements from the 8 individuals. The figure shows how the heart rate varies in interact-

ing dominant and subordinate rainbow trout, Oncorhynchus mykiss, from 30 minutes before to 9 hours after initi-ated interaction (marked with a dotted line). The dissimilar data resolution of pair 2 was caused by a program-ming error.


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In figure 5 the mean heart rates are shown, calculated from before and after the wall was elevat-ed, with the two values from one individual connect-ed by a dotted line. Dominant 1 and subordinate 1 represent the two individuals from pair 1. The angles of both dominant and subordinate lines show a clear pattern of increasing heart rates, although subordi-

nate 1 defies this pattern with a slightly lower mean heart rate after wall elevation. When comparing dom-inants (fig. 5a) against subordinates (fig. 5b) there is a tendency towards steeper lines for the subordi-nates, indicating a greater increase of mean heart rates. Conversely, this tendency is broken by domi-nant 4 and subordinate 1.

Figure 5. Mean heart rates before and after wall elevation. Mean heart rate in Oncorhynchus mykiss ranged from 27.3 bpm for dominant 4 to 65.9 bpm for subordinate 3. The separate mean values are calculated with different data amounts, between 9 and 47 values.

Figure 6 shows the average change in mean heart rate among dominants and among subordinates, from before to after wall elevation. The subordinates show a slightly higher change of plus 12.6 bpm, compared to the dominants showing plus 11.1 bpm. Indicated by the standard deviation, these two averages are based on groups with a very high variability, and are evidently not significantly different from each other.

Figure 6. Average heart rate change for dominants and sub-ordinates. The average increase in mean heart rate, from before to after the wall elevation, was 11.1(± S.D. 12.2) bpm for the 4 dominants and 12.6(± S.D. 9.8) bpm for the 4 subor-dinates of Oncorhynchus mykiss.

As mentioned in methods above, the video recordings were used to connect levels of activity to heart rate measurements. Table 2 gives values of all time points answering to certain behavioural criteria, such as low activity. First of all it shows that pair 3 and 4 have relatively few data points from periods of time with low activity. Secondly, it shows that all listed time points from pair 2, 3, and 4 have values where the measured heart rate is higher in the sub-ordinate individual. Furthermore, the positive differ-ence between each of these values and the calculated mean from before wall elevation is larger in each and every case for the subordinates, than that of the dom-inant individuals. This result differs from figure 5 that gives a greater heart rate increase for the dominant in pair 4.


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Time HR Dif. HR Dif. Time HR Dif. HR Dif. Time HR Dif. HR Dif. Time HR Dif. HR Dif.12:00 64 (+9) 62 (-2) 12:15 47 (+7) 68 (+29) 13:15 41 (±0) 75 (+23) 12:45 47 (+20) 75 (+30)13:30 62 (+7) 62 (-2) 13:00 40 (±0) 61 (+22) 15:15 31 (-10) 61 (+9) 13:00 49 (+22) 78 (+33)13:45 62 (+7) 64 (±0) 13:30 45 (+5) 67 (+28) 16:00 31 (-10) 70 (+18)14:00 65 (+10) 63 (-1) 14:15 41 (+1) 58 (+19)14:30 66 (+11) 64 (±0) 14:45 40 (±0) 61 (+22)14:45 64 (+9) 63 (-1) 15:15 43 (+3) 58 (+19)15:30 63 (+8) 61 (-3) 15:30 45 (+5) 61 (+22)15:45 62 (+7) 59 (-5) 15:45 37 (-3) 57 (+18)16:00 63 (+8) 63 (-1) 16:30 44 (+4) 58 (+19)16:30 63 (+8) 63 (-1) 16:45 47 (+7) 67 (+28)17:00 65 (+10) 63 (-1) 17:00 45 (+5) 61 (+22)17:30 65 (+10) 63 (-1) 17:30 50 (+10) 57 (+18)18:00 63 (+8) 63 (-1) 17:45 40 (±0) 61 (+22)

18:00 45 (+5) 60 (+21)

PAIR 1 PAIR 2 PAIR 3 PAIR 4DOM SUB DOM SUB

MB = 55 MB = 64DOM SUB

MB = 40 MB = 39 MB = 41 MB = 52DOM SUB

MB = 27 MB = 45

Discussion Utility of procedures Since these bio-loggers were never before used in studies observing behaviour in salmonids, it was unclear if the invasiveness of the implant would re-sult in either no interaction at all, or in a changed scoring outcome. All individuals displayed the same social status before and after the surgery, suggesting that the implant was not invasive enough to alter the outcome of agonistic interactions. This result, added to the observation of unchanged general behaviour, makes the application of these bio-loggers relevant for future studies related to behaviour. This result can nonetheless be questioned if the used scoring method is irrelevant when assessing social domi-nance in the subject animals. However, the scoring of variables like spatial activity and initiated agonistic interactions, has been successfully used in preceding studies on Oncorhynchus mykiss (Abbott and Dill, 1989; Höjesjö et al., 2007) and Oncorhynchus masou ishikawai (Nakano, 1995). Also colouration is com-monly observed and used in dominance scoring (Keenleyside and Yamamoto, 1962; Abbott and Dill, 1989; O'Connor et al., 1999; Höjesjö et al., 2007), but was excluded as a variable from the current study due to low levels of light. Regarding the connection of low quality data points to certain behaviours, 81 % of the defected

data points were close in time to high activity events. This indicates that the bio-loggers may be negatively affected when measuring heart rates during high physical activity. On the other hand, if one is mainly interested in measurements acquired at low activity, this negative effect on quality is a drawback of lower impact. Apart from activity affecting measurement quality there is also the placement of the logger, which appears to be of great importance in this issue (Ponganis, 2007; Boman, 2014). The fact that the video scoring turned out to give the same scoring results in all scoring rounds opens up for this alternative scoring method, that may be more time efficient since it enables higher flexibility in when to score the animals. Although there were certain disadvantages such as problems perceiving details of behaviour and tag colours, the method is sufficient if the main purpose is to assess relative dominance. With higher resolution and inte-gration of computer programs, camera surveillance looks like a promising tool in both behavioural and physiological studies. Heart rate and behavioural responses The most distinct result achieved in this pilot study was that mean heart rate increased in 7 out of 8 indi-vidual from before to after wall elevation. This sug-gests that an interaction between two rainbow trout, Oncorhynchus mykiss, leads to an increased heart rate

Table 2. The table contains all data points that 1: had values received from both individuals, 2: had values received from after settled dominance and 3: had a period of low activity 30 seconds before and after the given measurement time. “A period of low activity” was defined as a period with either total motionlessness or slow swimming by the rainbow trout, Oncorhynchus mykiss. All values are in the unit beats per minute. DOM=dominant, SUB=subordinate, MB=mean heart rate calculated from before wall elevation, HR=heart rate, Dif.=HR minus MB.


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for both dominants and subordinates, indicating a somewhat stressful situation. On the other hand, this result may be affected by the possibly stressful wall elevation itself. Nonetheless, the result conforms to findings in several preceding studies of physiological responses to hierarchic interactions. Heart rate has been shown to increase in rainbow trout, Oncorhyn-chus mykiss, visually exposed to dominant conspecif-ics (Höjesjö et al., 2007), and in individuals of the same species that were grouped (Boman, 2014). Moreover this result can be connected to the post-interactional increase of plasma cortisol (Øverli et al., 1999) since this in turn is connected to sympathetic neural activities. Returning to the questions phrased in the introduction, the temporarily increased heart rate suggests an effect of interactions on heart rate, at least caused by interactions in general, although not necessarily connected to social status. A factor that potentially can distort these re-sults is the possibility of olfactory signalling, caused by water circulating between the two compartments in the experiment tank. However, recent researches suggest the visual signalling to be of greater im-portance in interacting rainbow trout, Oncorhynchus mykiss (Höjesjö et al., 2007; Höjesjö et al., 2015). Although no particularly distinct differences between dominant and subordinate individuals where found in this pilot study, the observed trend will be discussed. The vagueness of the trend towards a greater heart rate increase among subordinates (fig. 5 and fig. 6), could be caused by several circumstanc-es. Firstly, interfering with this trend stands the data received from pair 1, of which the credibility can be questioned. The heart rate measurements from the subordinate individual show remarkably high values prior to the wall elevation. The mean value of these data, 65 bpm, can be compared to the values 40 to 45 pbm, which were the stabilized heart rates in rain-bow trout (Oncorhynchus mykiss) of similar size and after the same implant surgery (Boman, 2014). This relatively high mean heart rate indicates that this individual was not fully recovered from surgery, or that it for unknown reasons already had an elevated heart rate prior to surgery, and that these data points therefore are of questionable representativeness. Curiously, pair 1 was given approximately 69 hours of recovery whereas the three remaining pairs were given about 48 hours. Recent research showed that stabilisation of heart rate after a similar incision oc-curred about two days after surgery (Gräns et al., 2014) and already one day after surgery implanting

the same bio-logger from STAR-ODDI (Boman, 2014). This possible lack of recovery for pair 1 could also be explained by the fact that these individuals were anesthetised during a slightly longer time when op-erated on. A second circumstance levelling out a thinka-ble difference between dominant and subordinate individuals is the choice of values in calculations of mean heart rates after the wall elevation. The mean heart rates of dominants after wall elevation are, like all other mean values in this study, calculated using all available data from after (or before) raising the wall (see fig. 4 and 5). The choice of a broader aim and the choice of programmed intervals led to a large number of heart rate values densely positioned short-ly after the elevation, before the point of settled dom-inance. If one is interested particularly in the effect of social status, it might be wiser to only include the data attained after the settlement of social status. Especially when viewing the data from pair 2 and 3, showing many high values close to the wall elevation, one can suspect that the mentioned alteration might reveal different results in future studies. Lastly, the possible trend is naturally disturbed by individual variation. When responding to stress, individual variation in physiological coping styles may collide with the ambition to connect certain physiological characteristics to certain behavioural characteristics (reviewed by Koolhaas et al., 1999). The consequences of individual variation are natural-ly also worsened by the small sample size used in the current pilot study. If a difference is to be statistically detected in future research one important step would be to increase the sample size. If there is an actual difference between dominants’ and subordinates’ change in mean heart rate, this could arguably be connected to the results of earlier studies. Research-ers have found that the change in standard metabolic rate, based on oxygen consumption, is significantly greater and positive for the subordinate individual of brown trout, Salmo trutta (Sloman et al., 2000b). In a study on juvenile Atlantic salmon (Salmo salar), using ventilation frequency as an indicator, smaller indi-viduals were shown to double their resting metabo-lism in presence of a larger individual, whereas the larger individual’s resting metabolism decreased (Millidine et al., 2009). Furthermore, although meas-urements were not simultaneously run on the domi-nant individual, a study on rainbow trout (Oncorhyn-chus mykiss) revealed a postponed heart rate increase


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in individuals visually exposed to dominant individu-als (Höjesjö et al., 2007). So, do the results from the current study sup-port the hypothesis that there is an effect of social status on heart rate? No, not unambiguously. The results are too few with too high variability and con-tradiction in order to statistically support or logically argue for such conclusions. However, the observed trend motivates further investigation that, with de-veloped methodology, may lead to such conclusions. The procedure of connecting behaviour to heart rate responses, using video recordings, is one aspect that needs to be developed. To successfully do this a higher resolution of data points is essential. This would not only enable a more detailed and ro-bust type of analysis, but also heavily increase the likelihood of attaining sufficient amounts of qualita-tive heart rate data. The results in table 2 might have looked very different with a higher resolution. Cur-rently it suffers from data shortage which prohibits any robust conclusions to be drawn from the data that appears to be relatively uniform, suggesting a higher heart rate for subordinate individuals. Shorter intervals between heart rate measurements would most likely yield abundant data points in all columns and possibly enable an analysis based exclusively on periods of low activity. A higher resolution of physio-logical data combined with a higher resolution of behavioural data, such as scoring, would also allow further correlation analyses between certain types of behaviour and heart rate responses. It is important to recall that the animals in this study were not wild but raised at a hatchery. Alt-hough farm and laboratory animals to a certain ex-tent display patterns of behaviour that are similar to those of wild conspecifics (see Koolhaas et al., 1999), there are in this debate arguments pointing towards the importance of behavioural differences connected to domestication (Johnsson and Abrahams, 1991; Koolhaas et al., 1999; Sloman and Armstrong, 2002; Johnsson et al., 2006). For this reason the biological relevance of the current study, performed on domes-ticated fish, should be evaluated with caution. Con-cerning the resemblance to natural habitat, this study has been carried out with experimental settings that actually are more “natural” than previous laboratory studies investigating heart rate responses, with either hardwired or space limiting methods. While freely swimming animals is a step in the right direction, it is still highly relevant to pay notice to the fact that the subordinate individuals are prohibited from escaping

the tank, and that their stress response therefore may be overestimated (discussed by Sloman and Armstrong, 2002). There are evolutionarily logical arguments for why the subordinate individual might respond to the interaction in the fashion shown by the discussed trend. A greater increase of heart rate in the subordi-nate is most likely connected to higher stress levels, and higher stress levels can press this individual to escape the location holding a dominant. Under en-closed laboratory conditions this response is almost certainly a costly response for this individual. A re-sponse resulting in a passive behaviour, but instead with a lower and stable heart rate, would seem like a more fitness enhancing alternative. In a natural habi-tat, however, where an escape often is possible, a cost-benefit analysis would look quite different. Here a stress response pressing the subordinate to take flight is arguably the more fitness enhancing alterna-tive, since it drives the subordinate to locations that might yield more accessible food resources and more accessible reproductive mates. During this study the hypothesis and general approach has been limited to the perspective of social interaction as a possible factor leading to physiologi-cal responses such as change of heart rate, and not the other way round. This unidirectional causality should of course not be assumed as true, and it should be kept in mind that physiological and behav-ioural responses interact in highly complex and mul-tidirectional ways (Øverli et al., 2004; Gilmour et al., 2005; Johnsson et al., 2006). Conclusions and future research In conclusion, this study has shown that bio-logging as a method has great potential in the investigation of heart rate responses in interacting animals. The re-sults also indicate that social interactions cause heart rate to temporarily increase in rainbow trout (On-corhynchus mykiss), but that the variability in this study was too high for conclusions to be drawn re-garding differences between subordinates and domi-nants. For future research further method testing evaluating implant position would be valuable in the pursuit of qualitative data. Furthermore, to unambig-uously test the effect of social status on heart rate, it is necessary to develop the procedures to more firmly control for undesired factors such as the physical activity of subjected animals, and for the effect of human disturbance of the interacting animals. To-


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gether with the results from this pilot study, recent research speaks for promising potential in future behavioural and physiological studies. Here bio-logging can play an important role in the understand-ing of interactions between physiological and behav-ioural responses, as well as in the understanding of costs involved in social conflicts.

Acknowledgements I thank Johan Höjesjö and Michael Axelsson for su-pervising this study, and Albin Gräns for providing expertise in the specific surgical procedure and pro-gramming of the loggers. Moreover, I would like to thank my co-student Josefin Roos for the collabora-tion during the period of data collection.

References

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Appendix A – Sample of scoring protocol SCORING PROTOCOL PAIR 3 - SOCIAL STATUS AFTER SURGERY - APRIL 24 - start 11:00

WHITE 3 PURPLE 3

Seconds after start

Initiated attack

Completed attack

Initiated interaction

Most central position (1/0)

Seconds after start

Initiated attack

Completed attack

Initiated interaction

Most central position (1/0)

30 30 160 0 60 3 190 90 1 1

120 0 120 2 1150 1 150 1 0180 180 1210 210 3240 0 240 1 1 1270 270300 300330 1 330 1 0360 0 360 1 1390 0 390 1420 0 420 1 1450 0 450 1 1480 480 2510 0 510 4 1540 0 540 2 1570 0 570 1600 0 600 2 1630 630 3660 0 660 2 1690 0 690 1720 0 720 1750 0 750 1780 780810 0 810 1 1840 840 2870 870900 0 900 1 1930 0 930 1960 0 960 1990 990 1

1020 1020 41050 0 1050 11080 10801110 1 1110 1 01140 11401170 1 1170 3 01200 1200 1TOT 0 0 0 4 TOT 2 0 46 20

% 0 0 100 % 100 100 100

DOMINANT SCORING ROUND BY PURPLE - SECOND CONSECUTIVE - SOCIAL STATUS DETERMINED

heart rate in dominant and subordinate rainbow trout ...fish. these conflicts yield dominance and...

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