Impact of Ambient Temperature on Heart Rate and Oxygen Consumption in the Red Junglefowl (Gallus Gallus)

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Linköping University | Department of Physics, Chemistry and Biology Bachelor’s Thesis, 16 hp | Educational Program: Biology Spring term 2020 | LITH-IFM-G-EX—20/3862--SE

Impact of Ambient Temperature on Heart Rate and Oxygen Consumption in the Red Junglefowl (Gallus gallus)

Maryam Chaid

Examiner: Hanne Lovlie Supervisor: Jordi Altimiras

Datum

Avdelning, institution Date 2020-06-11 Division, Department

Department of Physics , Chemistry and Biology Linköping University

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Svenska/Swedish Licentiatavhandling ISRN: LITH-IFM-G-EX--20/3862--SE Engelska/English Examensarbete ______C-uppsats D-uppsats Serietitel och serienummer ISSN Engelska/English Övrig rapport Title of series, numbering

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URL för elektronisk version

Titel Title Impact of Ambient Temperature on Heart Rate and Oxygen Consumption in the Red Junglefowl (Gallus gallus)

Författare Authort’s name Maryam Chaid

Sammanfattning Abstract

Ambient temperature has an impact on the energetic requirements of all animals, causing changes in oxygen consumption and heart rate. Considering the impact that external factors have on adjusting physiological functions, it is important to know how natural alterations in temperature affect the body. The objective of this study was to examine the change in heart rate and oxygen consumption of Red Junglefowl (Gallus gallus) when exposed to naturally occurring low temperatures and if there was a difference in these parameters between the sexes. Furthermore, there was an interest in investigating the occurrence of circadian rhythms in heart rate. Heart rate was measured for indoor-kept animals, exposed to a constant temperature of 20 °C, and outdoor-kept animals that were subjected to variations in ambient temperature. Exposing the chickens to low ambient temperatures, oxygen consumption was monitored using a respirometer while simultaneously measuring their body temperature. An elevated heart rate could be observed during the day. No difference in heart rate was found between males and females, but oxygen consumption was higher for females and body temperature was higher for males. An inverse, linear relationship occurred between heart rate and oxygen consumption in relation to ambient temperature, causing them to increase in response to a decreasing temperature. These results indicate that the energy demand rises when animals are exposed to cold environments, followed by an elevation in oxygen consumption and heart rate in order to supply the body with the energy needed to maintain a stable body temperature.

Nyckelord Keyword

Ambient temperature, Basal metabolic rate, Biotelemetry, Energetic requirement, Heart rate, Oxygen consumption, Red Junglefowl Contents 1 Abstract ...... 4 2 Introduction ...... 4 3 Materials and Methods ...... 7 3.1 Animals and experimental set-up ...... 7 3.2 Data ...... 8 3.3 Statistics ...... 8 4 Results ...... 9 4.1 Heart rate and ambient temperature ...... 9 4.2 Diurnal pattern of heart rate ...... 12 4.3 Oxygen consumption and ambient temperature ...... 13 4.4 Body temperature ...... 15 5 Discussion ...... 15 5.1 Temperature impact and energy requirement ...... 15 5.2 Difference between sexes ...... 16 5.3 Diurnal pattern of heart rate ...... 18 5.4 Final comparison between heart rate and oxygen consumption ...... 19 5.5 Conclusions ...... 20 6 Societal and ethical considerations ...... 21 7 Acknowledgements ...... 22 8 References ...... 23

Impact of Ambient Temperature on Heart Rate and Oxygen Consumption in the Red Junglefowl (Gallus gallus)

1 Abstract

2 Introduction

Heart rate is the rate at which the heart pumps blood to the body. Oxygen consumption, also called metabolic rate, is the use of oxygen per unit of time. Heart rate can be used to predict the change in energy requirements of animals, while oxygen consumption is an indirect

measurement of metabolism, the energy that an animal uses. Measuring oxygen consumption gives an indirect clue about the energy requirement because metabolic reactions are oxygen dependent. According to the Fick’s equation, oxygen consumption is related to the cardiac output, because an organism can only consume as much oxygen as is in the blood that the heart pumps out (Grubb, 1983; Bishop & Butler, 1995).

The cost of metabolism changes with variation in ambient temperature (Chatelain et al., 2013). Birds and mammals change their heart rate and oxygen consumption in relation to ambient temperature due to them being endotherms, defined as the ability of maintaining a constant body temperature (Walter & Seebacher, 2007). Endothermy is developed during embryogenesis and include changes in internal heat production in response to variability in the environment that the animals are exposed to (Walter & Seebacher, 2007). Poor-Wills (Phalaenoptilus nuttallii) are able to have a stable body temperature of 42 °C when exposed to ambient temperatures as high as 44 and 45 °C, at a considerable energy expense (Bartholomew et al., 1962).

A thermoneutral zone (TNZ) exists for all endotherms, in which the metabolic cost of maintaining a stable body temperature is at its minimum (Chatelain et al., 2013). The TNZ range for Ross 308 broiler breeders (Gallus gallus domesticus) that are 14–17 days old is between 30 and 36 °C (Lindholm et al., 2017). When endothermic animals are exposed to temperatures below or above their defined thermoneutral zone, the thermoregulatory mechanisms are activated (Chatelain et al., 2013). If the ambient temperature is well below the thermoneutral zone, the energetic requirements increase, along with metabolic rate and heart rate. European starlings (Sturnus vulgaris) start consuming huge amounts of toxic prey when they get exposed to decreased temperatures in winter, not paying attention to the harmful intake of this food because of the high energy demand (Chatelain et al., 2013).

Birds have different ways of changing their body temperature. They increase evaporative cooling by initiating strong gular flutter, vibrations of the throat skin, in response to high ambient temperatures and shiver in response to low ambient temperatures (Bartholomew et al., 1962). Furthermore, heat loss is also induced by gular flutter. As mentioned before, the reason for these physiological changes is because the birds are exposed to temperatures that do not lay in their TNZ range. For example, the metabolic rate at 5 °C is more than three times higher than the metabolic rate at 35 °C because of the greater energy that is required to manage the thermoregulation at the lower temperature (Bartholomew et al., 1962).

Time of the day, age and sex of the animal are all elements affecting the energetic demand differently. For instance, heart rate follows a diurnal pattern. In most birds, and especially for older individuals, the heart rate is higher at day and decreases steadily at dawn (Cain & Abbott, 1970). The diurnal pattern is influenced by light conditions and activity (Nichelmann et al., 1999 in Moriya et al., 2004). Observing the feather pecking from two different lines of hens, one low and the other high, researchers have determined that the higher activity at day leads to higher heart rate, and the lower activity at night leads to a decrease in heart rate (Korte et al., 1998).

Oxygen consumption and heart rate display a positive, linear relationship with each other (Bevan et al., 1995; Green et al., 2001). In contrast, both have been found to have an inverse relationship with low ambient temperature (Vogel et al., 1963; Lasiewski & Dawson, 1964; Khandoker et al., 2004). However, most studies have been conducted using induced temperature changes in order to see the relationship between these two parameters and ambient temperature. Other studies have concerned the response of embryos or hatchlings to either induced or natural temperature changes, and monitoring has been done on restrained animals (Khandoker et al., 2004; Janke et al., 2002). It was the aim of this study to show how different ambient temperatures affect the energetic requirements of free-ranging Red Junglefowl, the ancestor of the domestic chicken (Gallus gallus domesticus). Measuring oxygen consumption

requires restraining animals in a chamber. The least invasive way of assessing metabolism in free-ranging animals is by monitoring the heart rate. Heart rate, like oxygen consumption, is related to cardiac output, which in turn is related to how much oxygen the body receive (Bishop & Butler, 1995).

Two experimental set-ups have been conducted. First, oxygen consumption was monitored using a respirometer while the chickens were exposed to decreasing temperatures. Second, heart rate was measured via biotelemetry in free-ranging chickens, exposed to natural changes in ambient temperature. The metabolic rate of interest is the basal metabolic rate, which is the rate of resting animals in a fasting, or postabsorptive, state (Hulbert & Else, 2004). Decreasing temperature was hypothesized to be associated with an increased heart rate and thus increased oxygen consumption.

3 Materials and Methods

3.1 Animals and experimental set-up

To obtain data on heart rate, six Red Junglefowls (Gallus gallus) were used; three hens and three roosters. Each animal was implanted with a logger prior to the study. The first pair of animals, one rooster and one hen, was studied for 8 days and 2 hours, starting 2019-11-28. The heart rate was recorded every 5 minutes. The other pair of animals was studied for 9 days and 12 hours, starting from 2019-12-29. Heart rate was again recorded every 5 minutes. The final pair of animals was studied for 8 days and 23 hours, starting from 2020-02-20. Heart rate was recorded every 10 minutes. All animals were fed ad libitum. Ethical permission was done under permit 2492-2019.

To expose the animals to different temperatures, they were kept indoors for a few days and then moved outdoors. While kept indoors, they were exposed to a constant temperature of

approximately 20 °C. Two animals, one hen and one rooster, were excluded from the study because not enough heart rate values had been obtained from the logger. Temperature corresponding to days of measurement was downloaded from SMHI (the metrological and hydrological institute of Sweden). The downloaded temperature regarded Malmslätt (58° 24´ 50´´ N, 15° 31´ 10´´ E), which is the nearest place to where the experiment took place, Vreta (58° 28´58´´N, 15° 31´2´´E).

To obtain data on oxygen consumption, 21 Red Junglefowls were used; 13 hens and 8 roosters. Data was obtained at different days, starting from 2017-12-22 to 2017-12-23 for the first animal and continuing to 2018-02-24 for the last animal. The animals were outdoor-kept before they were moved to a respiration chamber where oxygen consumption was monitored while exposing them to low ambient temperatures (for details, see Lindholm et al., 2017). Measurement of oxygen consumption for every animal was done during the night. Two animals, one hen and one rooster, were also excluded from the study. At the same time, the body temperature of 15 of the 21 animals was measured by inserting PIT tags subcutaneously. The temperature was read continuously with a PIT tag reader.

All animals were fed ad libitum. Ethical permission was done under permit Dnr.9-13.

3.2 Data

Data was obtained from my supervisor’s group that followed the abovementioned protocol and was then processed in Microsoft Excel 2016.

3.3 Statistics

The heart rate data was not normally distributed, as was seen by examining it with a histogram. Difference in heart rate due to sex was thus analyzed using a non-parametric statistical method,

a Mann-Whitney U-test. To examine the interaction effects between heart rate obtained at day and night and outdoor- and indoor kept animals, two-way ANOVA was used. The same test was also used to see if there was any difference in heart rate when the animals were kept indoors and outdoors, and if there was a difference in heart rate at day and night.

Histograms were also made to demonstrate the existence of normal distribution for the data on oxygen consumption and body temperature. Subsequently, difference between males and females in oxygen consumption and body temperature was analyzed with a parametric statistical method, a two-sample t-test: equal variances. Finally, to investigate the correlation between heart rate and ambient temperature, and oxygen consumption and ambient temperature, simple linear regression was used.

All tests were done using Microsoft Excel 2016. The significance (α) was 0.05, and data is presented as mean (M) ± standard deviation (SD).

4 Results

4.1 Heart rate and ambient temperature

Heart rate was measured at two different conditions, starting when the Red Junglefowl were indoors and later when they were moved outdoors. The indoor temperature was constant, hold at approximately 20 °C (Figure 1a). During the day, the lowest average heart rate for all animals was 242 bpm (SD = 11.79), and the highest was 271 bpm. The lowest average heart rate for all animals when kept indoors during the night was 165 bpm (SD = 35.65) and the highest was 247 bpm.

The average outdoor temperature for a whole day varied between -6 and 8 °C (SD = 4.45). However, to make the visualization of heart rate relative to ambient temperature more comparable with the relationship between oxygen consumption and ambient temperature, the

average heart rate for outdoor-kept animals was taken for every 3 °C. The lowest average heart rate during this interval was 274 bpm (SD = 13.23) at day and 227 bpm (SD = 15.3) at night. In contrast, the heart rate went as high as 305 bpm (SD = 13.23) during the day and 265 bpm (SD = 15.3) during the night (figure 1b).

a 300

250

200

150 Day

100 Night Heartrate(bpm)

0 0 5 10 15 20 25 Temperature (°C)

b 350

300

250

200

150 Day Night 100 Heartrate(bpm)

0 -6 -4 -2 0 2 4 6 8 10 Temperature (°C)

Figure 1. Heart rate when Red Junglefowl were held indoors (a) and outdoors (b). The indoor temperature was held constant at 20 °C, and every point for the indoor values shows the average heart rate for an animal at day and night, respectively. The outdoor temperature varied and was pooled in 3 °C along with the corresponding heart rate values. The lines for the outdoor heart rate shows the result of simple linear regression (day: Y=-2.5011x + 295.56, r2=0.87, p=0.02; night: Y=-3.0931x + 246.91, r2=0.82, p=0.013). Error bars represent standard deviation. n=4.

Since the outdoor temperature varied and the indoor temperature did not, the relationship between heart rate and ambient temperature could be analyzed statistically when the animals were held outdoors. There was a strong relationship between ambient temperature and heart rate. Around 85% of the variation in heart rate could be explained by the variation in temperature (day: r2 = 0.87, p = 0.02; night: r2 = 0.82, p = 0.013). Heart rate increased with decreasing temperature (Figure 1b).

The heart rate of the animals was higher when they were outdoors (M = 265 bpm, SD = 28.8,

F1,15 = 6.24, p = 0.02). A visualization of the comparison in heart rate between indoor- and outdoor kept animals can be seen in Figure 2.

Figure 2. Comparison of heart rate when Red Junglefowl were kept indoors and outdoors. The bold dark line in every boxplot shows the average heart rate, and the dot in the lower right corner is an outlier. Bars represent standard deviation. n=4.

No difference in heart rate could be found between males and females (U = -476.3, N = 4, p = 0.5).

4.2 Diurnal pattern of heart rate

A diurnal pattern in heart rate was also noticed (Figure 3). The heart rate was higher at day (M = 275 bpm, SD = 22.3) and became lower at night (M = 231.8 bpm, SD = 27.5), with a significant difference between these two conditions (F1,15 = 10.7, p = 0.007). Time of the day was from sunrise to sunset for the corresponding day of measurement, and time of the night was from sunset to sunrise. The average heart rate for all animals at day and night, respectively, was taken and is visualized below (Figure 3).

Figure 3. Heart rate of Red Junglefowl at day and night, respectively. The dark bold line in the middle of every boxplot is the average heart rate, and the dot in the lower left corner is an outlier. Bars represent standard deviation. n=4.

4.3 Oxygen consumption and ambient temperature

-1 -1 Oxygen consumption was higher for females (M = 1.13 ml O2 g h , SD = 0.27) than for males -1 -1 (M = 0.9 ml O2 g h , SD = 0.21) (t = 4.48, df = 17, p = 0.0003). The relationship between oxygen consumption and ambient temperature was the same as the relationship between heart rate and ambient temperature; as the temperature decreased, the oxygen consumption increased (figure 4). The temperature range varied, on average, between -8 and 20 °C (SD = 8.1), -1 -1 corresponding to 1.4–0.6 ml O2 g h (SD = 0.27).

2 1) - 1,8 1 h 1 - 1,6 1,4 1,2 1 Females 0,8 Males 0,6 0,4 0,2

Oxygen consumption (ml O2 O2 g (ml consumption Oxygen 0 -9 -4 1 6 11 16 21 Temperature (°C)

Figure 4. Oxygen consumption of males and females of Red Junglefowl relative to ambient temperature. Females (n=12) had a slightly higher oxygen consumption than males (n=7), but animals of both sexes showed an increase in oxygen consumption when the ambient temperature decreased. The temperature here, as in the analysis of the heart rate, shows the average values of every 3 °C change, corresponding to the average values in oxygen consumption. The lines shows the result of a simple linear regression (females: Y=-0.0265x + 1,3191, r2=0.69, p=2.4 * 10-29; males: Y=-0,0246x + 1.1156, r2=0,8, p=1.7 * 10-22). Error bars represent standard deviation.

68% of the variation in oxygen consumption of females could be explained by the variation in ambient temperature (r2 = 0.68, p <<0.05). In contrast, 80% of the variation in oxygen consumption of males could be explained by the variation in ambient temperature (r2 = 0.8, p <<0.05). The variation in oxygen consumption of all animals (not shown) could be explained by the variation in ambient temperature of approximately 64% (r2 = 0.64, p <<0.05). In either case, temperature had a strong impact on the oxygen consumption of Red Junglefowl (Figure 4).

4.4 Body temperature

The body temperature of 15 animals, two of which were excluded from the analysis because not enough data had been obtained, was measured along with the temperature they were exposed to during the study. There was no significant impact of ambient temperature on the body temperature (not shown) (r2 = 0.036, p = 0.5). Nonetheless, males had a significantly higher body temperature (M = 40.4 °C, SD = 0.27, n = 6) than females (M = 39.87 °C, SD = 0.59, n = 7) (t = 2.05, df = 11, p = 0.03).

5 Discussion

5.1 Temperature impact and energy requirement

Variation in ambient temperature had the same impact on heart rate and oxygen consumption; when the temperature decreased, heart rate and oxygen consumption increased (Figure 1b, Figure 4). When measuring the heart rate, the indoor temperature, kept at 20 °C, was essentially higher than the outdoor temperature, that never reached an average value above 10 °C. The outdoor heart rate was significantly higher than the indoor value (Figure 2). Similar to this, the oxygen consumption at -8 °C was significantly higher than the oxygen consumption at 20 °C (Figure 4).

Exposing domestic fowl to abrupt changes in ambient temperature, declining temperature was associated with an elevated heart rate and oxygen consumption (Harrison & Biellier, 1969). The same response has been observed in the common nighthawk (Chordeiles minor) (Lasiewski & Dawson, 1964), European starlings (de Bruijn & Romero, 2011), tufted duck (Aythya fuligula) (Bevan & Butler, 1992) and Przevalski’s Partridge (Alectoris magna) (Liu & Li, 2005). The important question is what happens in the body when the temperature increases or

declines? In other words, what physiological functions can give rise to the observed changes in heart rate and oxygen consumption?

An endothermic animal must maintain a stable body temperature. In this experiment, no correlation was found between body temperature and ambient temperature, which means that the body temperature of the chickens was held constant while they were exposed to low ambient temperatures. The thermoregulatory mechanisms that occur when the temperature lies above or below the TNZ range require energy (Batholomew et al., 1962; Chatelain et al., 2013). In order to meet the higher energy demand of the body when exposed to very high or very low temperatures, oxygen consumption increase. Since oxygen needs to be distributed throughout the body, the heart rate escalates to pump blood to the tissues and meet the elevated oxygen demand (Chatelain et al., 2013).

Energy requirement does increase in cold winters, which is met by a higher metabolic rate (Cooper, 2000; Nzama et al., 2010; Chatelain et al., 2013). When the energy demand increases, the body utilizes stored reserves and streamline the digestive system in order to acquire more energy (Broggi et al., 2004). This response to a decline in ambient air temperature leads to elevated levels in basal metabolic rate in order to meet the energetic costs that follows thermoregulation. Subsequently, heart rate also increases to ensure that tissues receive the oxygen they need to maintain a stable body temperature.

5.2 Difference between sexes

No difference in heart rate could be found between males and females. However, oxygen consumption was significantly higher in females (Figure 4), and body temperature was higher in males.

Males and females of Red Junglefowl, like other animals, differ in their body composition. Females have heavier spleens, liver, reproductive and peritoneal organs (Hammond et al.,

2000). In contrast, males have larger heart, lungs and pectoralis. Since the organs responsible for uptake and distribution of oxygen are the heart and lungs, and those are heavier in males, the aerobic scope and capacity is higher for males than females (Hammond et al., 2000). Higher aerobic capacity, meaning more efficient utilization of oxygen, gives rise to enhanced citrate synthase activity and lower basal metabolic rate (Hammond et al., 2000).

In contrast to the observed increase in oxygen consumption of females as opposed to males, there was no difference in heart rate between the sexes. Two possible explanation exists. First, the number of animals for which heart rate was measured could simply have been so small that no significant difference between males and females could be observed. After all, the test subjects consisted of two males and two females. Nevertheless, there could also be a physiological explanation behind it.

Females have a higher parasympathetic control of the heart than males have (Carter et al., 2003). This could be the result of developmental differences or sex hormones (Kuo et al., 1999; Dart et al., 2002). For example, oestrogen, the sex hormone usually found in females, inhibit vasoconstrictory responses of the heart and enhances parasympathetic activity (Dart et al., 2002). When the parasympathetic activity increases and the sympathetic activity decreases, a reduction in heart rate can be observed (Carter et al., 2003). Following this statement, there must have been a smaller increase in heart rate relative to oxygen consumption in females compared with males. Males, having lower oxygen consumption, did have a less pronounced increase in heart rate than if their oxygen consumption was higher than females. Thus, no significant difference in heart rate could be observed between the sexes.

Another interesting observation is the fact that in this study, females had lower body temperature than males. Since body temperature is the balance between heat production and heat loss, it is directly related to resting metabolic rate and body mass (Clarke & Rothery, 2007). In birds, body temperature has an inverse relationship with body mass and resting metabolic

rate. In contrast, there is a positive relationship between activity and body temperature (Long et al., 2005). Males of Red Junglefowl are known to cooperate in several daily activities, like fighting individuals of the same sex, that females do not take part of, causing them to have higher body temperature (Hammond et al., 2000). Other species, like arctic ground squirrels (Spermophilus parryii), have also been found to have a peak in body temperature at increased activity (Long et al., 2005).

5.3 Diurnal pattern of heart rate

Heart rate showed a clear diurnal pattern, with a higher rate at day and lower values at night (Figure 3). The relationship between time of the day and heart rate could not be coupled to temperature, since the temperature decreases at night and it has already been noted that heart rate had an inverse relationship with low ambient temperature. A plausible suggestion for the diurnality is the change in activity between day and night. Red Junglefowls are diurnal animals, which means they have higher activity at day and fall asleep at night.

Activity is positively correlated with energy demand. For example, energetic requirements of birds increase at flight and decrease at walking (Ward et al., 2002). Chickens, especially older individuals, have a peak in heart rate just as the sun goes up (Cain & Abbott, 1970). The heart rate continues to be high during the day and decrease slowly to its lowest values when it becomes dark.

Light conditions and metabolism are also factors affecting the circadian rhythm (Shimada & Korde, 1978; Nichelmann et al., 1999). Light has been found to cause a decrease in the vagal nerve activity and a higher excitation of the sympathetic system, leading to increased heart rate (Moriya et al., 2004). When the activity of the vagal nerve, being a part of the parasympathetic system, decline during the day, a higher heart rate can be observed. A similar system has been suggested for birds, involving daily variation in a specific gene, per2, that resides in the

suprachiasmatic nucleus. Variation in the gene and the subsequent interaction of the suprachiasmatic nucleus with the sympathetic and parasympathetic autonomic nerve system gives rise to the diurnality of heart rate observed in chicken and most birds (Okabayashi et al., 2003 in Moriya et al., 2004).

This mechanism, the change in activity of the sympathetic and parasympathetic system, could also be the result of the change in activity of the body. It has already been noted that Junglefowl are diurnal animals. Thus, when the activity of the body increases at day, the activity of the sympathetic system also increases, the parasympathetic activity decreases and the heart rate gets higher.

5.4 Final comparison between heart rate and oxygen consumption

It is difficult to draw a general conclusion about the relationship between heart rate and oxygen consumption because they have been measured in different individuals. Also, different parameters have been studied for heart rate and oxygen consumption. The diurnality of heart rate has been measured, as well as the change that appears when Red Junglefowl were kept indoors and then moved outdoors, which was done for the sake of observing how the change in temperature would affect the heart rate. In contrast, when measuring the oxygen consumption, the chickens were placed in a respirometer while being exposed to a steady decrease in ambient temperature. Despite these differences, a comparison between heart rate and oxygen consumption can be done by comparing the impact of ambient temperature on each parameter.

The heart rate and oxygen consumption of Red Junglefowl were affected by the ambient temperature in similar ways. A decrease in air temperature led to an increase in basal metabolic and heart rate (Figure 1b, Figure 4). However, the degree at which both parameters increased in order to keep the body temperature constant differed. The slope of the regression line between

heart rate and ambient temperature was -2.8, and that between oxygen consumption and ambient temperature was -0.025.

The smaller slope of the regression line when comparing heart rate and ambient temperature indicate that heart rate increased more than oxygen consumption in order to maintain a stable body temperature. Also, 85 % of the variation in heart rate could be explained by variation in ambient temperature, while the regression coefficient of oxygen consumption and ambient temperature was only 64 %. A generally drawn conclusion is thus a higher impact of ambient temperature on heart rate than that on oxygen consumption.

Other studies have shown the existence of a linear, positive relationship between heart rate and oxygen consumption (Lasiewski & Dawson, 1964; Bevan et al., 1995; Bishop & Butler, 1995; Green et al., 2001; Ward et al., 2002). The differing results in this experiment could be, as mentioned earlier, because of the fact that heart rate and oxygen consumption were not measured on the same individuals. This could have led to dissimilar slopes and regression coefficients.

In the future, it would be interesting to measure the heart rate and oxygen consumption on the same animals, ruling out the differences in body mass, condition etc. It would also be interesting to see if other bird species react to the natural changes in ambient temperature like Red Junglefowl do, and if the same pattern occurs in mammals as well. Nevertheless, given this experiment, heart rate and oxygen consumption appear to increase in response to a decreasing temperature, but there is a higher increase in heart rate than oxygen consumption.

5.5 Conclusions

When exposed to cold temperatures, endotherms must work harder to maintain a stable body temperature. As a result, energy demand increases to allow the thermoregulatory mechanisms, for example shivering, to take place (Bartholomew et al., 1962). Oxygen consumption rises in

order to provide the body with the energy needed. The inhaled oxygen is distributed throughout the body by augmenting the heart rate, thus increasing the amount of blood that the heart pumps out and can reach the tissues that demand oxygen. This proposed mechanism follows the observation that oxygen consumption and heart rate increased at decreasing temperatures. The relationship between temperature, heart rate and oxygen consumption is in line with the hypothesis stated prior to the study; decreasing temperature was associated with an increased heart rate and oxygen consumption.

Greater mass of the organs responsible for distribution and transport of oxygen rises the aerobic capacity and lower the oxygen consumption that follows a decrease in ambient temperature (Hammond et al., 2000). At the same time, a proposed correlation between sex hormones and heart rate has been suggested, leading to a higher activity of parasympathetic system and the observed relationship between heart rate and oxygen consumption in males and females (Dart et al., 2002; Carter et al., 2003). Finally, activity is also suggested to have an impact on energy demand and the subsequent change in heart rate, such as activity, that increases during the day, lead to a higher demand of energy and thus elevated heart rate.

6 Societal and ethical considerations

All subjects included in the study have been handled in line with their animal rights in accordance with the European Community Directive 2010/63/EU. The experiment when obtaining heart rate data was ethically permitted under permit 2492-2019. The experiment when obtaining oxygen consumption and body temperature data was ethically permitted under permit Dnr.9-13. Both experiments were approved by the Ethical Board of Agriculture, Sweden.

This study, though decreasing the comfort of the subjects involved in it, gives valuable insights into the impact of external factors on the physiology of animals. External factors are something that an animal continuously comes in contact with and understanding how they affect

physiological factors makes it easy to predict what challenges the animal will face when set in different conditions. Heart rate and oxygen consumption are directly coupled to the survival of animals, since they are the mechanisms by which the body is supplied with oxygen, one of the most essential factors in keeping the animal alive.

Understanding how the body changes its oxygen requirement due to a change in ambient temperature and how this change varies between sexes is fundamental in knowing how the animal keeps itself alive when exposed to a stressful condition. Red Junglefowl are endotherms, as are human beings, which makes it possible to draw indirect conclusions about the physiological changes occurring in humans when met with the same conditions as the subjects in this experiment.

7 Acknowledgements

I would like to thank Jordi Altimiras for supervision, care taking of the experimental set-ups and provision of data. Thanks also to all the teachers and examiners in coordinating the course, including Hanne Lovlie for support and advice, Matthias Laska for advice about report writing and Anders Hargeby for input about figure making. And lastly, thanks to my opponents, Martin Larsson, Filip Henriksson and Emelie Karlsson for giving me advice about how to improve the thesis.

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