Department of Physics, Chemistry and Biology Examensarbete 16 hp
Phenotypic plasticity of the heart and skeletal muscles in
cold acclimated Red Junglefowls (Gallus gallus)
Emma Ingeström
LiTH-IFM- Ex--15/3021--SE
Supervisor: Jordi Altimiras, Linköping University Examiner: Anders Hargeby, Linköping University
Department of Physics, Chemistry and Biology Linköpings universitet
Rapporttyp Report category Examensarbete C-uppsats Språk/Language Engelska/English Titel/Title:
Phenotypic plasticity of heart and skeletal muscles in cold acclimated Red Junglefowls (Gallus gallus)
Författare/Author:
Emma Ingeström Sammanfattning/Abstract:
The ability of the heart and skeletal muscles to remodel to environmental demands, their plasticity, is of interest when studying animals’ adaptation to environment changes. Temperature variation due to seasonal change seems to lead to the development of a cold acclimated phenotype in small birds. To endure cold conditions a higher metabolism is required for shivering thermogenesis in aerobic skeletal muscles. This in turn leads to several physiological changes, including a heart and muscle hypertrophy as well as an increased oxygen carrying capacity of the blood. In this study were Red Junglefowls (Gallus
gallus) bred indoors and outdoors and physiological aspects such as body size, growth rate, relative size
of heart and skeletal muscles (pectoralis major and gastrocnemius maximus) as well as hematocrit and hemoglobin concentrations of the blood were compared between the groups. Observed significant differences included a slower growth rate in fowls bred outdoors, 2.5 (0.7) g/day than indoors, 3.8 (0.4) g/day, as well as a larger relative size of the heart and gastrocnemius muscle. The average relative size of the heart was more than twice as big in fowls bred outdoors, 0.97 (0.08) %, than indoors, 0.40-42 (0.05) %. The average relative size of the gastrocnemius muscle for the fowl bred outdoor was significantly larger than for fowl bred indoors (0.95 (0.11) %, vs. 0.58-0,63 (0.09) %). In addition, fowl bred outdoors showed an increased capacity for oxygen transportation, with blood hematocrit values of 43 (3) % higher than 35-37 (3) % for the indoor animals. Fowls bred outdoors also showed higher hemoglobin concentrations in the blood, 127 (7) g/l, than fowls bred indoors, 113 (7) g/l. Findings indicate a cold acclimated phenotype among the outdoor bred fowls.
ISBN
LITH-IFM-A-EX—15/3021—SE
__________________________________________________ ISRN
__________________________________________________
Serietitel och serienummer ISSN
Title of series, numbering
Handledare/Supervisor Jordi Altimiras Ort/Location: Linköping
Nyckelord/Keyword:
Cold acclimation, heart, gastrocnemius, pectoralis, plasticity, Red Jungelfowl
Datum/Date
2015-06-08
URL för elektronisk version
Institutionen för fysik, kemi och biologi
Department of Physics, Chemistry and Biology
Avdelningen för biologi
Instutitionen för fysik och mätteknik
Content 1 Abstract ... 3 2 Introduction ... 4 2.1 Background ... 4 2.2 Aim ... 5 2.3 Social aspects ... 5
3 Material & methods... 6
3.1 Animals, management and experimental set-up ... 6
3.2 Animal grouping... 6 3.3 Measurement procedures ... 6 3.4 Statistical analysis ... 7 3.5 Ethical aspects ... 7 4 Results ... 8 4.1 Temperatures ... 8 4.2 Growth ... 8
4.3 Relative heart size ... 10
4.4 Relative skeletal muscle size ... 11
4.5 Blood parameters ... 13
5 Discussion ... 14
5.1 Growth rate... 14
5.2 Heart plasticity ... 14
5.3 Skeletal muscle plasticity ... 15
5.5 Conclusions ... 16
5.6 Further studies ... 16
6 Acknowledgement ... 17
7 References ... 17
1 Abstract
The ability of the heart and skeletal muscles to remodel to environmental demands, their plasticity, is of interest when studying animals’ adaptation to environment changes. Temperature variation due to seasonal change seems to lead to the development of a cold acclimated phenotype in small birds. To endure cold conditions a higher metabolism is required for shivering thermogenesis in aerobic skeletal muscles. This in turn leads to several physiological changes, including a heart and muscle hypertrophy as well as an increased oxygen carrying capacity of the blood. In this study Red Junglefowls (Gallus gallus) were bred indoors and outdoors and physiological aspects such as body size, growth rate, relative size of heart and skeletal muscles (pectoralis major and gastrocnemius maximus) as well as hematocrit and hemoglobin concentrations of the blood were compared between the groups. Observed significant differences included a slower growth rate in fowls bred outdoors, 2.5 (0.7) g/day than indoors, 3.8 (0.4) g/day, as well as a larger relative size of the heart and gastrocnemius muscle. The average relative size of the heart was more than twice as big in fowls bred outdoors, 0.97 (0.08) %, than indoors, 0.40-42 (0.05) %. The average relative size of the gastrocnemius muscle for the fowl bred outdoor was significantly larger than for fowl bred indoors (0.95 (0.11) %, vs. 0.58-0,63 (0.09) %). In addition, fowl bred outdoors showed an increased capacity for oxygen transportation, with blood hematocrit values of 43 (3) % higher than 35-37 (3) % for the indoor animals. Fowls bred outdoors also showed higher hemoglobin concentrations in the blood, 127 (7) g/l, than fowls bred indoors, 113 (7) g/l. Findings indicate a cold acclimated phenotype among the outdoor bred fowls.
2 Introduction
2.1 Background
The heart is responsible for pumping blood, containing oxygen and essential nutrients throughout the animal body. Animals need more energy and oxygen, a higher metabolism, to endure cold conditions, to keep up their body temperature and other body functions. This cold acclimated phenotype result in a larger work load of the heart, which may contribute to an increase in relative heart size to account for the increased demand (Green et al., 2008). It has been reported that the relative heart mass of dark-eyed juncos is larger in individuals exposed to a cold climate in comparison to individuals living in warmer climates (Swanson et al., 2014b). However in the case of hoopoe larks there was no significant difference in relative heart size found when comparing larks exposed to
high and low temperatures respectively (Williams and Tieleman, 2000).
Phenotypic plasticity refers to morphological or physiological remodeling to environmental stimuli or demands. These changes are temporary, reversible and repeatable. (Piersma and Drent, 2003; Starck and Rahmaan, 2003; McKechnie, 2008). It has been reported that small birds show such a plasticity to endure temperature changes due to seasonal variation (Swanson, 2010). It has also been demonstrated that temperature is an essential factor for seasonal thermoregulation in birds and that their body mass and basal metabolic rate are affected by temperature (Swanson, 1993, 2001; Tieleman et al., 2003; Vézina et al., 2006; McKechnie et al., 2007; Williams and Tieleman, 2000; Zheng et al., 2013).
The main thermogenic mechanism, to produce heat, in small birds is shivering in skeletal muscles, primary in the pectoral muscle, but the gastrocnemius muscles are found to be particularly important in shivering thermogenesis in some Galliform chicks (Hohtola et al., 1998; Marjoniemi and Hohtala, 1999). The muscles involved in shivering thermogenesis are expected to increase in relative size due to cold phenotypic plasticity. Muscle hypertrophy occurs as a result of a more intense usage in this case due to the increased demand of heat production and shivering thermogenesis. As a result, muscle mass increases and a higher thermogenic capacity and cellular metabolic intensity should be achieved (Swanson et al., 2014b). It has been reported that passerine birds show larger relative masses of heart and pectoral muscle when exposed to winter-like conditions (Liknes and Swanson, 2011). Another recent study showed that American goldfinches in winter have significantly greater pectoral and heart masses than in summer, whereas black-capped
chickadees showed no significant seasonal variation (Swanson et al., 2014a).
A higher metabolism means an increased oxygen consumption and therefore would a more efficient oxygen transport capacity fit the cold acclimation phenotype of small birds (Gaesser and Brooks, 1984). Hematocrit is a measurement of the amount of erythrocytes, red blood cells, in the blood. Erythrocytes contain the oxygen carrying agent hemoglobin. Hemoglobin is also found solved in the blood plasma. Blood from cold acclimated birds is therefore expected to contain higher amounts of erythrocytes and higher concentrations of hemoglobin. In conclusion, changes associated with an increased metabolic capacity are expected in cold acclimated Red Junglefowls. This includes improved heat production in the muscles and oxygen transportation to those tissues by the cardiovascular system.
2.2 Aim
The aim of this study is to investigate if young Red Junglefowl (Gallus gallus) bred outdoors during fall show a cold acclimated phenotype and to compare body size, growth rate, relative size of heart and skeletal muscles and blood parameters such as hematocrit and hemoglobin concentrations with Red Junglefowl bred indoors.
The hypothesis is that Red Junglefowls bred outdoors will show a cold acclimated phenotype with features indicating an increased metabolic capacity. These phenotypic features include a slower growth rate, higher amounts of erythrocytes and hemoglobin in the blood, a larger relative heart size and an increase in relative size of skeletal muscles involved in shivering thermogenesis.
2.3 Social and ethical aspects
The heart’s capacity to remodel in response to stimuli or environmental demands, its plasticity, can induce growth of the heart. It is possible to distinguish two different types of heart plasticity that leads to heart growth, a physiological growth that has been associated with exercise, pregnancy and postnatal development and a pathologic hypertrophic growth associated with neurohumoral activation, hypertension and myocardial injury, that when persistent leads to heart failure and ventricular arrhythmia (Hill and Olson, 2008). A proven correlation between metabolism and relative heart and skeletal muscle size in Red Junglefowl makes a genetic analysis possible, since the whole sequence of the Red Junglefowl genome is known (Hillier et al., 2004). Hence the study contributes to the
understanding of the fundamental physiological and genetically mechanisms that leads to a physiological growth of heart and muscles. Ethical implications of this study include the need to use animals to understand these physiological processes. As for today there is no other equivalent method available than to use live animals when studying the plasticity of the heart and muscles in vertebrates. The most relevant parameters for comparisons between groups are the relative heart and muscle sizes which most accurately are obtained by euthanization and dissections. Animals used in this study were handled with care and with as few handlings as possible. Animals were kept and handled under as stress free conditions as possible and euthanized by decapitation, a relatively quick and painless method. The study was performed accordingly to the permission mentioned in section 3.5 Ethical aspects.
3 Material & methods
3.1 Animals, management and experimental set-up
Red Junglefowl, Gallus gallus, were bred indoors at the hatchery at Linköping University or outdoors in aviaries at Vreta, a farm approximately 16 km north west of Linköping (~58° 28′ N, 15° 31′ E), from September to November 2014. Hatchery fowls were exposed to a constant temperature of 28°C, while aviary fowls were exposed to seasonal variation as shown in the appendix (Figure A1). Maternal care is essential for the survival of young fowls at lower temperatures, therefore were the outdoor chicks raised along with a mother hen. All fowls had ad libitum access to food and water.
3.2 Animal grouping
Red Junglefowl bred outdoors (n = 30) were allowed to reach a target body weight of 70 ± 10 g. Two indoor groups were then bred and matched in size (n = 30) or age (n = 35) with the outdoor group.
3.3 Measurement procedures
Body masses of outdoor (n = 30) and indoor (n = 38) fowl were recorded continuously during the upbringing as a measurement of growth rate. Fowls were collected and brought to the laboratory after reaching a specific target weight or age. Blood samples for duplicate measurements of hematocrit values and hemoglobin concentrations were collected in capillary tubes and microcuvettes by a small incision at the branchial vein. Capillary tubes were centrifuged at 10 000 rpm for five minutes and hematocrit values were obtained with a hematocrit reader. Hemoglobin concentrations were obtained by measurements with the HemoCue
HB201+ system (HemoCue AB). Hematocrit values and hemoglobin concentrations were measured to the significance of 1 % and 1 g/l respectively.
Fowls were weighed and thereafter euthanized by cervical dislocation or a pentobarbital overdose. The right pectoral muscle (Pectoralis major), right gastrocnemius muscle (Gastrocnemius maximus) and the heart were isolated, dissected out and weighed. Heart mass is here defined as ventricular mass. Significant digits of total body mass, heart weight, pectoral and gastrocnemius muscle weights are 0.1 g, 1 mg, 0.01 g and 1 mg respectively.
3.4 Statistical analysis
Data were statistically tested using one way ANOVA tests of the general linear model in MiniTab. Tukey’s model was used for significant grouping. The same method was used when comparing differences between genders. Body mass and growth rate were not analyzed for gender differences. All tests were set at the significance of α = 0.05. Calculations of means and variation (standard derivation), as well as individual value plots were also made in MiniTab. The graph containing average body weight (Figure 1A) was made in GraphPad.
3.5 Ethical aspects
This study follows the direction of animal experimentation according to the laws of Sweden and European Union (EU). The study is included in the ethical permission of the Development Programming of Cardiovascular Disease, Genetic and Physiological Mechanisms Involved in Cardiac Growth and Regulation of Cardiovascular Function in Chicken project Dnr.9-13 admitted by the Regional Ethical Review Board in Linköping, Sweden. The study is financed by Linköping University.
4 Results
Results are presented as average and standard derivation, i.e. mean (SD). No significant differences were found between the genders when analyzing any physiological aspect, hematocrit, hemoglobin concentrations and relative heart and skeletal muscle sizes, and are therefore not presented.
4.1 Temperatures
Red Junglefowl bred outdoor hatched between 10 – 23 of September and were euthanized 6 – 15 October 2014. Temperature were measured at Vreta (~58° 28′ N, 15° 31′ E), from the earliest hatchling to the latest
euthanization, and an average temperature of 11.3 (3.6) °C was calculated.
Temperature variations according to temperature measurements at Vreta are presented in the appendix (Figure A1). The temperature at the closest measurement point of the meteorological and hydrological institute of Sweden (SMHI, Sveriges meteorologiska och hydrologiska institut,), in Malmslätt (~58° 40′ N, 15° 53′ E) 7.5 km away, was 10.7 (3.8) °C for the same period. Temperature fluxions according to the measurements of SMHI, are presented in the appendix (Figure A1).
4.2 Growth
Outdoor fowls (n = 30) reached the average target weight 73.3 (5.8) g at the average age of 26 (3) days. Indoor size matched fowls (n = 30) reached body mass 71.9 (4.9) g at the age of 16 (1) days. Indoor age matched fowls (n = 35), average age 26 (3) days reached a body mass of 132.5 (13.3) g. There is a significant difference in growth rate between Red Junglefowls bred outdoor (n = 30) and indoor (n = 38). Total body mass increased more rapidly in fowls bred indoors than outdoors, average growth rates are 3.8 (0.4) g/day and 2.5 (0.7) g/day, respectively (Figure 1).
Hatchling weights did not differ between the indoor (n = 10) and outdoor bred fowls (n = 10), P = 0.859. The average body weight of two days old indoor and outdoor fowls was 22.9 (3.2) and 22.6 (1.3) g, respectively (Figure 1A).
Figure 1. (A) Average body mass of Red Junglefowls bred outdoor (n =
30) or indoor (n = 38) at different ages. Fowls bred outdoor are showed as white dots, while indoor fowls are symbolized by black dots. Growth rate is estimated by nonlinear exponential regressions. Estimated growth rates for indoor and outdoor bred fowls are represented by a solid and dashed line respectively. Error bars represent standard derivation. (B) Calculated average growth rates for indoor, 3.8 (0.4) g/day, and outdoor, 2.5 (0.7) g/day, bred Red Junglefowls. There is a significant difference in growth rate between fowls bred outdoor and indoor. Black dots symbolize individual growth rates g/day. Asterisk indicate statistically significant differences, *** represent P < 0.001.
A
Living conditions
4.3 Relative heart size
Significant differences in relative heart size were observed between all groups (Figure 2). Outdoor fowls with a heart size approximately 708 (87) mg showed a relative heart size of 0.97 (0.08) %. Average heart size in the indoor, size and age matched, groups were 319 (39) mg and 531 (90) mg respectively, corresponding to a relative heart size of 0.44 (0.05) % and 0.40 (0.05) %.
Figure 2. Relative heart size of Red Junglefowls bred outdoor 0.97 (0.08)
% in comparison to size, 0.44 (0.05) %, and age, 0.40 (0.05) %, matched fowls bred indoors. Bars, black dots and error bars characterize average relative heart size, individual values and standard error, respectively. Asterisk indicate statistically significant differences, *** represent P < 0.001 and ** represent P < 0.01.
4.4 Relative skeletal muscle size
The smallest pectoral muscles 1.99 (0.27) g were observed in the younger indoor, size matched, fowls corresponding to a relative pectoral size of 2.76 (0.27) %. The relative pectoralis size was significantly different from the older indoor, age matched, and outdoor fowls which had a relative pectoralis size of 3.52 (0.50) % and 3.37 (0.42) % respectively, corresponding to average pectoral weights of 4.69 (0.93) g and 2.47 (0.37) g. There is no significant difference in relative pectoralis size between fowls of the same age bred indoor or outdoor, P = 0.384 (Figure 3).
Figure 3. Relative size if the right pectoral muscle in Red Junglefowls
living outdoor 3.37 (0.42) % in comparison to size, 2.76 (0.27) % and age, 3.52 (0.50) %, matched fowls bred indoors. Bars, black dots and error bars characterize average relative muscle size, individual values and standard error, respectively. Asterisk indicate statistically significant differences, *** represent P < 0.001.
Relatively larger gastrocnemius muscles were found among the outdoor fowls 695 (92) mg corresponding to a relative size of 0.95 (0.11) %. The relative gastrocnemius size of the outdoor fowls was significantly larger than those for both indoor groups. Indoor size and age matched fowls showed relative gastrocnemius sizes of 0.58 (0.09) % and 0.63 (0.06) % respectively, corresponding to muscle weights of 425 (74) mg and 842 (134) mg. No significant differences were observed in relative gastrocnemius size between the indoor groups, P = 0.191 (Figure 4).
Figure 4. Relative size if the right gastrocnemius muscle in Red
Junglefowls living outdoor 0.95 (0.11) % in comparison to size, 0.58 (0.09) %, and age, 0.63 (0.06) %, matched fowls bred indoors. Bars, black dots and error bars characterize average relative muscle size, individual values and standard error, respectively. Asterisk indicate statistically significant differences, *** represent P < 0.001.
4.5 Blood parameters
Outdoor fowls showed higher hematocrit values 43 (3) % than fowls bred indoor, were the younger, size matched fowls, and older, age matched, fowls had hematocrit values of 37 (3) % and 35 (3) % respectively. When statistically comparing hematocrit values were all groups significantly different, P = 0.026 between indoor groups (Figure 5A). Outdoor fowls also showed significantly higher hemoglobin concentrations 127 (7) g/l than fowls bred indoor, for which the average hemoglobin concentration was measured to 114 (7) and 111 (7) g/l for the younger and older indoor fowls respectively. There was no significant difference in blood hemoglobin concentrations between the indoor groups, P = 0.299 (Figure 5B).
Figure 5. Hematocrit values (A) and hemoglobin concentrations (B) for
Red Junglefowls bred outdoors in comparison to size and age matched fowls bred indoors. Bars, black dots and error bars characterize average values, individual values and standard error, respectively. Individual plots are averages of duplicate measurements. Asterisk indicate statistically significant differences, *** represent P < 0.001 and * represent P < 0.05. (A) Average hematocrit values for fowls bred outdoor 43 (3) %, in comparison to size and age matched fowls bred indoors, for which measured hematocrit are 37 (3) % and 35 (3) %, respectively. (B) Average hemoglobin concentrations for fowls bred outdoor 127 (7) g/l, in comparison to size and age matched fowls bred indoors, for which measured hemoglobin concentrations are 114 (7) and 111 (7) g/l respectively.
Living conditions
A B
5 Discussion
5.1 Growth rate
Red Junglefowl bred indoors with a constant temperature, 28 ° C, showed a higher growth rate than fowls bred in aviaries outdoors, which were exposed to seasonal temperature variations (Figure A1 and A2). Average ambient temperatures for the outdoor fowls of this study was 11.3 (3.6) °C. Indoor and outdoor fowls increase in body mass at significantly different rates (Figure 1).
Red Junglefowl have a thermoneutral zone between 27.5-37.5 °C (Van Kampen et al., 1979). Within this temperature range the basal rate of heat production is equal to the heat loss to the external environment, the body temperature is therefore adjusted with mechanisms requiring little metabolic energy (Meltzer, 1983). The differences in growth rate between Red Junglefowl bred indoor and outdoor can be explained by taking notion to that the indoor fowls have no need for thermoregulation and therefore have more metabolic energy available for mechanisms related to growth and increase in body weight. Basal metabolic rate is higher in cold-acclimated birds (Maldonado et al., 2009).
5.2 Heart plasticity
Red Junglefowl bred outdoors have significantly larger hearts than fowls of the same body weight bred indoors, corresponding to a relative heart size more than twice as big (Figure 2). Relative heart size seems to decrease with age in indoor bred fowls, since the age matched indoor group showed significantly smaller relative heart size, than the younger indoor fowls and outdoor fowls of the same age.
Greater heart mass of birds living in colder climates has previously been reported (Liu and Li, 2006; Liknes and Swanson, 2011; Swanson et al., 2014b). A larger heart fit to the cold acclimated phenotype in small birds. A larger heart presumably contributes to an increased ability of oxygen transport and more efficient transportation of metabolic nutrients and substrates required for shivering thermogenesis (Liknes and Swanson, 2011; Swanson et al., 2014a). The increase in relative heart size might therefore be an acclimation to increase the metabolic rate to endure increased demands of a colder conditions. A larger heart has previously been associated with increases in maximum metabolic output in birds (Swanson et al., 2014a).
5.3 Skeletal muscle plasticity
Comparisons between size matched Red Junglefowls bred indoor and outdoor showed a significant difference in relative pectoralis size (Figure 3). The relative pectoral size of these younger indoor fowls, 16 (1) days, were also statistically significantly smaller than for the older indoor fowls, 26 (3) days, age matched with the outdoor group. Nonetheless between the age matched indoor and outdoor groups, both groups being 26 days old, no significant differences were found when comparing relative size of the pectoral muscle (Figure 3).
The pectoralis major of Red Junglefowls is an anaerobic muscle responsible for upwards movement, lifting, of the wing. In other birds is pectoralis an aerobic muscle involved in shivering thermogenesis and flying, but Red Junglefowls are not a flying bird species (Petit and Vézina, 2014). Shivering thermogenesis primarily occur in aerobic muscles and secondarily in anaerobic muscles (Dietz et al., 1997). The lack of significant differences in relative size of the pectoral muscle of age matched fowls bred indoor and outdoor leads to the proposition that the pectoralis major is most likely not involved in shivering thermogenesis to that extinct that it provides to the cold acclimation in Red Junglefowls. The explanation of the differences in relative pectoralis between the age and size matched indoor groups might be as easy that the size matched fowls are younger, and therefore have had less muscular growth. The gastrocnemius maximus of Red Junglefowl is an oxidative skeletal muscle responsible for the upward rotation of the ankle and is involved in walking as well as standing. The gastrocnemius muscle is found to be particularly important in shivering thermogenesis in some Galliform chicks (Marjoniemi and Hohtala, 1999). Large gastrocnemius muscles therefore fit a cold acclimation phenotype for Red Junglefowl. Outdoor fowls showed a relative gastrocnemius size of almost twice as big as those for the indoor groups (Figure 4).
5.4 Blood parameters
Hematocrit is a measure of the volume percentage of erythrocytes, red blood cells, in the blood. Erythrocytes contain hemoglobin, which is the oxygen transporting agent in the circulatory system. Hemoglobin is also present directly solved in the blood plasma. Higher values of hematocrit and hemoglobin indicates a higher oxygen transport capacity which can be associated with a higher metabolic demand and are therefore to be expected
significantly higher hematocrit and hemoglobin concentration versus the indoor fowls (Figure 5).
5.5 Conclusions
Red Junglefowl bred outdoors during the Swedish fall (1.7-21.6 °C) show a cold acclimation phenotype including slower growth rate, larger oxidative muscles (gastrocnemius maximus), an improved oxygen transport by a relatively larger heart size, greater volume of blood erythrocytes and higher hemoglobin concentrations. All these physiological aspects are associated with a higher metabolic rate needed for shivering thermogenesis at cold temperatures.
Pectoralis major seems to not be involved in shivering thermogenesis to the extent that it leads to a cold acclimated phenotype in young Red Junglefowls.
5.6 Further studies
The experimental set up of this study makes it difficult to conclude if differences solely depends on living condition, indoor or outdoor, since the outdoor fowls were bred along with a mother hen, when the indoor fowls were not. To exclude the potential effects of maternal presence and care I suggest to breed the indoor fowls along with the hen as well. Excluding the hens completely is not an option when using this set up, since maternal care is essential for the survival of young Red Junglefowls at lower temperatures.
It is not necessary to include two different indoor groups, size and age matched, in further studies of Red Junglefowl since the physiological changes show no correlation with neither age nor body size. I recommend the usage of age matched groups for comparisons based on the results of the relative skeletal muscle sizes, which would make eventual differences of both aerobic and anaerobic skeletal muscles visible.
It would be interesting to include an ethological study of the Red Junglefowl, since there might be a difference in behavior and activity of fowls living indoor or outdoor. It would also be of interest follow the young fowls to mature adulthood and see if the differences are kept.
The advantages of a genomic analysis of which genes that are involved in the physiological mechanisms providing the cold acclimated phenotype, which can be compared with the athletic phenotype of athletes have been mentioned in section 2.3 Social aspects.
6 Acknowledgement
Thanks to Jordi Altimiras for his supervision and partition in animal euthanizing and collecting the blood data. Thanks to Jordi Altimiras, Yu-Ying Chang and Mikael Danielson for assistance with dissecting the outdoor bred animals. Finally, thanks to Andreas Calais for valid input on figures showed in this report.
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8 Appendix
Figure A1. Temperature variation measured at the outdoor aviaries at
Vreta (~58° 28′ N, 15° 31′ E) and at the closest measurement point of the meteorological and hydrological institute of Sweden (SMHI, Sveriges meteorologiska och hydrologiska institut,), in Malmslätt (~58° 40′ N, 15° 53′ E). The figure present temperatures measured from the earliest hatchling, September 10th, to the latest euthanization, October
15th. Temperatures measured at Vreta and Malmslätt are represented by a
solid and dashed line, respectively and corresponding average temperatures for the period of interest were 11.3 (3.6) °C at Vreta and 10.7 (3.8) °C at the measure point in Malmslätt.
