Institutionen för fysik, kemi och biologi
Examenarbete
Effects of postnatal stress on tonic immobility in White
Leghorn chicks (Gallus gallus domesticus).
Mia Persson
Examensarbetet utfört vid Linköping universitet
2010-06-01
LITH-IFM-G-EX--10/2336—SE
Linköpings universitet Institutionen för fysik, kemi och biologi 581 83 Linköping
Rapporttyp Report category Licentiatavhandling x Examensarbete C-uppsats D-uppsats Övrig rapport _______________ Språk Language Svenska/Swedish x Engelska/English ________________ Titel Title
Effects of postnatal stress on tonic immobility in White Leghorn chicks (Gallus gallus domesticus).
Författare Author Mia Persson
Sammanfattning Abstract
Early life stress is something that animals used in production often have to experience. What we do not know is if there are any consequences of this treatment later on in life. Zebra finches postnatal treated with the stress hormone corticosterone showed an exaggerated and prolonged stress response later on. To examine the effects of early life stress 77 White Leghorn chicks were used, half of them was stressed from postnatal day 1-14 and then tested between 47-63 days of age. The tonic immobility (TI) test is a commonly used test to evaluate the fearfulness and stress reaction in fowl. The chicks were placed on their back in a V-shaped wooden cradle and TI was induced by applying light pressure on the breast and neck. The number of inductions required to induce TI was recorded as well as the time until the first alert head movement and the total duration of the TI. The birds were tested in a calm environment but also after a stressful situation. There were no differences in the total duration of the TI reactions. However, stressed animals tended to need more induction attempts than the control animals. While looking at the time elapsed until the first head movement stressed chicks had a significantly lower duration. This indicates a dullness or shift in the stress response of the treated birds and there seem to be a more exaggerated response in the males.
ISBN ____________________________________________ ______ ISRN ____________________________________________ ______
Serietitel och serienummer ISSN Title of series, numbering
LITH-IFM-G-Ex—10/2336--SE
Nyckelord Keyword
Postnatal, stress, White Leghorn, tonic immobility, fowl.
Datum
Date 2010-06-01
URL för elektronisk version
Avdelning, Institution
Division, Department Avdelningen för biologi
Contents
1 Abstract ... 1
2 Introduction ... 2
3 Material and methods ... 3
3.1 Animals and housing ... 3
3.2 TI material ... 3 3.3 Method ... 4 3.4 Statistics ... 5 4 Results ... 5 4.1 Induction ... 5 4.2 First movement ... 7 4.3 Righting ... 10 5 Discussion ... 10 6 References ... 13 7 Appendix ... 15
1
1 Abstract
Early life stress is something that animals used in production often have to experience. What we do not know is if there are any consequences of this treatment later on in life. Zebra finches postnatal treated with the stress hormone corticosterone showed an exaggerated and prolonged stress response later on. To examine the effects of early life stress 77 White Leghorn chicks were used, half of them was stressed from postnatal day 1-14 and then tested between 47-63 days of age. The tonic immobility (TI) test is a commonly used test to evaluate the fearfulness and stress reaction in fowl. The chicks were placed on their back in a V-shaped wooden cradle and TI was induced by applying light pressure on the breast and neck. The number of inductions required to induce TI was recorded as well as the time until the first alert head movement and the total duration of the TI. The birds were tested in a calm environment but also after a stressful situation. There were no differences in the total duration of the TI reactions. However, stressed animals tended to need more induction attempts than the control animals. While looking at the time elapsed until the first head movement stressed chicks had a significantly lower duration. This indicates a dullness or shift in the stress response of the treated birds and there seem to be a more exaggerated response in the males.
1.1 Keywords
2
2 Introduction
Animals like pigs, cattle and fowl used in production are experiencing stressful situations early on in life due to for example early weaning, change of environment, change in diet and regrouping. This way of handling the animals at an early age may be motivated by an economical profit but the early stress might bring consequences later on, compromising the welfare and health of the animals. Recent research even suggests that Zebra finch chicks that is postnatal treated with the stress hormone CORT, corticosterone, show an exaggerated and extended response to stress at a higher age [1]. These findings show that early stress can lead to long-term physiological changes in stress handling. Another study made in rats where they were exposed to maternal deprivation at post natal day 9, showed physical and behavioural defects in the rats when examined at day 69-90 [2]. These animals compared to control animals had less body weight, increased anxiety-like disorders and elevated levels of plasma corticosterone. The maternal deprivation resulted in a long term modification in the HPA axis and also in the serotonergic activity that evidently signifying a relationship between stress early in life and the development of anxiety like behaviours later in life.
To understand the natural behaviour of the White Leghorn chicks, their ancestors, the red jungle fowl have been studied [3]. Naturally the fowl lives in small and relatively stable groups of several females, their chicks and one male. Each group got its own home range with their usual foraging areas and regular roosting sites and the group stays together for the different activities. The fowl is very social and communication both within and between groups is important to be able to keep up a stable group with its pecking order as well as defending the home area from other flocks. The group serves primarily as an important protection against predators and therefore the safety of the individual lies in the group. For this reason, the method used to cause postnatal stress in the chicks is by social isolation. To be left alone must be, according to the natural behaviour of the birds, a very frightening and therefore stressful situation for the chick. Tonic immobility (TI) is an antipredator response where the prey animal is ―death feigning‖ [4]. TI is known to occur in many vertebrate species like birds, lizards and rabbits [5]. This is an adaptive behaviour that may give the animal an opportunity to escape a predator when/if it gets less concentrated on its prey. It has also been shown that quails using TI are less likely to get predated by cats [3]. TI can also be induced by physical restraint and is commonly used as a method to estimate the fearfulness of an animal [5]. Behavioural tests have shown that quails that have a long duration on TI are more fearful than those with a shorter TI response [6]. The physiological changes that occur during the induction and duration of the TI response have been studied [5]. During the restrain period heart rate, blood pressure and the temperature of the legs increase while the core temperature drops. After the period of restraint the respiratory rate and heart rate decrease during the duration of TI. Stressful treatments like electric shocks or aversive handling prior to the tonic immobility test will increase the duration of it. These and other fear related treatments are known to have a positive correlation to the duration of TI. Therefore we can use the duration of the TI response and the number of attempts it takes to induce it as a display of how fearful an animal is when exposed to a stressful situation.
The aim of the present study is to investigate whether postnatal stress in chickens will influence their tonic immobility response later in life but still at an early age compared to birds reared in a less stressful environment.
3
3 Material and methods 3.1 Animals and housing
The chickens used were of the breed White Leghorn HyLine white. In total 80 animals were bred for the test but three of them could not be used and therefore the final amount of animals used were 77. Out of these there were 37 control animals and 40 animals that had been stressed. The birds were held as a single group except during the days when the testing took place, since it eased the sorting of the animals already tested from those not yet tested to split the group into two parts. The housing boxes during the first set of tests were 3x1.5m split into 1.5x1.5m for each group. During the second and third set of test the housing box were 3x3m and split into 3x1.5m for each part of the group. As the animals were mixed the observers did not know which chickens that had been treated and which that were control animals. The sex ratio was 32 hens to 45 roosters. If separated by sex and treatment there were 13 stressed hens, 19 control hens, 21 stressed roosters and 24 control roosters. To identify the individuals the chickens were given a wing clip with an identification number.
3.2 TI material
The tonic immobility tests took place in two separate rooms with subdued light and with as little disturbance from sound as possible. In each testing room there was a table with a V-shaped wooden cradle placed upon it (Figure 1) (cradles have in an earlier comparison to cloth and table to require fewer inductions of TI [7]. Two cradles were used, one for each experimenter. The cradles were, as mentioned, V-shaped with a 90 degree angle. The distance for the head of the chicken to hang before it reached the table was 2 cm and the length of the cradles was 23 cm. The observer were standing still 60-70 cm from the cradle. Stop-watches of the brand Rucanor were used. Also two cardboard boxes measured to 576x346x407 mm were used to put the birds into prior to the TI-test and it was also these boxes that were used during the transportation of the animals to their new environment.
Figure 1. A chick in tonic immobility, laying on its back in a V-shaped wooden cradle with its head hanging off the edge of the cradle.
4
3.3 Method
The stress treated chicks were stressed for one hour every day starting on postnatal day 4, two hours each day during the upcoming week and for three hours every day the third week. The method used to stress the chickens was social isolation where each chick was picked out of the group, and placed in a box by itself. During the social isolation the chick were deprived from food and water and were also exposed to a lower temperature then in the housing boxes. The control animals were also picked out of the group but placed into boxes with other control chicks in it. This way the treated and non-treated animals were handled the same amount of time.
Prior to the tonic immobility test, two test animals were caught by the same experimenter and placed into each of the cardboard boxes in the separate rooms. While catching the birds the light was turned off so that as little stress as possible was caused to the animals. They were held in the closed boxes for 3 min before the TI-test. A maximum of three inductions of TI were used and each induction had the duration of 10 sec. The chicken was placed on its back in the cradle with its head hanging off the edge of it and the testing person applied pressure on the chest with the right hand while holding down the head of the bird with the left hand. After the duration of 10 sec, the testing person gently removed the hands from the animal and took a step back from the table. The time of the first head movement was recorded and defined as the first conscious movement of the head. Also the time until righting, when they had fully left the state of tonic immobility and were standing up, was measured. The maximum time before TI was manually terminated was set to 10 min. When the TI test ended the animals were given plastic leg rings so that the tested animals could be told from the not yet tested. After testing and marking a bird, it was introduced back into the group. The same method was applied to all TI-tests made.
Three sets of tonic immobility tests were made. First a test where the animals were considered to be unstressed, secondly a test where the animals had been stressed and last a test where the birds were considered as being unstressed again. The first one was done on all animals at the location where they had been hatched and hold from the day of the hatching. This was considered as a non-stressful environment and the animals were not stressed prior to the TI tests, except for a group of 28 individuals that had been moved to a new environment on the second day, for a couple of hours, and then reintroduced to the group. These animals were tested on the third day and were regarded as stressed the day before testing. These three days of testing were done when the animals were at the age of 47, 48 and 49 days. The TI testing method follows the exact pattern of what was previously explained.
For the second set of tonic immobility tests all animals were transported in cardboard boxes, by car, to a different location where they were introduced into a new box. This was regarded as a stressful experience for the birds and the TI-tests were made during the first, second and third day after the introduction into the new environment. All birds were tested. At these three days of testing the animals were at the age of 56, 57 and 58 days. The TI testing method follows the exact pattern of what was previously explained.
The third set of tonic immobility tests were taking place the following week after the introduction of the birds into their new environment. At this point the animals were considered as unstressed since they had had time to get used to the new environment. The tests were split between 2 days and the age of the animals was 62 and 63 days. Due to logistical complications, the box could not be divided into two parts. Therefore only 40 out of 77 animals were caught and tested before it
5
was too hard to find untested individuals without causing too much stress to the other animals. The TI testing method follows the exact pattern of what was previously explained.
3.4 Statistics
The collected data was inserted and arranged in Excel 2007. To analyze the data STATISTICA 9 Trial Version were used. All of the measured variables were analyzed with one-way ANOVA to assure that there were no influences on the data from the fact that there were two different observers performing the tonic immobility tests. To get an overview of the data a t-test, independent sorted by group, were made and presented in box plots. Further analyzation of the data was done using repeated measures ANOVA. This method were used to investigate if there were any differences between treatments and/or sex in the numbers of inductions required, the time until the first movement and the time until rightning. Also a general linear model (GLM) were used to analyze the data from the second set of tonic immobility tests to see if there were any differences between the three days after the introduction into the new box. GLM were further used to separately study the data from the third set of TI-tests since there were only 40 individuals observed.
4 Results 4.1 Induction
The number of inductions required inducing tonic immobility in the first, second and third set of TI-tests are presented in box plots below in Figure 2 and in Figure 10-11 in the appendix. These graphs show a t-test comparison between treatments. Using the one-way ANOVA, no statistical significant differences were found between the control animals and the stressed ones, but a trend can be seen (p<0.1) in Figure 2 where stressed birds tend to require a slightly higher number of TI-inductions to enter TI. In Figure 12-14 in the appendix show box plots of a t-test comparison between the numbers of inductions required to induce tonic immobility and sex is presented. Analyzation with one-way ANOVA show no statistical significant difference between males and females (p>0.05).
6
Figure 2. Box & Whisker Plot from t-test of the numbers of inductions needed to induce tonic immobility in the first set of tests. The comparison is made between the control group and stress group of animals. There is no statistical significant difference between the groups, though a trend can be noticed where the stressed animals tend to need an increased number of inductions, 0.1>p>0.05 and 0.95 confidence intervals.
Below, in Figure 3, are the results from repeated measurements ANOVA showing a trend (0.1>p>0.05) in difference between treatments in all three sets of tonic immobility tests. The next graph, Figure 4, is from the same test showing a statistical significant difference in the required number of inductions to induce TI between the third set and the other two sets of TI-tests (p<0.05)
Figure 3. Number of inductions required to induce TI in stressed (S) and non-stressed (C) chickens. There is not a statistical significant difference between treatments but there is a trend showing that stressed animals require an increased number of attempts to enter tonic immobility, 0.1>p>0.05 and 0.95 confidence intervals.
Mean Mean±SE Mean±1,96*SE C S Treatment 0,95 1,00 1,05 1,10 1,15 1,20 1,25 1,30 1,35 1,40 N u m b e r o f i n d u ct io n s C S Treatment 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 N umber of induc tio ns
7
Figure 4. Comparison between the three different sets of the numbers of inductions needed to induce TI. There is a statistical significant difference between the third sets and the other two sets, p<0.05 and 0.95 confidence intervals.
4.2 First movement
Box plots of time until the first alert head movement are presented below in Figure 5 and in Figure 15-16 in the appendix. These represent the outcome of a t-test comparison between treatments. There is no significant difference between treatments, though a trend can be seen in Figure 5 where stressed animals tend to have a shorter time until their first conscious head movement during tonic immobility than control animals (p<0.1).
Figure 5. Box & Whisker Plot from t-test of the recorded time until first alert head movement during tonic immobility in the third set of tests. The comparison is made between the control and stress group. There is no statistical significant difference but a trend can be noticed, 0.1>p>0.05 and 0.95 confidence intervals.
Induc.1 Induc. 2 Induc. 3
Sets 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 N um ber of indu cti ons Mean Mean±SE Mean±1,96*SE C S Treatment 20 40 60 80 100 120 140 160 180 200 220 240 260 280 F ir st m o ve m e n t (s )
8
Figure 6 below and Figure 17-18 in the appendix show box plots of the first alert head movements. These graphs represent the t-test results of a comparison between males and females. No statistical significant difference were shown by the test, however in Figure 13 a trend can be noticed where males tend to lay still for a longer time than females (0.1>p>0.05).
Mean Mean±SE Mean±1,96*SE F M Sex 100 120 140 160 180 200 220 240 260 280 300 320 340 Fi rs t m o v e m e n t (s )
Figure 6. Box & Whisker Plot from t-test of the recorded time until first alert head movement during tonic immobility in the second set of tests. The comparison is made between females and males. There is no statistical significant difference, however a trend can be noticed, 0.1>p>0.05 and 0.95 confidence intervals.
The results from analyzing the data recorded, of the time until the first alert head movement, with repeated measurements ANOVA is presented in Figure 7 and 8 below. Figure 7 show a statistical significant difference between treatments where stressed animals move their head earlier than control animals (p<0.05). In Figure 8 a comparison between both sexes and treatments has been made. Here we see a statistical significant difference between sexes and treatments where control males lay still for a longer time then stressed males (p<0.05). When analyzing whether there was any effect on the data by the two different experimenters a significant difference were found in the third set. However, when analyzed with repeated measurements ANOVA and compared to both sex and treatment the effect from the experimenters were not significant.
9
Figure 7. Time until the first alert head movement in stressed (S) and non-stressed (C) chickens. There is a statistical significant difference between treatments where stressed animals lay still for a shorter time, before the first head movement, than the control animals, p<0.05 and 0.95 confidence intervals.
Figure 8. Time until the first alert head movement in stressed (S) and non-stressed (C) chickens comparison between males (m) and females (f). There is a statistical significant difference between treatment and sexes where control males lay still for a longer time than stresses males, p<0.05 and 0.95 confidence intervals.
10
4.3 Righting
The recorded time until righting is presented in box plots in Figure 19-21 in the appendix. These graphs show the t-test comparison of tonic immobility duration between the control group and the stress group of animals. There is no statistical significant differences between treatments (p>0.05). Figure 22-24 presented in the appendix are box plots from t-test comparing the recorded time until righting between males and females. There is no statistical significant differences (p>0.05). Presented in Figure 9 is the repeated measurements ANOVA from all three sets of righting compared against treatment, sex and sets. There is a statistical significant difference between righting 3 and righting 1 and 2, where the animals had a shorter duration if tonic immobility in the third set of tests (p<0.05).
Right. 1 Right. 2 Right. 3
Sets 0 50 100 150 200 250 300 350 400 450 R ig h ti n g ( s )
Figure 9. Comparison between the three different sets of the time until righting. There is a statistical significant difference between the third set and the other two sets, p<0.05 and 0.95 confidence intervals.
5 Discussion
The results show that stressed chicks tend to require a higher number of inductions to enter tonic immobility. When they are in TI the stressed animals have a shorter duration of time until the first alert head movement. The largest difference is between control and stressed males where stressed males have a shorter duration until the first alert head movement than control males. If comparing the time between sexes, males tend to have a longer duration until the first alert head movement than females. There were no significant differences found in the total duration of TI (righting). Though, looking at the difference between the separate sets of testing the animals in the third require a higher number of inductions then in the previous sets and also have a shorter duration of TI than in the first two sets.
11
The tonic immobility test is a commonly used method to evaluate fearfulness in fowl [4]. Many tests have previously shown that fearful and stressful situations like aversive handling prior to the TI induction will increase the duration of the tonic immobility (time until righting) [8] and also increase the susceptibility to the induction of TI [4]. On the other hand, regular handling seems to decrease the susceptibility and the duration of TI [9]. Our results show that the stressed animals tended to have a lower susceptibility to TI than the control birds even though it is not significant. This could indicate that the stressed chicks are less fearful than the control animals which are contradictory to what could be expected from the results of previous studies of stress and TI [10, 11, 12]. Only the third set differed from the other two sets, both in inductions and the time until righting. This might indicate that the birds were stressed in the housing during the first set of tests, or the birds did not get stressed enough from the environmental changes that came from the change of housing environment. What yet is unknown is whether the age difference between the first and the third set of tests might play a role in the differences between the two sets. The most important sets to compare could be the second and third since there was some more activity going on around the birds during and before the first set of testing, but between the second and third set of tests the animals were left alone to get used to the new housing environment. At least the significant differences between the second and third sets indicate that there probably was a difference in how the animals perceived the situations.
The results of the time until the first alert head movement showed that the stressed birds had a shorter duration until the first movement. Males tended to have a longer duration than females which could indicate that males were more fearful than females. The comparison of all three sets showed a significant difference between treatments where the stressed animals had a shorter duration of time before the first head movement. This, similar to the induction results, indicated that the control animals are more fearful than the stress treated ones. There is also a difference between stressed males and control males where stressed males have a shorter duration before the first movement. No differences were found between females of the different treatments. These results could signify that males are more sensitive to this kind of stress early on in life than females and that their ability to handle stressful situation later in life get affected. These long-term effects on the fear response might be explained by a shift in the HPA axis response to fear or stress, as previously mentioned, in a study made on rats [2]. We can also hypothesize that the early stress on the treated animals creates a dullness in their fear response, making them more resistant to stress and therefore require a higher amount of inductions and have a shorter duration until the first alert head movement.
A study have been made with quail chicks that in several generations have been selected for high stress reaction (exaggerated plasma corticosterone levels as a response to stress), that are more susceptible to tonic immobility inductions (fewer attempts needed) than quail selected for a low stress reaction [10]. These high stress birds are thought to be more fearful than the low stress birds and also react with higher concentrations of plasma corticosterone which is explained by an elevated adrenocortical activity. This study also shows that low stress birds have a shorter duration until the first head movement than the high stress line. These findings are contradictory to our study where the stressed birds actually needed more TI inductions and also had a shorter duration to the first head movement. The first alert head movement is thought to be the one of the first actions the animal make before righting itself and the purpose of the behaviour may be to investigate whether the surroundings are threatening or not [10]. It has been proposed that the low stress chicks perceive the experimenter as less of a threat than high stress chicks do and
12
therefore low stress chicks were expected to have had a shorter duration of TI than high stress chicks. If that could be an explanation then why would our stressed birds perceive the experimenters and the procedure as less threatening then the control animals do? However, the results of the TI duration, in the case with the quails, did not show a statistical significant difference like it did with the number of inductions and the time to first head movement. Another study have had an increase in susceptibility, increase in first head movement duration and an increased duration of TI in high stressed quails in comparison to low stressed quails [11]. Another study comparing the TI reaction between White Leghorn and the red jungle fowl, only had a difference in induction attempts [12]. The pattern we can see is that the birds in the studies often differ in induction attempts even if the duration to the first head movement and the total duration of the TI are not significant. There is also a study with contradictive results that only had a difference in the total duration of TI and not in the other variables [13]. This study was made on offspring of stressed hens. Are we focusing on the wrong variable when concentrating on the total duration of TI? That might be the case since the differences between the TI reactions only were found in the number of inductions and the time until the first head movement. The results may also have been affected by the fact the gaze of the experimenter was not standardized and a previous study have shown that it might have an effect on the TI duration [14].
Another study done on quails is investigating the changes in heart rate during tonic immobility [5]. They saw, amongst other things, that the heart rate of the bird increased during the induction of TI and then decreased during the duration of TI. The conclusion was that the susceptibility to TI could not be explained by autonomic reflex changes alone, but also related to the perception of the test by the quail. They also concluded that the physiological response they saw during TI, were closely related to the physiological changes that occurred during the induction (shift towards sympathetic dominance). What they could not answer was if the physiological changes were what caused the tonic immobility reaction or if the increase in sympathetic dominance was a result from the handling of the bird during the induction attempts. An additional study made on quails show that the duration of TI is not related to the corticosterone levels in the birds [6]. However the birds that had a short duration of TI also had higher levels of corticosterone. The conclusion was on the other hand that the increase of corticosterone during the induction of TI was due to the handling of the birds. Maybe the results from the previously mentioned study can explain the differences in the corticosterone levels as the difference in how the birds perceive the situation of being restrained.
In conclusion, the differences seen in the present study may be explained by a mixture of physiological and behavioural means. There might be a dullness in the fear response of the chicks, that affect their perceiving of the experimenter and the test. They might see it as less of a threat and therefore the duration until the first head movement was shorter. These results may also/instead be the outcome of a shift in the HPA axis or indicate that the stressed birds are more resistant to stress later in life. We also saw that males might be more affected by this kind of treatment then females. It would be interesting to study the effects of this early life stress on physiological mechanisms like heart rate, blood pressure and hormone levels and it might also be interesting to perform TI tests directly after stressing each animal.
13
6 References
1. Spencer K.A., Evans N.P., Monaghan P. 2009. Postnatal stress in birds: a novel model of glucocorticoid programming of the hypothalamic-pituitary-adrenal axis. Endocrinology. v. 150; Apr 2009; p. 1931-1934.
2. Rentesi, G., Antoniou, K., Marselos, M., Fotopoulos, A., Jihad, A., Konstandi, M. 2010. Long-term consequences of maternal deprivation in serotonergic activity and HPA function in adult rat. Neurosci Lett; 2010 May 5. [Epub ahead of print]
3. Appleby, M. C., Mench, J. A., Hughes, B. O.; Poultry behaviour and welfare; 2004, CABI Publishing, Wallington, UK; p. 71-77.
4. Forkman B., Boissy A., Meunier-Salaün M.-C., E. Canali E. and Jones R.B. 2007. A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology & Behavior. v. 92; 22 Oct 2007; p. 340-374.
5. Valance, D., Després, G., Richard, S., Constantin, P., Mignon-Grasteau, S., Leman, S.,
Boissy, A., Faure, J. M., Leterrier, C. 2007. Changes in Heart Rate Variability during a
tonic immobility test in quail. Physiol Behav. v. 27; 2008 Feb; p. 512-20. Epub 2007 Oct 25. 6. Hazard D., Couty M, S., Guémené R. and D. 2007. Intensity and duration of
corticosterone response to stressful situations in Japanese quail divergently selected for tonic immobility. General and Comparative Endocrinology, v. 155; Jan 2008; p 288-297. 7. Jones, R. B., Faure, J. M. 1981. Sex and strain comparisons of tonic immobility (―righting
time‖) in the domestic fowl and the effects of various methods of induction. Behav Processes; v. 6; 1981; p. 47–55.
8. Marin, R. H., Freytes, P., Guzman, D., Jones, R. B. 2000. Effects of an acute stressor on fear and on the social reinstatement responses of domestic chicks to cagement and strangers; Applied Animal Behaviour Science; v. 71; Feb 2001; p. 57-66.
9. Jones, R. B., Faure, J. M. 1981. The effects of regular handling on fear responses in the domestic chick. Behav Processes; v. 6; 1981; p.135–48.
10. Davis, K. A., Schmidt, J. B., Doescher, R. M., Satterlee, D. G. 2008. Fear responses of offspring from divergent quail stress response line hens treated with corticosterone during egg formation. Poult Sci. v. 87; 2008 Jul; p. 1303–1313.
11. Jones, R. B., Satterlee, D. G., Ryder, F. H. 1992. Fear and distress in Japanese quail chicks of two lines genetically selected for low or high adrenocortical response to immobilization stress. Horm Behav; v. 26; 1992 Sep; p. 385-393.
12. Schütz, K. E., Forkman, B., Jensen, P. 2001. Domestication effects on foraging strategy, social behaviour and different fear responses: a comparison between the red junglefowl
14
(Gallus gallus) and a modern layer strain; Applied Animal Behaviour Science; v. 74; Sep 2001; p. 1-14.
13. Janczak, A. M., Torjesen, P., Palme, R., Bakken, M. 2007. Effects of stress in hens on the behaviour of their offspring; Applied Animal Behaviour Science; v. 107; Oct 2007; p. 66-77.
14. Gallup, Jr. G. G., Cummings, W. H., Nash, R. F. 1972. The experimenter as an
independent variable in studies of animal hypnosis in chickens (Gallus gallus). Anim Behav; v. 20; 1972; p. 166–9.
15 7 Appendix Mean Mean±SE Mean±1,96*SE C S Treatment 0,9 1,0 1,1 1,2 1,3 1,4 1,5 1,6 N u m b e r o f in d u cti o n s
Figure 10. Box & Whisker Plot from t-test of the numbers of inductions needed to induce tonic immobility in the second set of tests. The comparison is made between the control group and stress group of animals. There is no statistical significant difference between the groups, p>0.05 and 0.95 confidence intervals.
Mean Mean±SE Mean±1,96*SE C S Treatment 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 2,8 N u m b e r o f in d u c ti o n s
Figure 11. Box & Whisker Plot from t-test of the numbers of inductions needed to induce tonic immobility in the third set of tests. The comparison is made between the control group and stress group of animals. There is no statistical significant difference between the groups, p>0.05 and 0.95 confidence intervals.
16 Mean Mean±SE Mean±1,96*SE F M Sex 0,95 1,00 1,05 1,10 1,15 1,20 1,25 1,30 N u m b e r o f in d u c ti o n s
Figure 12. Box & Whisker Plot from t-test of the numbers of inductions needed to induce tonic immobility in the first set of tests. The comparison is made between females and males. There is no statistical significant difference between sexes, p>0.05 and 0.95 confidence intervals.
Mean Mean±SE Mean±1,96*SE F M Sex 0,95 1,00 1,05 1,10 1,15 1,20 1,25 1,30 1,35 1,40 1,45 N u m b e r o f in d u ct io n s
Figure 13. Box & Whisker Plot from t-test of the numbers of inductions needed to induce tonic immobility in the second set of tests. The comparison is made between females and males. There is no statistical significant difference between sexes, p>0.05 and 0.95 confidence intervals.
17 Mean Mean±SE Mean±1,96*SE F M Sex 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 2,8 N u m b e r o f in d u c ti o n s
Figure 14. Box & Whisker Plot from t-test of the numbers of inductions needed to induce tonic immobility in the third set of tests. The comparison is made between females and males. There is no statistical significant difference between sexes, p>0.05 and 0.95 confidence intervals.
Mean Mean±SE Mean±1,96*SE C S Treatment 100 120 140 160 180 200 220 240 260 280 F irst m o ve m e n t (s)
Figure 15. Box & Whisker Plot from t-test of the recorded time until first alert head movement during tonic immobility in the first set of tests. The comparison is made between the control and stress group. There is no statistical significant difference, p>0.05 and 0.95 confidence intervals.
18 Mean Mean±SE Mean±1,96*SE C S Treatment 100 120 140 160 180 200 220 240 260 280 300 320 340 F ir s t m o v e m e n t (s )
Figure 16. Box & Whisker Plot from t-test of the recorded time until first alert head movement during tonic immobility in the second set of tests. The comparison is made between the control and stress group. There is no statistical significant difference, p>0.05 and 0.95 confidence intervals. Mean Mean±SE Mean±1,96*SE F M Sex 100 120 140 160 180 200 220 240 260 F ir s t m o v e m e n t (s )
Figure 17. Box & Whisker Plot from t-test of the recorded time until first alert head movement during tonic immobility in the first set of tests. The comparison is made between females and males. There is no statistical significant difference, p>0.05 and 0.95 confidence intervals.
19 Mean Mean±SE Mean±1,96*SE F M Sex 20 40 60 80 100 120 140 160 180 200 220 240 260 F ir s t m o v e m e n t (s )
Figure 18. Box & Whisker Plot from t-test of the recorded time until first alert head movement during tonic immobility in the third set of tests. The comparison is made between females and males. There is no statistical significant difference, p>0.05 and 0.95 confidence intervals.
Mean Mean±SE Mean±1,96*SE C S Treatment 300 320 340 360 380 400 420 440 460 R ig h ti n g (s)
Figure 19. Box & Whisker Plot from t-test of the recorded time until rightning, the end of tonic immobility, in the first set of tests. The comparison is made between the control and stress group. There is no statistical significant difference, p>0.05 and 0.95 confidence intervals.
20 Mean Mean±SE Mean±1,96*SE C S Treatment 260 280 300 320 340 360 380 400 420 440 460 480 R ig h ti n g ( s )
Figure 20. Box & Whisker Plot from t-test of the recorded time until rightning, the end of tonic immobility, in the second set of tests. The comparison is made between the control and stress group. There is no statistical significant difference, p>0.05 and 0.95 confidence intervals.
Mean Mean±SE Mean±1,96*SE C S Treatment 80 100 120 140 160 180 200 220 240 260 280 300 320 340 R ig h ti n g ( s )
Figure 21. Box & Whisker Plot from t-test of the recorded time until rightning, the end of tonic immobility, in the third set of tests. The comparison is made between the control and stress group. There is no statistical significant difference, p>0.05 and 0.95 confidence intervals.
21 Mean Mean±SE Mean±1,96*SE F M Sex 280 300 320 340 360 380 400 420 440 460 480 R ig h ti n g ( s )
Figure 22. Box & Whisker Plot from t-test of the recorded time until rightning, the end of tonic immobility, in the first set of tests. The comparison is made between females and males. There is no statistical significant difference between sexes, p>0.05 and 0.95 confidence intervals.
Mean Mean±SE Mean±1,96*SE F M Sex 240 260 280 300 320 340 360 380 400 420 440 460 480 R ig h ti n g ( s )
Figure 23. Box & Whisker Plot from t-test of the recorded time until rightning, the end of tonic immobility, in the second set of tests. The comparison is made between females and males. There is no statistical significant difference, p>0.05 and 0.95 confidence intervals.
22 Mean Mean±SE Mean±1,96*SE F M Sex 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 R ig h ti n g (s )
Figure 24. Box & Whisker Plot from t-test of the recorded time until rightning, the end of tonic immobility, in the third set of tests. The comparison is made between females and males. There is no statistical significant difference, p>0.05 and 0.95 confidence intervals.
