Heart Rate Response to Light in the Embryo of the Japanese Quail (Coturnix coturnix) ELIZABETH M. OCKLEFORD AND MARGARET A. VINCE Psychological Laboratory, Downing Street, Cambridge CB2 3EB
ABSTRACT The effects of exposure to light were investigated during and shortly after the period of light stimulation. A particular objective was to evaluate any responses to the type of illumination conditions generally used for observation of avian embryos. Twenty 16-day Japanese quail embryos were illuminated for 15 minutes each during a total recording period of 55 minutes. Heart rate, respiration rate and an approximate measure of activity were recorded. Mean heart rate was found to be higher during stimulation than before and did not return to the prestimulation level during the recording period after stimulation ceased. The lack of an immediate heart rate response to the onset of stimulation separates the effects of light from those of touch, rotation, odours, tastes and maternal calls. The possibility that light may act to stimulate accelerated development is discussed. A simple technique used in studies of the behaviour of avian embryos involves the removal of part of the shell to allow direct observation or manipulation of the embryo. This technique was first described by Kuo (‘32) and has been used routinely by many working in this field (for example Orr and Windle, ’34; Hamburger and Oppenheim, ’67; Balaban and Hill, ’69; Oppenheim et al., ’73; Vince et al., ’76). Eggs are maintained in an observation incubator and often brightly illuminated with a heat filtered light to facilitate observation. A behavioural response to light was reported by Bursian (‘64) who found intense illumination increased motility in embryos of the domestic fowl as early as four days of i n cubation and that solitary movements were grouped on exposure to light earlier than otherwise. This effect could probably not have been mediated by the specific light sensitive receptors of the visual system since this system is immature a t this stage of development. Increased motility was also found in 11.5-day pigeon embryos in response to light and was attributed to a dermal sensitivity (Heaton and Harth, ’74). This altricial species hatches after about 17.5 days and the visual system does not appear to function until about day 15. Responses of the developing visual system to light have also been studied. Peters et al. (‘58) recorded evoked potentials in optic lobe J. EXP. ZOOL., 201: 439-444.
of precocial domestic fowl embryos and also first found electroretinograms about day 18. More recently Heaton (‘73) has shown that Japanese quail (also a precocial species and the one used in this investigation) showed a pupillary constriction reflex reliably on day 11.5 of incubation, indicating that a t least retina, optic nerve and the pretectal central visual areas are functional several days before hatching occurs on day 17. Heaton points out that the sunlight intensity reaching quail embryos within the shell is sufficient to elicit a visually mediated reflex. This reinforces the suggestion made by Impekoven and Gold (‘73) that light, associated with other sensory stimulation, during absence of the incubating parent bird may be important for development. Two previous studies of responsiveness of late stage avian embryos to light have produced conflicting results. Kuo (‘32) observed that 17-day domestic fowl embryos opened and closed their eyes in response to itlumination but did not show any altered pattern of movements of other parts of the body whereas Oppenheim (‘68) found beak clapping increased (a typical response to stimulation a t this stage) in chick and duck embryos exposed to one minute of intense light stimulation. Beak clapping also increased sometimes when stimulation ceased. The present experiment was designed to in-
439
440
ELIZABETH M. OCKLEFORD AND MARGARET A. VINCE
vestigate further the immediate effects of short term exposure to light. There is a possibility which is important when embryos are observed directly, that the illumination used to facilitate observation may affect the activity measured. The experiment to be described attempted to outline possible artifacts in such experimental situations. The conditions of illumination were chosen so as to approximate those which could occur in the wild. However other features of the environment such as temperature and egg position, which would be altered in association with onset and offset of illumination in the wild, were kept constant. MATERIALS AND METHODS
Japanese quail (Coturnix coturnixl eggs were incubated in a forced draught “Westernette” incubator a t 37.8”C for 13 days. On the fourteenth day of incubation they were transfered to a “Curfew” observation incubator (expected day of hatching, day 17). On day 15 the position of the air space and the embryo within the shell were identified by candling and three holes each approximately 2 mm in diameter were drilled in the shell in preparation for the insertion of electrodes and a pressure recording device. On day 16, silver wire electrodes were inserted into the egg for recording the EKG and
EMG (Laughlin et al., ’75;Tolhurst and Vince, ’76). In order to measure embryonic movements the air space of the egg was joined to a Mercury electronics modified M8 pressure transducer by means of a polypropylene tube (Dawes, ’76). The shell over part of the airspace was removed and the opening resealed with cellophane glued in place with a ring of Bostic number 1 clear adhesive (Vince, ’77). This preparation is illustrated in figure 1.The egg was then placed on a tripod of sound transducers (Vince and Salter, ’67) in the observation incubator. The egg was undisturbed for a t least one hour before any measurements were made.
Light stimulation An olympus microscope light with a heat absorbing filter was held approximately 25 cm above the egg. Background illumination was dim. A stimulation consisted of the microscope light being turned up manually (silently) until the light intensity a t the surface of the egg was approximately 0.089 lumens cm-2 as measured with an SEI photometer. Recording After the acclimatisation period, recordings of EKG, EMG, respiratory and other movements (pressure changes) and sound were
tube to pressure transducer
cellophane window electrode over neck
outline of airspace
electrode over R. tarsal joint
Fig. 1 Diagram of egg showing electrode positions, pressure recording tube and window cut in shell over airspace and resealed with cellophane.
44 1
EMBRYONIC RESPONSE TO LIGHT
tinued during 15minutes of illumination with the stimulus light. Measurements of the air temperature in the incubator under a cellophane film were also made over a similar period. RESULTS
temperature probe through hole drilled in shell
Fig. 2 Diagram showing position of temperature probe in relation to embryo one day before hatching. The temperature probe is gently pushed through the slit the embryo has made in the membranes.
made on a “Mingograf”’recorder. Recordings lasted for 55 minutes during which a 15minute period of stimulation was given. The time of onset of the stimulation within the 55minute recording period was randomised with the constraints that there were a minimum of 13-minuterecording before stimulation began and 3-minute recording after the end of stimulation. Notes were made throughout the recording period of the nature of any sounds produced by the embryo and of when the incubator heater switched on and off. The position of the embryo was noted after recording had finished, particularly whether the right eye was clearly visible through the cellophane window or whether i t was obscured by the right wing. Heart rate and respiration rate were scored by inspection of the Mingograf paper records. Temperature measurements Control temperature measurements were made using a Jenway platinum resistance probe inserted into the opened egg as shown in figure 2. Temperature measurement was con-
1. Heart rate Mean heart rates in beats per minute for each individual before, during and after stimulation are given in table 1. Figure 3 shows differences in mean heart rates during and after stimulation from means before stimulation. Mean heart rate during stimulation was significantly higher than before stimulation began (Wilcoxon matched pairs signed ranks test T=15, p < 0.01). Mean heart rate after stimulation was turned off was also higher than the prestimulation level (Wilcoxon test T=27, p < 0.01). There was no significant difference between mean heart rates during and after stimulation. No immediate effects of light stimulation on heart rate were found when rates during the minute or part of one minute (the smallest period considered was that occupied by 10 beats) immediately prior to or immediately following the onset of stimulation were compared. Similarly no immediate “off” effects were found. 2. Respiration rate
Mean respiration rates in breaths per minute before, during and after stimulation are given in table 1 for the 16 eggs from which pressure records were obtained. There were no significant differences in mean respiration rates between the different periods. Figure 4 shows differences in mean respiration rates during and after stimulation from mean rates before stimulation. 3. Movements Very few movements other than breathing movements were detected in any of the subjects with the methods used. 4. Temperature Internal egg temperature fluctuated between 36.5”C and 38’C. No differences in the maximum temperature attained by the egg or the period of incubator temperature fluctuations were recorded with the light on. Similarly no differences in air temperature were recorded. DISCUSSION
The results show that mean heart rate of
442
ELIZABETH M. OCKLEFORD AND MARGARET A. VINCE TABLE 1
Mean heart rates in beats per minute and respiration rates (Resp) in breaths per minute for individual embryos before, during and after stimulation with light Before Egg
After
During
Heart rate
Resp
Heart rate
Resp
Heart rate
Resp
~~
1
2 3 4 5 6
7 8 9 10 11 12 13 14 15 16 17 18 19 20 Grand mean
n
M
13 26 34 16 36 15 36 27 37
418 330 397 286 329 418 427 391 298 358 417 366 360 356 403 325 288 312 381 383
35 37 30 17 36 18 26 21 13 24 18
362
S
D
18 15 11 11 17 15 16 17 8 1
8 9 12 15 18 14 9 9 26 12
M
S
D
133 36 29
13 13 15
38 42 68 39 8 22 90
8 16 7 6 4 3 9
71 39 53
3 5 10
18 42 62
6 10 9
n
M
15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15
440 342 406 289 349 425 430 410 315 366 436 364 369 368 402 329 285 306 403 388
15
49
15 15 15
371
S
D
12 20 15 11 15 13 14 14 6 9 13 16 24 18 17 14 13 9 25 17
M
S
D
145 43 12
7 14 3
36 21 52 33 9 19 96
8 14 6 4 1 6 7
71 32 53
10
17 18 63
6 1 10
45
4 3
n
M
27 14 6 24 4 25 4 13 3 5 3 10 23 4 22 14 19 27 16 22
452 334 408 281 352 433 433 421 304 364 429 366 372 365 396 336 283 307 371 383 370
S
D
11 15 17 15 19 18 20 16 18 17 3 17 19 18 5 9 9 8 9 18
M
S
D
132 36 11
5 13 13
67 13 59 33 8 17 104
17 4 7 5 4 2 10
70 30 64
9 1 14
13 13 65
7 3 13
46
n is the number of minutes of recording included in each mean value Bven Standard deviations from the mean are also shown
avian embryos can be increased during illumination. An increase in heart rate would be expected if the light source were contributing heat but temperature measurements showed that this was not occuring in this case. The lack of any immediate heart rate response to the light stimulus separates the effect of this form of stimulation from some stimuli in other modalities. Heart rate responds by a brief increase to olfactory stimulation (Tolhurst and Vince, '76), taste (Vince, '77), rotation (Vince, Clark and Reader, unpublished observations) and maternal calls (Gottlieb, '71). Heart rate is known to rise markedly during the last 24 hours before hatching in the domestic fowl (Laughlin et al., '76) and in the quail (unpublished observations). In the experiment described here, mean heart rate remained higher during the period of recording after stimulation ceased than it had been before any light stimulus was given. Heart rate was not however higher during the second half of the prestimulation recording than the first half. Nor was it higher after the end of stimulation than during the stimulation period. These findings prompt the suggestion that stimulation may have brought forward or triggered the prehatching period of de-
velopment where the heart rate is increasing. Several experiments investigating the longer term effects of light on the embryo have pointed to the possibility that light may act as an accelerating stimulus by bringing forward hatching time. Of particular interest is the finding that hatching time was brought forward after brief periods of stimulation with light on the seventeenth or nineteenth day of incubation in the domestic fowl (Adam and Dimond, '71). Adam and Dimond also found hatchability improved with exposure to light. Other work has not been in agreement with these findings. For example Shutze et al. ('69) found development accelerated if light was given in the first week but not the last, with hatchability reduced if light was given throughout incubation. Tamimie and Fox ('67) found hatchability reduced in light treated eggs and hatching time delayed. Some of the inconsistencies may be explained by different levels and schedules of illumination but taken together the results indicate that light can alter the rate of development. This aspect needs to be investigated further. In this respect there are similarities between light and certain forms of vibratory or auditory stimulation (Vince, '66). Fifteen-minute periods of stimulation with clicks known to
EMBRYONIC RESPONSE TO LIGHT
443
Fig. 3 Comparison of mean heart rates in beats per minute before, during and after stimulation. Shaded areas indicate differences in mean heart rates before stimulation from those during stimulation. Open areas indicate differences in mean heart rates before stimulation from after stimulation ceased.
Fig. 4 Comparison of mean respiration rates in breaths per minute before, during and after stimulation. Shaded areas indicate differences in mean respiration rates before stimulation from those during stimulation. Open areas indicate differences in mean respiration rates before stimulation from those after stimulation ceased.
accelerate hatching did not significantly alter the heart rate of domestic fowl embryos over the period of stimulation (Vince et al., ‘76). In the present experiment with a light stimulus however, effects were found over a similar time period. The effect on respiration rate reported by Vince et al. (‘76) is similar to the effect on heart rate reported here in that the rate increased during stimulation and neither re-
turned to the previous level nor continued to increase after stimulation ceased. Light stimulation on the other hand did not have a s i g nificant effect on respiration rate, but this may be explained by the fact that several of the embryos were only just beginning to make respiratory movements a t the time of testing. Beak clapping often occurs between respiratory movements early in the parafoetal period when respiration is irregular (Freeman
444
ELIZABETH M. OCKLEFORD AND MARGARET A. VINCE
and Vince, '74). For this reason, a reduction in respiration rate a t this stage may represent an increase in beak clapping. Oppenheim ('68) found beak clapping rate was higher during one minute of stimulation with light than during the 10 minutes before stimulation. If the respiration rate data from the present experiment are treated in the same way as the beak clapping data obtained by Oppenheim (the rate during the first minute of stimulation compared with the rate during the preceding 10 minutes), i t may be seen that of nine embryos breathing slowly (below 50 breaths per minute), six showed reduced respiration rates when stimulated. Six out of seven embryos breathing more rapidly showed an increase in respiration rate on stimulation. The reduction in respiration rate of the slow breathing embryos probably indicates increased beak clapping activity and so this result offers indirect support for Oppenheim's findings of an increase in beak clapping as a response to light. The main finding that mean heart rate may be higher during exposure to light is important for experiments where heart rate is used as a measure of responsiveness. There is growing evidence that heart rate is a reliable indicator of responsiveness (Tolhurst and Vince, '76) but that acceleratory heart rate responses are more marked during periods of low or gradually decreasing heart rate (Wilder, '67; Graham and Jackson, '70). The illumination necessary for performing an experiment on an avian embryo may, by raising the heart rate, induce a state which masks or reduces a response to experimental treatment. ACKNOWLEDGMENTS
The authors would like to thank Doctor Phillip Poldon for his help with the measurement of light intensity. This work was carried out with the support of the Medical Research Council. LITERATURE CITED Adam, J. H., and S. J. Dimond 1971 Influence of light and the time of hatching in the domestic chick. Anim. Behav., 19: 226-229. Balaban, M., and J. Hill 1969 Perihatching behaviour patterns of chick embryos (Gallus domesticus). Anim. Behav., 17: 430-439. Bursian, A. V. 1964 The influence of light and the spontaneous movements of chick embryos. Bull. Exp. Biol. Med., 58: 767-770 (English translation from Russian). Dawes, C. M. 1976 A method for recording the respiratory and hatching movements of the chick embryo. J. Exp. Biol., 64: 379-383. Freeman, B. M., and M. A. Vince 1974 Development of the Avian Embryo. Chapman and Hall, London.
Gottlieb, G. 1971 Development of Species Identification in Birds. University of Chicago Press. Chicago and London. Graham, F. K., and J. C. Jackson 1970 Arousal systems and infant heart rate responses. In: Advances in Child Development and Behaviour. Vol. 5. H. W. Reese and L. P. Lipsitt, eds. Academic Press, New York and London. Hamburger, V., and R. Oppenheim 1967 Prehatching motility and hatching behaviour in the chick. J. Exp. Zool., 166: 171-204. Heaton, M. B. 1973 Early visual function in bobwhite and Japanese quail embryos a s reflected by pupillary reflex. J. Comp. Physiol. Psychol., 84: 134-139. Heaton, M. B.,and M. S. Harth 1974 Non-visual light responsiveness in the pigeon: developmental and comparative consideration. J. Exp. Zool., 188: 251-264. Impekoven, M., and P. S. Gold 1973 Prenatal origins of parent-young interactions in birds: a naturalistic approach. In: Behavioural Embryology. Vol. 1. G. Gottlieb, ed. Academic Press, New York, pp. 325-356. Kuo, 2. Y. 1932 Ontogeny of embryonic behaviour in aves 1. The chronology and general behaviour of the chick embryo. J. Exp. Zool., 61: 395-429. Laughlin, K. F., H. Lundy and J. A. Tait 1975 A method for monitoring avian embryonic heart rate during the last week of incubation. J. Physiol. Lond., 244: 8P-9P. Laughlin, K. F., H. Lundy and J. A. Tait 1976 Chick embryo heart rate during the last week of incubation: population studies. Br. Poult. Sci., 17: 293-301. Oppenheim, R. W. 1968 Light responsivity in chick and duck embryos just prior to hatching. Anim. Behav., 16: 276-280. Oppenheim, R. W., H. L. Levin and M. S. Harth 1973 An investigation of various eggopening techniques for use in avian behavioural embryology. Develop. Psychobiol., 6: 53-68. Orr, D. W., and W. F. Windle 1934 The development of behaviour in chick embryos: the appearance of somatic movements. J. Comp. neur., 60: 271-285. Peters, J. J., A. R. Vonderahe and T. H. Powers 1958 Electrical studies of functional development of the eye and optic lobes in the chick embryo. J. Exp. Zool., 139: 459-468. Shutze, J., J. K. Lauber, M. Kato and W. Wilson 1962 Influence of incandescent and coloured light on chicken embryos during incubation. Nature (London), 196: 594595. Siegel, P. B., S. T. Isakson, F. N. Coleman and B. J. Huffman 1969 Photo-acceleration of development in chick embryos. Comp. Biochem. Physiol., 28: 753-758. Tamimie, H. S., and M. W. Fox 1967 Effect of continuous and intermittent light exposure on the embryonic development of chicken eggs. Comp. Biochem. Physiol., 20: 793-799. Tolhurst, B. E., and M. A. Vince 1976 Sensitivity to odours in the embryo of the domestic fowl. Anim. Behav., 24: 772-779. Vince, M. A. 1966 Artificial acceleration of hatching in quail embryos. Anim. Behav., 14: 389-394. Vince, M. A. 1977 Taste sensitivity in the embryo of the domestic fowl. Anim. Behav., in press. Vince, M. A., M. R. Reader and B. E. Tolhurst 1976 Effects of stimulation on embryonic activity in the chick. J. Comp. Physiol. Psychol., 90: 221-230. Vince, M. A., and S. M. Salter 1967 Respiration and clicking in quail embryos. Nature, Lond., 216: 582-583. Walter, J. H., and R. A. Voitle 1972 Effects of photoperiod during incubation on embryonic and post-embryonic development of broilers. Poult. Sci., 51: 1122-1126. Wilder, J. 1967 Stimulus and Response. The Law of Initial Value. Write. Bristol, England.
ABSTRACT The effects of exposure to light were investigated during and shortly after the period of light stimulation. A particular objective was to evaluate any responses to the type of illumination conditions generally used for observation of avian embryos. Twenty 16-day Japanese quail embryos were illuminated for 15 minutes each during a total recording period of 55 minutes. Heart rate, respiration rate and an approximate measure of activity were recorded. Mean heart rate was found to be higher during stimulation than before and did not return to the prestimulation level during the recording period after stimulation ceased. The lack of an immediate heart rate response to the onset of stimulation separates the effects of light from those of touch, rotation, odours, tastes and maternal calls. The possibility that light may act to stimulate accelerated development is discussed. A simple technique used in studies of the behaviour of avian embryos involves the removal of part of the shell to allow direct observation or manipulation of the embryo. This technique was first described by Kuo (‘32) and has been used routinely by many working in this field (for example Orr and Windle, ’34; Hamburger and Oppenheim, ’67; Balaban and Hill, ’69; Oppenheim et al., ’73; Vince et al., ’76). Eggs are maintained in an observation incubator and often brightly illuminated with a heat filtered light to facilitate observation. A behavioural response to light was reported by Bursian (‘64) who found intense illumination increased motility in embryos of the domestic fowl as early as four days of i n cubation and that solitary movements were grouped on exposure to light earlier than otherwise. This effect could probably not have been mediated by the specific light sensitive receptors of the visual system since this system is immature a t this stage of development. Increased motility was also found in 11.5-day pigeon embryos in response to light and was attributed to a dermal sensitivity (Heaton and Harth, ’74). This altricial species hatches after about 17.5 days and the visual system does not appear to function until about day 15. Responses of the developing visual system to light have also been studied. Peters et al. (‘58) recorded evoked potentials in optic lobe J. EXP. ZOOL., 201: 439-444.
of precocial domestic fowl embryos and also first found electroretinograms about day 18. More recently Heaton (‘73) has shown that Japanese quail (also a precocial species and the one used in this investigation) showed a pupillary constriction reflex reliably on day 11.5 of incubation, indicating that a t least retina, optic nerve and the pretectal central visual areas are functional several days before hatching occurs on day 17. Heaton points out that the sunlight intensity reaching quail embryos within the shell is sufficient to elicit a visually mediated reflex. This reinforces the suggestion made by Impekoven and Gold (‘73) that light, associated with other sensory stimulation, during absence of the incubating parent bird may be important for development. Two previous studies of responsiveness of late stage avian embryos to light have produced conflicting results. Kuo (‘32) observed that 17-day domestic fowl embryos opened and closed their eyes in response to itlumination but did not show any altered pattern of movements of other parts of the body whereas Oppenheim (‘68) found beak clapping increased (a typical response to stimulation a t this stage) in chick and duck embryos exposed to one minute of intense light stimulation. Beak clapping also increased sometimes when stimulation ceased. The present experiment was designed to in-
439
440
ELIZABETH M. OCKLEFORD AND MARGARET A. VINCE
vestigate further the immediate effects of short term exposure to light. There is a possibility which is important when embryos are observed directly, that the illumination used to facilitate observation may affect the activity measured. The experiment to be described attempted to outline possible artifacts in such experimental situations. The conditions of illumination were chosen so as to approximate those which could occur in the wild. However other features of the environment such as temperature and egg position, which would be altered in association with onset and offset of illumination in the wild, were kept constant. MATERIALS AND METHODS
Japanese quail (Coturnix coturnixl eggs were incubated in a forced draught “Westernette” incubator a t 37.8”C for 13 days. On the fourteenth day of incubation they were transfered to a “Curfew” observation incubator (expected day of hatching, day 17). On day 15 the position of the air space and the embryo within the shell were identified by candling and three holes each approximately 2 mm in diameter were drilled in the shell in preparation for the insertion of electrodes and a pressure recording device. On day 16, silver wire electrodes were inserted into the egg for recording the EKG and
EMG (Laughlin et al., ’75;Tolhurst and Vince, ’76). In order to measure embryonic movements the air space of the egg was joined to a Mercury electronics modified M8 pressure transducer by means of a polypropylene tube (Dawes, ’76). The shell over part of the airspace was removed and the opening resealed with cellophane glued in place with a ring of Bostic number 1 clear adhesive (Vince, ’77). This preparation is illustrated in figure 1.The egg was then placed on a tripod of sound transducers (Vince and Salter, ’67) in the observation incubator. The egg was undisturbed for a t least one hour before any measurements were made.
Light stimulation An olympus microscope light with a heat absorbing filter was held approximately 25 cm above the egg. Background illumination was dim. A stimulation consisted of the microscope light being turned up manually (silently) until the light intensity a t the surface of the egg was approximately 0.089 lumens cm-2 as measured with an SEI photometer. Recording After the acclimatisation period, recordings of EKG, EMG, respiratory and other movements (pressure changes) and sound were
tube to pressure transducer
cellophane window electrode over neck
outline of airspace
electrode over R. tarsal joint
Fig. 1 Diagram of egg showing electrode positions, pressure recording tube and window cut in shell over airspace and resealed with cellophane.
44 1
EMBRYONIC RESPONSE TO LIGHT
tinued during 15minutes of illumination with the stimulus light. Measurements of the air temperature in the incubator under a cellophane film were also made over a similar period. RESULTS
temperature probe through hole drilled in shell
Fig. 2 Diagram showing position of temperature probe in relation to embryo one day before hatching. The temperature probe is gently pushed through the slit the embryo has made in the membranes.
made on a “Mingograf”’recorder. Recordings lasted for 55 minutes during which a 15minute period of stimulation was given. The time of onset of the stimulation within the 55minute recording period was randomised with the constraints that there were a minimum of 13-minuterecording before stimulation began and 3-minute recording after the end of stimulation. Notes were made throughout the recording period of the nature of any sounds produced by the embryo and of when the incubator heater switched on and off. The position of the embryo was noted after recording had finished, particularly whether the right eye was clearly visible through the cellophane window or whether i t was obscured by the right wing. Heart rate and respiration rate were scored by inspection of the Mingograf paper records. Temperature measurements Control temperature measurements were made using a Jenway platinum resistance probe inserted into the opened egg as shown in figure 2. Temperature measurement was con-
1. Heart rate Mean heart rates in beats per minute for each individual before, during and after stimulation are given in table 1. Figure 3 shows differences in mean heart rates during and after stimulation from means before stimulation. Mean heart rate during stimulation was significantly higher than before stimulation began (Wilcoxon matched pairs signed ranks test T=15, p < 0.01). Mean heart rate after stimulation was turned off was also higher than the prestimulation level (Wilcoxon test T=27, p < 0.01). There was no significant difference between mean heart rates during and after stimulation. No immediate effects of light stimulation on heart rate were found when rates during the minute or part of one minute (the smallest period considered was that occupied by 10 beats) immediately prior to or immediately following the onset of stimulation were compared. Similarly no immediate “off” effects were found. 2. Respiration rate
Mean respiration rates in breaths per minute before, during and after stimulation are given in table 1 for the 16 eggs from which pressure records were obtained. There were no significant differences in mean respiration rates between the different periods. Figure 4 shows differences in mean respiration rates during and after stimulation from mean rates before stimulation. 3. Movements Very few movements other than breathing movements were detected in any of the subjects with the methods used. 4. Temperature Internal egg temperature fluctuated between 36.5”C and 38’C. No differences in the maximum temperature attained by the egg or the period of incubator temperature fluctuations were recorded with the light on. Similarly no differences in air temperature were recorded. DISCUSSION
The results show that mean heart rate of
442
ELIZABETH M. OCKLEFORD AND MARGARET A. VINCE TABLE 1
Mean heart rates in beats per minute and respiration rates (Resp) in breaths per minute for individual embryos before, during and after stimulation with light Before Egg
After
During
Heart rate
Resp
Heart rate
Resp
Heart rate
Resp
~~
1
2 3 4 5 6
7 8 9 10 11 12 13 14 15 16 17 18 19 20 Grand mean
n
M
13 26 34 16 36 15 36 27 37
418 330 397 286 329 418 427 391 298 358 417 366 360 356 403 325 288 312 381 383
35 37 30 17 36 18 26 21 13 24 18
362
S
D
18 15 11 11 17 15 16 17 8 1
8 9 12 15 18 14 9 9 26 12
M
S
D
133 36 29
13 13 15
38 42 68 39 8 22 90
8 16 7 6 4 3 9
71 39 53
3 5 10
18 42 62
6 10 9
n
M
15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15
440 342 406 289 349 425 430 410 315 366 436 364 369 368 402 329 285 306 403 388
15
49
15 15 15
371
S
D
12 20 15 11 15 13 14 14 6 9 13 16 24 18 17 14 13 9 25 17
M
S
D
145 43 12
7 14 3
36 21 52 33 9 19 96
8 14 6 4 1 6 7
71 32 53
10
17 18 63
6 1 10
45
4 3
n
M
27 14 6 24 4 25 4 13 3 5 3 10 23 4 22 14 19 27 16 22
452 334 408 281 352 433 433 421 304 364 429 366 372 365 396 336 283 307 371 383 370
S
D
11 15 17 15 19 18 20 16 18 17 3 17 19 18 5 9 9 8 9 18
M
S
D
132 36 11
5 13 13
67 13 59 33 8 17 104
17 4 7 5 4 2 10
70 30 64
9 1 14
13 13 65
7 3 13
46
n is the number of minutes of recording included in each mean value Bven Standard deviations from the mean are also shown
avian embryos can be increased during illumination. An increase in heart rate would be expected if the light source were contributing heat but temperature measurements showed that this was not occuring in this case. The lack of any immediate heart rate response to the light stimulus separates the effect of this form of stimulation from some stimuli in other modalities. Heart rate responds by a brief increase to olfactory stimulation (Tolhurst and Vince, '76), taste (Vince, '77), rotation (Vince, Clark and Reader, unpublished observations) and maternal calls (Gottlieb, '71). Heart rate is known to rise markedly during the last 24 hours before hatching in the domestic fowl (Laughlin et al., '76) and in the quail (unpublished observations). In the experiment described here, mean heart rate remained higher during the period of recording after stimulation ceased than it had been before any light stimulus was given. Heart rate was not however higher during the second half of the prestimulation recording than the first half. Nor was it higher after the end of stimulation than during the stimulation period. These findings prompt the suggestion that stimulation may have brought forward or triggered the prehatching period of de-
velopment where the heart rate is increasing. Several experiments investigating the longer term effects of light on the embryo have pointed to the possibility that light may act as an accelerating stimulus by bringing forward hatching time. Of particular interest is the finding that hatching time was brought forward after brief periods of stimulation with light on the seventeenth or nineteenth day of incubation in the domestic fowl (Adam and Dimond, '71). Adam and Dimond also found hatchability improved with exposure to light. Other work has not been in agreement with these findings. For example Shutze et al. ('69) found development accelerated if light was given in the first week but not the last, with hatchability reduced if light was given throughout incubation. Tamimie and Fox ('67) found hatchability reduced in light treated eggs and hatching time delayed. Some of the inconsistencies may be explained by different levels and schedules of illumination but taken together the results indicate that light can alter the rate of development. This aspect needs to be investigated further. In this respect there are similarities between light and certain forms of vibratory or auditory stimulation (Vince, '66). Fifteen-minute periods of stimulation with clicks known to
EMBRYONIC RESPONSE TO LIGHT
443
Fig. 3 Comparison of mean heart rates in beats per minute before, during and after stimulation. Shaded areas indicate differences in mean heart rates before stimulation from those during stimulation. Open areas indicate differences in mean heart rates before stimulation from after stimulation ceased.
Fig. 4 Comparison of mean respiration rates in breaths per minute before, during and after stimulation. Shaded areas indicate differences in mean respiration rates before stimulation from those during stimulation. Open areas indicate differences in mean respiration rates before stimulation from those after stimulation ceased.
accelerate hatching did not significantly alter the heart rate of domestic fowl embryos over the period of stimulation (Vince et al., ‘76). In the present experiment with a light stimulus however, effects were found over a similar time period. The effect on respiration rate reported by Vince et al. (‘76) is similar to the effect on heart rate reported here in that the rate increased during stimulation and neither re-
turned to the previous level nor continued to increase after stimulation ceased. Light stimulation on the other hand did not have a s i g nificant effect on respiration rate, but this may be explained by the fact that several of the embryos were only just beginning to make respiratory movements a t the time of testing. Beak clapping often occurs between respiratory movements early in the parafoetal period when respiration is irregular (Freeman
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ELIZABETH M. OCKLEFORD AND MARGARET A. VINCE
and Vince, '74). For this reason, a reduction in respiration rate a t this stage may represent an increase in beak clapping. Oppenheim ('68) found beak clapping rate was higher during one minute of stimulation with light than during the 10 minutes before stimulation. If the respiration rate data from the present experiment are treated in the same way as the beak clapping data obtained by Oppenheim (the rate during the first minute of stimulation compared with the rate during the preceding 10 minutes), i t may be seen that of nine embryos breathing slowly (below 50 breaths per minute), six showed reduced respiration rates when stimulated. Six out of seven embryos breathing more rapidly showed an increase in respiration rate on stimulation. The reduction in respiration rate of the slow breathing embryos probably indicates increased beak clapping activity and so this result offers indirect support for Oppenheim's findings of an increase in beak clapping as a response to light. The main finding that mean heart rate may be higher during exposure to light is important for experiments where heart rate is used as a measure of responsiveness. There is growing evidence that heart rate is a reliable indicator of responsiveness (Tolhurst and Vince, '76) but that acceleratory heart rate responses are more marked during periods of low or gradually decreasing heart rate (Wilder, '67; Graham and Jackson, '70). The illumination necessary for performing an experiment on an avian embryo may, by raising the heart rate, induce a state which masks or reduces a response to experimental treatment. ACKNOWLEDGMENTS
The authors would like to thank Doctor Phillip Poldon for his help with the measurement of light intensity. This work was carried out with the support of the Medical Research Council. LITERATURE CITED Adam, J. H., and S. J. Dimond 1971 Influence of light and the time of hatching in the domestic chick. Anim. Behav., 19: 226-229. Balaban, M., and J. Hill 1969 Perihatching behaviour patterns of chick embryos (Gallus domesticus). Anim. Behav., 17: 430-439. Bursian, A. V. 1964 The influence of light and the spontaneous movements of chick embryos. Bull. Exp. Biol. Med., 58: 767-770 (English translation from Russian). Dawes, C. M. 1976 A method for recording the respiratory and hatching movements of the chick embryo. J. Exp. Biol., 64: 379-383. Freeman, B. M., and M. A. Vince 1974 Development of the Avian Embryo. Chapman and Hall, London.
Gottlieb, G. 1971 Development of Species Identification in Birds. University of Chicago Press. Chicago and London. Graham, F. K., and J. C. Jackson 1970 Arousal systems and infant heart rate responses. In: Advances in Child Development and Behaviour. Vol. 5. H. W. Reese and L. P. Lipsitt, eds. Academic Press, New York and London. Hamburger, V., and R. Oppenheim 1967 Prehatching motility and hatching behaviour in the chick. J. Exp. Zool., 166: 171-204. Heaton, M. B. 1973 Early visual function in bobwhite and Japanese quail embryos a s reflected by pupillary reflex. J. Comp. Physiol. Psychol., 84: 134-139. Heaton, M. B.,and M. S. Harth 1974 Non-visual light responsiveness in the pigeon: developmental and comparative consideration. J. Exp. Zool., 188: 251-264. Impekoven, M., and P. S. Gold 1973 Prenatal origins of parent-young interactions in birds: a naturalistic approach. In: Behavioural Embryology. Vol. 1. G. Gottlieb, ed. Academic Press, New York, pp. 325-356. Kuo, 2. Y. 1932 Ontogeny of embryonic behaviour in aves 1. The chronology and general behaviour of the chick embryo. J. Exp. Zool., 61: 395-429. Laughlin, K. F., H. Lundy and J. A. Tait 1975 A method for monitoring avian embryonic heart rate during the last week of incubation. J. Physiol. Lond., 244: 8P-9P. Laughlin, K. F., H. Lundy and J. A. Tait 1976 Chick embryo heart rate during the last week of incubation: population studies. Br. Poult. Sci., 17: 293-301. Oppenheim, R. W. 1968 Light responsivity in chick and duck embryos just prior to hatching. Anim. Behav., 16: 276-280. Oppenheim, R. W., H. L. Levin and M. S. Harth 1973 An investigation of various eggopening techniques for use in avian behavioural embryology. Develop. Psychobiol., 6: 53-68. Orr, D. W., and W. F. Windle 1934 The development of behaviour in chick embryos: the appearance of somatic movements. J. Comp. neur., 60: 271-285. Peters, J. J., A. R. Vonderahe and T. H. Powers 1958 Electrical studies of functional development of the eye and optic lobes in the chick embryo. J. Exp. Zool., 139: 459-468. Shutze, J., J. K. Lauber, M. Kato and W. Wilson 1962 Influence of incandescent and coloured light on chicken embryos during incubation. Nature (London), 196: 594595. Siegel, P. B., S. T. Isakson, F. N. Coleman and B. J. Huffman 1969 Photo-acceleration of development in chick embryos. Comp. Biochem. Physiol., 28: 753-758. Tamimie, H. S., and M. W. Fox 1967 Effect of continuous and intermittent light exposure on the embryonic development of chicken eggs. Comp. Biochem. Physiol., 20: 793-799. Tolhurst, B. E., and M. A. Vince 1976 Sensitivity to odours in the embryo of the domestic fowl. Anim. Behav., 24: 772-779. Vince, M. A. 1966 Artificial acceleration of hatching in quail embryos. Anim. Behav., 14: 389-394. Vince, M. A. 1977 Taste sensitivity in the embryo of the domestic fowl. Anim. Behav., in press. Vince, M. A., M. R. Reader and B. E. Tolhurst 1976 Effects of stimulation on embryonic activity in the chick. J. Comp. Physiol. Psychol., 90: 221-230. Vince, M. A., and S. M. Salter 1967 Respiration and clicking in quail embryos. Nature, Lond., 216: 582-583. Walter, J. H., and R. A. Voitle 1972 Effects of photoperiod during incubation on embryonic and post-embryonic development of broilers. Poult. Sci., 51: 1122-1126. Wilder, J. 1967 Stimulus and Response. The Law of Initial Value. Write. Bristol, England.
