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Grouping and respiratory behavior induced in rats and quails by LD12:12 illumination
Physiology & Behavior, Vol. 25, pp. 439--447. Pergamon Press and Brain Research Publ., 1980. Printed in the U.S.A.
Grouping and Respiratory Behavior Induced in Rats and Quails by LD12:12Illumination M A U R I C E S T U P F E L , .1 A R T H U R P E R R A M O N , ¢ P H I L I P P E Mt~RAT,t V I C T O R - H U G O D E M A R I A P E S C E , * Ht~LI~NE MASSIf* A N D V I ~ R O N I Q U E G O U R L E T *
*French National Institute of Health (INSERM) Physiopathological Mechanisms of Environmental Nuisances Research Group, Le Vdsinet, 78, France tFrench National Institute of Agronomical Research (INRA) Factorial Genetic Laboratory, Jouy en Josas, 78, France Received 19 D e c e m b e r 1979 STUPFEL, M., A. PERRAMON, P. MI~RAT, V.-H. DEMARIA PESCE, H. MASSI~ AND V. GOURLET. Grouping and respiratory behavior induced in rats and quails by LD12:12 illumination. PHYSIOL. BEHAV. 25(3) 439--447, 1980.Continuous recordings of the respiratory VCO2 and VOz of isolated, separated or grouped rats (Sprague-Dawley) and quails (Coturnix coturnix japonica) in a LDI~:n (L=100 lux) lighting regimen, under controlled environmental conditions of temperature, humidity and noise, and with food ad lib, show circadian rhythmicity with marked increases in respiratory amplitude at L--~D in rats and at D--*L in quails. Grouping increases these respiratory amplitude variations at L---~Dand D---~L in rats but not in quails. Continuous illumination (LL; 100 lux) provokes respiratory interindividual desynchronization after 13 days in grouped rats and after 3 days in quails. The differences in grouping effects shown between these two species are mostly due to interindividual contacts which, under the above experimental conditions, are closer in rats than in quails. Respiration
Motor activity
Grouping
Rats
THE importance of social synchronization in many human physiological activities is well appreciated. And yet with a few exceptions [3, 7, 12], human chronobiological studies have been performed on isolated individuals. This arises more through difficulties in recruitment, for a sufficiently long time, of a great enough number of volunteers or paid subjects having the same age and genetic origin, than from the task of submitting them to identical synchronizers under controlled environmental parameters. Animal experimentation, on the other hand, enables studies of groups of individuals of the same age, from a genetically defined origin and kept for a long time (considering the short life span of usual laboratory animals) under standardised environmental conditions. Another difficulty in studying groups arises from the necessity of having a great number of apparatus recording simultaneously physiological and biochemical parameters, and from the constraints of calculations involving a large quantity of data recorded for a long time on a large number of iridividuals. Measurement of the variations in respiratory gases emitted by a colony of animals, appears at first sight to be the simplest approach to the simultaneous study of a great number of individuals, since it aims to discover whether the total gas variations measured or recorded correspond to the sum of the respiratory exchanges of each individual. This leads one to investigate whether the resultant concentrations
Quails
Circadian rhythms
Illumination
Darkness
are merely a sum, or a biological addition, in other word whether or not a "group effect" is present. This study considers how respiratory behavior of two species of laboratory vertebrates, diurnal birds and nocturnal rodents, of approximately the same ages and kept under the same environmental conditions (temperature, humidity, ventilation, noise, food and water ad lib) react to alternating L (light) and D (dark). Quails and rats have been compared for, though they have predominant either diurnal or nocturnal activities, they are of similar size and therefore their activity could be compared under the same housing (cages) conditions. A 100 lux lighting and a LDI2:1.2(L=06:00-18:00) regimen has been chosen since it is probably the one most often used in animal houses [11]. METHOD
Animals Rats (Rattus norvegicus) were SPF (specific pathogen free) OFE from the breeding farm of IFFA-CREDO, Les Oncins, 69, France. They were fed with a special sterile commercial food (NoAo 3, UAR, Villemoisson sur Orge, 91, France), the composition of which has been described [9]. The Japanese quails (Coturnix coturnix japonica) were issued from the flock of the Factorial Genetics Laboratory (INRA, Jouy en Josas, 78, France). They were fed with
1Send reprint requests to Dr. Maurice Stupfel, Environmental Nuisances Research Group, INSERM, 44, Chemin de Ronde, 78110, Le
Vrsinet, France.
Copyright © 1980 Brain Research Publications Inc.--0031-9384/80/090439-09502.00/0
S T U P F E L ET AL.
440 INRA quail granules, the composition of which has been previously given [10]. The animals arrived at the I N S E R M Research Group of Le Vrsinet, 5-15 days before physiological measurements were made. On arrival they were placed in air conditioned (temperature 20-21°C; humidity 50-70%) animal rooms, LD,~:,~ (L=06:00-18:00 hr) lighted with 100 lux. All animals were randomly 5-6 grouped, sex separated, in wire mesh cages of either type A (42 cm long, 28 cm wide and 18 cm high), or type B (50 cm long, 40 cm wide and 29 cm high); isolated and separated animals were placed in wire mesh cages of type C (26 cm long, 20 cm wide and 16 cm high). Food and tap water were given ad lib.
Respiratory Measurements Several type A cages, each containing 5-6 grouped animals or several type C cages, each containing one single animal, were enclosed in two translucent lucite chambers (Nos. 1 and 2) (2 m long, 1 m wide and 1 m high) which were placed side by side but separated by blackened opaque faces. The use of these two similar chambers enabled simultaneous measurements and hence comparisons to be made (e.g. in one chamber groups of rats and in the other chamber separate rats, see below). Measurements of the respiratory exchanges of a single animal were made in a translucent lucite chamber (47 cm long, 43 cm wide and 38 cm high). All these "respiratory chambers" were placed in an air conditioned room. Inside them temperature, humidity and atmospheric pressure were continuously recorded (for details see Stupfel and Bouley [8]). The air of each chamber was renewed at the same rate measured with Pitot tubes or flowmeters. The carbon dioxide concentration inside each chamber varied between 0.10 and 0.25% depending on the COs emission of the animal or animals which were enclosed into these "respiratory chambers". Light was provided by fluorescent tubes "blanc (white) industrie" yelding 100 lux at 5 cm above the floor of each of the lucite chambers. Light was either alternating LD,~:,~ (06:00-18:00 hr) or continuously on (LL), or continuously off (DD). Inside the respiratory chambers the temperature range was 20.0-20.5°C and the humidity 60-80%. The level of background noise mainly due to climatiser and analysers was measured with a dB meter, and found to be 76 dB in each chamber. The concentrations of carbon dioxide and oxygen inside the respiratory chambers, sampled 25 cm from the roofs, were measured with two ONERA differential infrared carbon dioxide analysers (0-0.2% CO2) and a Pauling oxygen Beckman F:~ oxygen analyser (20-21% O2). Respiratory measurements were continuously made over periods lasting from 3 to 26 days, gas (O2, COz) concentrations being continuously recorded (MECI recorder) on the same paper. VCO2 and VO2 were expressed STPD, either per animal or per kg body weight.
Activity Measurements An Animex, type DSE activity meter (LKB), was used to measure displacements of isolated rats, placed isolated, in translucent polycarbonate cages (39 cm long, 25 cm wide and 18 cm high) which could be enclosed inside the "respiratory chambers." Numbers of counts for a 1-20 minute time inter-
val were recorded on a paper printer. Measurement of activity of grouped rats was performed by placing an inductance sensor (geophone) on the floor of the large respiratory chamber; vibrations were registered with a Brush recorder. As both types of these activity measurements appear to be not reliable for quails, observation and counts of the number of active and resting (lying down) individuals were performed, from I0:00 to 16:00 at 2-hour intervals, in separated and grouped rats and quails (15 separated and 3 groups of 5 of each species) during 3 days in LD and during 3 days in LL.
Group Effect Determination To compare the effects of interindividual reactions on respiratory activity the following procedure was designed. In one (No. 1) of the 2 identical large respiratory chambers (2 m long, 1 m wide, 1 m high) were placed n wire mesh cages of type A, each containing n' (5--6) rats or quails. In the second (No. 2) respiratory chamber were placed the same number n × n' of rats or quails, but each animal was placed in a separate wire mesh cage of type C. Carbon dioxide and oxygen concentrations were continuously and simultaneously recorded in both chambers. One LKB actometer was placed in each chamber, each recording the displacement activity of a single rat or quail. This procedure enables comparison of the VCO2 and VO2 of both chambers containing the same number of animals but whilst in chamber No. 1 animals have interindividual contacts and so will be called " g r o u p e d " animals, in chamber No. 2 animals have no interindividual contacts and so will be called " s e p a r a t e d " animals. In several experiments " g r o u p e d " animals were placed in chamber No. 2 and " s e p a r a t e d " animals in chamber No. 1, and it was proved that no "chamber effect" was affecting the observed results.
Statistics In expressing the results the arithmetic mean is followed by the standard deviation: mean _+ SD. Assuming that in LD VCOz recordings in L or in D can be considered as straight lines Student's t-test has been used. Furthermore, two-way analysis of variance has been used to compare " g r o u p e d " and " s e p a r a t e d " individuals at different times of LD, L L and DD exposures. Chi-square testing was used to compare active and resting animals during L L and LD exposure. p is the probability, and statistical significance has been assessed for at least p<0.05. RESULTS
Respiratory Activity in Isolated Rats and Quails in LD,2:,2, LL and DD Continuous recordings of VCO2 and of VO2 of an isolated Sprague-Dawley rat and of an isolated Japanese quail were performed on 2-4 individuals of each species placed either in a LD,2:,2, L L or DD regimen. In LD,2:,2 (Fig. 1) t-testing of the mean VCO,., and VO,, taken every 20 minutes on the records, shows that for the rat the respiratory activity is statistically (p<0.001) greater during the dark (D) period than during the light (L) period and that conversely for the quail the respiratory activity is statistically (p<0.001) greater during L than during D. In isolated
GROUPING AND RESPIRATORY BEHAVIOR ~°2
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17
07
18
19
FIG. 2. Parts of the recording of the carbon dioxide percentages measured in a respiratory chamber containing one male Japanese quail. Peaks A and C are CO.2%increases when light is on at 06:00 (D-~L) and when light is off at 18:00 (L-~D). B and D are CO~% decreases followingpeaks A and C.
Vco2 .... 3.0 1
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individuals of both species sharp VCOz and VO2 increases are noted at 06:00 (D--*L) and 18:00 (L---*D) (portions A and C in gra.phs of Fig. 2). Circadian VCO2 rhythmicity is still observed in an isolated rat or quail maintained for 5 days in an LL or DD exposure. The results of 2 experiments performed with 2 male rats (93 day old) and 2 male quails (167 day old) show changes in r which corroborate data of the literature (i.e. ~'>24 hr in nocturnal rodents in LL and in diurnal birds in DD, and r<24 hr in nocturnal rodents in DD and in diurnal birds in LL).
06
5 GROUPS OF 5 RATS 25 SEPARATED RATS
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FIG. 3. 36-hour VCO2 variations in Sprague-Dawley male rats, 25 separated individuals (dotted line) and 5 groups of 5 (solid line) in LD,~:12.
Respiratory Activity in Separated and Grouped Rats and Quails in LD12:12 As previously mentioned under methods, an experimental procedure was designed to simultaneously record respiratory activity of grouped and separated animals of the same species. Two such experiments were performed in duplicate, two with separated and grouped rats, two with separated and grouped quails. For rats the first experiment (a) used 5 groups of 5 males (chamber No. 1) compared to 25 isolated males (chamber No. 2); the rats were 36 day old at the beginning of the experiment which lasted 24 days. The second experiment (b) used 3 groups of 5 males compared to 15 isolated males; these rats were 81 day old at the beginning of the experiment which lasted 8 days. For quails the first experiment (c) was done with 1 group of 6 and I group of 5 males compared to 11 separated males; the quails were 37 day old at the beginning of the experiment which lasted 3 days. The second experiment (d) was performed with 3 groups (2 groups of 5 males and 1 group of 6 females) compared to 16 (10 males and 6 females) separated quails; these quails were 58 day old at the beginning of the experiment which lasted 7 days.
Two points will be considered concerning the results of these 4 experiments. (1) In all these experiments VCO2 recordings (Figs. 3 and 4) show the same patterns as found in isolated animals (Fig. 1). Respiratory variations are observed at 06:00 and 18:00 when light is turned on and off. The VCO2 variations in 15 separated and in 15 grouped rats, and in 16 separated and in 16 grouped quails are shown in Table 1. Column 1 shows VCO2 increases (+~VCO2) at 06:00 when light is turned on (D-~L; Fig. 2: part A of the VCO2 recording) and column 5 shows CO2 increases (+AVCO2) at 18:00 when light is turned off (L-~D; Fig. 2 i part C); the duration of these light on and light off induced VCO2 increases are given in columns 2 and 6. The after-effects which follow ~'CO~ increases at the beginning and end of illumination and which appear as VCO,, decreases (-AVCO2; Fig. 2: parts B and D) are shown in columns 3 and 7, and their respective durations in columns 4 and 8. t-testing (statistical results in lines 3 and 6 of Table (1) shows statistically significant differences between separated and grouped rats (at 06:00 for the 2nd VCO2 variation,
442
STUPFEL ET AL. TABLE 1
COMPARISON IN 15 SEPARATED AND IN 15 GROUPED RATS, AND IN 16 SEPARATED AND IN 16 GROUPED QUAILS OF THE FIRST A N D SECOND VCOz VARIATIONS (COLUMNS 1, 3, 5, 7) AND OF THE DURATIONS OF THESE VARIATIONS (COLUMNS 2, 4, 6, 8) AT 06:00 WHEN LIGHT (100 lux) IS TURNED ON (I)-~L) AND AT 18:00 WHEN LIGHT IS TURNED OFF (L--*D); i ± SD (n): MEAN ± STANDARD DEVIATION AND (NUMBER OF EXPERIMENTS)
Species
D--~L (06:00)
(animals number)
Rats (15)
1st variation +A VCO.~ t % min
1st variation +A VCO2 t % min
2nd variation - A VCO~ t % min
5.50 ± 1.52 15.00 ± 0.00 17.83 ± 7.30 34.17 ± 10.21 12.00 ± 2.74 18.00 ± 2.74 2.20 ± 1.10 11.00 ± 2.24
Separated
(6)
Grouped
Quails (16)
L--,D (18:00)
2nd variation - A VCO~ t % min
(6)
(6)
(6)
(5)
(5)
(5)
(5)
5.67 ± 2.16 10.83 ± 2.04 25.55 ± 6.41 37.50 ± II.73 28.80 _ 4.44 25.00 ± 6.12 11.80 ± 3.96 19.00 ± 7.42 (6) (6) (6) (6) (5) (5) (5) (5)
t-test separ-group
NS
"t
Separated
24.20 _+ 5.00 (5)
8.00 _+ 2.74 (5)
Grouped
23.75 _+ 1.09 9.00 _ 2.24
t-test separ-group
*
NS
"~
NS
2.00 _+ 0.71 4.20 _+ 3.49 4.00 ± 2.00 (5) (5) (5)
l-
*
7.00 _+ 2.74 30.60 ± 7.66 42.00 ± 10.37 (5) (5) (5)
2.20 ± 1.10 4.80 ± 3.27 3.20 ± 1.48 6.40 ± 3.51 29.40 ± 3.78 42.00 ± 5.70
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
NS
NS
NS
NS
NS
NS
NS
NS
*p<0.05; ep<0.01; NS non-significant.
p<0.05; at 18:00 for the 1st, p<0.01 and the 2nd, p<0.01 VCO2 variation). In quails no significant differences in ~VCO2% are observed between separated and grouped animals. The duration of these VCO2 variations are significantly shorter for the 1st VCO2 variation (/9<0.01) at 06:00 and significantly longer for the 2nd VCO2 variation (p<0.05) at 18:00 in grouped than in separated rats. N o significant differences of durations of VCO2 variations are observed between grouped and separated quails. (2) Secondly, as in isolated animals, mean respiratory levels of grouped and/or separated animals are significantly different in L and in D.
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F o r experiment a, figure 3 shows that the VCO2 levels during L and D are higher in the 25 separated rats than in the 5 batches of 5 grouped rats. This is confirmed by t-tests comparing VCOz (values taken every 20 minutes) during the L and D periods in these separated and grouped rats (p<0.001). For experiment c, Fig. 4 and t-testing show that t h e VCOz levels are higher in the grouped male quails than in the separated male quails (p< 0.001). For experiments b and d, once every 20 minute VCO,, sampling has been performed 72 times during 48 successive hours in 15 separated (347 - 17 g) and 3 groups of 5 (355 _+ 25 g) male rats, 81 day old (experiment b), and in 15 separated (165 _+ 17 g) and 3 groups of 5 (171 - 20 g) male quails, 58 day old (experiment d). This gives the followiflg VCO2 values expressed in ml.kg-Khr -1, during L and D:
1
12
I
18
FIG. 4.36-hour VCO~ variations in Japanese male quails: 1 group of 5 plus 1 group of 6 (solid line), and 11 separated individuals (dotted line) in LD1~:12.
: : : :
L=1177 L = 873 L=2318 L=2962
_+ 92 _+ 93 _+ 185 _ 279
(72) (72) (72) (72)
; D=1507 ; D=1259 ; D=1589 ; D=2267
_+ ± _+ _+
81 (72) 128 (72) 159(72) 194 (72)
t-tests show that separated rats have significantly higher (p<0.001) VCO2 than grouped rats either during L or during D, and that on the other hand separated quails have significantly lower VCO2 than grouped quails either during L (p<0.001) or during D (p<0.05). Moreover in rats (experiment b), both separated and grouped, VCOz is significantly (p<0.001) lower during L than during D. Conversely quails (experiment d), both separated and grouped, have a significantly (p<0.001) higher VCO2 during L than during D. Placing grouped rats or grouped quails in larger wire mesh cages (from cages A to cages B) does not change these differences between separated and grouped rats or quails.
GROUPING A N D RESPIRATORY B E H A V I O R 15/Vco2 • j
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FIG. 5.24-hour VCO2 in Sprague-Dawley male rats: 3 groups of 5 (solid line) and 15 separated (dotted line) individuals after 3 days and a half exposure either to LL (left side of figure) or to DD (right side of figure).
Respiratory Activity in Separated and Grouped Rats and Quails in LL and DD Exposure to continuous lighting (LL) or to continuous darkness (DD) suppresses the L--->D and D--~L induced VCO2 increases. L L decreases the mean VCO2 of rats (/9<0.001) and increases the mean VCO2 of quails (0.0124 hr), in separated quails (Fig. 6) an advance of approximately 2 hours (z<24 hr), in grouped quails (Fig. 6) no more circadian rhythmicity. On the last day of the 3 days and a half of DD exposure, this comparison of the overall VCO2 records with those previously obtained in LD shows: in separated and grouped rats (Fig. 5) an advance of approximately 2 hours (T<24 hr), in separated and grouped quails (Fig. 6) a lag of 2 hours (r>24 hr). To be more precise the different VCO2 curves recorded in separated and grouped animals in L L and DD have been compared by taking every 20 minutes the VCO2 values in each of these curves. Table 2 shows means and standard deviations of these VCO2, either between 06:00 and 18:00, or between 18:00 and 06:00, in 15 separated and in 15 grouped (3 groups of 5) rats, in 15 separated and in 15 grouped (3 groups of 5) quails, during 3 days and a half of 100 lux illumination or darkness. During L L (upper half of Table 2) t-test shows statistically (0.001
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FIG. 6.24-hour VCO2 in Japanese quails: 3 groups of 5 (solid line) and 15 separated (dotted line) individuals after 3 days and a half exposure either to LL (left side of figure) or to DD (right side of figure). 7) on the right of Table 2 (second 12 hours of third day and first 12 hours of fourth day of L L or DD) are statistically (p<0.001) different, with the exception of grouped quails in L L for which 2371 - 77 (36) does not statistically differ from 2402 __. 92 (36). A two-way variance analysis of the means has been performed on the 12-hour period (06-18 and 18-06) VCOz data of Table 2. Statistical results of this comparison of mean VCO., sampled either every 12 hours or every 24 hours are shown in Table 3. Significant differences are obtained for rats in all cases, with the exception of the three 18-06 periods during L L where the VCO2 differences between these 3 periods as well as between separated and grouped individuals are not statistically significant. In quails the VCO~ differences between separated and grouped individuals are statistically significant during L L but not significant during DD; moreover in these birds during L L the VCO2 differences between the four 06-18 and the three 18-06 periods are not statistically significant, and during DD the VCO2 differences between the three 06-18 periods are similarly not significant. To assess circadian self-sustainment of respiratory rhythms in grouped animals long term 100 lux L L experiments were performed in preference to long term DD experiments, since constant lighting appears to be more easy and more reliable to achieve than constant darkness. In 5 groups of 5 male rats, 422 day old, placed in respiratory chamber No. 1, disappearance of the whole group (5 x5 individuals) VCO2 circadian variations was observed after 13 days of I00 lux L L exposure. Under the same conditions in 1 group of 8 quails, 45 day old, no more circadian VCO2 variations of the group were obtained after 2 days of 100 lux L L exposure.
Motor Activity Observations and Measurements Concerning motor activity neither agonistic behavior, nor freezing and alarm responses were noted in grouped rats and in grouped quails. Huddling was usually noted in rats during L. Animex activity measurements of rats during 3 days and a half of L L exposure show that rats in a same chamber (whether they are separated or grouped in this chamber) have interindividual parallel records of activity displacement and VCO2, which shows interindividual "syncrhonization." Observations and counts from 10:00 to 16:00 of the number of rats active and resting during 3 days of exposure
S T U P F E L ET A L
444
TABLE 2 TWICE A DAY (06-18 AND 18-06 hr) VCO2, ml-kg-'.hr-' (MEAN -+ SD (n): MEAN LEVEL -+ STANDARD DEVIATION (NUMBER OF DATA)) OF SEPARATED OR GROUPED RATS AND QUAILS DURING 3 DAYS AND A HALF OF EXPOSURE TO CONSTANT 100 LUX ILLUMINATION (LL) OR CONSTANT DARKNESS (DD) 1st day
2nd day
06-18
18-06
Separated
837 ± 51 (36)
1052 ± 56 (33)
Grouped
766 ± 56 (36)
991 ± 92 (33)
t-test separ-grouped
:~
?
06-18
18-06
4th day 06-18
879± 166 1080± 56 (33) (36)
886± 64 (36)
1007 ± 81 (36)
907 ± 85 (36)
825 ± 89 (33)
982 ± 88 (36)
825 ± 49 (36)
988 ± 85 (36)
845 ± 88 (36)
~
$
06-18
3rd day 18-06 LL
Rats
Quails
NS
NS
?
Separated
2498 ± 137 2216 ± 295 (36) (36)
2260 ± 105 2054 ± 94 2188 ± 109 2056± 106 2256 ± 105 (32) (36) (29) (36) (36)
Grouped
2850 ± 154 2568 ± 180 2639 ± 154 2507 ± 153 2555 ± 128 2371 ± 77 2402 ± 92 (32) (36) (29) (36) (36) 36) (36)
t-test separ-group
:~
$
Separated
1353 ± 72 (36)
1158 ± 72 (36)
1448_+ 63 (36)
1185_+ 104 (36)
1434_+ 56 (36)
1186+_ 69 (36)
1452_+ 44 (36)
Grouped
1234 ± 78 (36)
987 ± 81 (36)
1329_+ 77 (36)
1007_+ 122 (36)
1329±- 68 (36)
1015 ± 102 (36)
1362 ± 68 (36)
t-test separ-group
:~
?
Separated
1561 ± 125 (36)
1901 ± 170 (36)
1764_+ 51 (36)
1970_+ 98 (36)
1830_+ 63 (36)
1994_+ 80 (36)
1826-+ 68 (36)
Grouped
1579 ± 101 (36)
1938 ± 127 (36)
1710_+ 64 2066_+ 277 (36) (36)
1909_+ 75 2170_+ 141 (36) (36)
1840-- 41 (36)
t-test separ-group
NS
NS
DD Rats
Quails
NS
*
NS
*
NS
*p<0.05; Cp<0.01; ~p<0.001; NS non-significant.
to L L and 3 days to L D corroborate the findings of no significant differences b e t w e e n separated and grouped rats (percentages of resting individuals 84--85%, n u m b e r of observations n=240-300). Concerning quails, percentages of resting individuals are: in in in in
LD LD LL LL
: : : :
53.33% 50.00% 66.11% 75.00%
(n=240) (n=240) (n= 180) (n= 180)
in in in in
separated individuals, grouped individuals, separated individuals, grouped individuals.
Chi-square testing shows greater (0.001
Correlations Between VCO.2 and (/0.2, (1C02 and Motor Activity Table 4 shows correlation coefficients in rats and quails b e t w e e n VCO2 and VOw, and b e t w e e n VCO2 and activity (either inductance or A n i m e x measurements) of rats measured e v e r y 20 minutes. The last 3 columns on the left of Table 4 give the correlation coefficients for the light (L), or dark (D) period, or for the whole circadian ( L + D ) period. All results shown are statistically significant (0.001
GROUPING AND RESPIRATORY BEHAVIOR
445
TABLE 3 TWO-WAYVARIANCEANALYSISOF SEPARATEDAND GROUPEDRATSAND QUAILSMEANVCO~ FROM TABLE 2. MEAN VCO~HAVE BEEN SAMPLEDEITHER EVERY24 hr, OR EVERY 12 hr DURING 3 DAYSAND A HALF OF EXPOSURETO CONSTANT100 LUX ILLUMINATION(LL) OR CONSTANTDARKNESS(DD); (n=NUMBEROF EXPERIMENTS) Mean VCO~ sampled every 12 hr
Mean VCO2 sampled every 24 hr
LL 06-18 (n=4)
18-06 (n=3)
06-06 (n=7)
Rats
between periods separ-group
F(3,3)= 82.36f F(1,3)= 315.95:~
F(2,2)= 0.76 NS F(I,2)= 6.76 NS
F(6,6)= 65.055 F(I,6)= 47.385
Quails
between periods separ-group
F(3,3)= 7.73 NS F(1,3)= 3 1 . 6 5 "
F(2,2)= 6.37 NS F(1,2)= 81.95'
F(6,6)= 9.819 F(1,6)= 89.48:~
DD 06--18 (n=3)
18-06 (n=4)
06-06 (n=7)
Rats
between periods separ-group
F(2,2)= 55.37* F(1,2)= 5518.36~:
F(3,3)= 5 3 . 4 1 7 F(1,3)= 244.66~
F(6,6)= 71.46:~ F(1,6)= 98.93:~
Quails
between periods separ-group
F(2,2)= F(1,2)=
F(3,3)= 24.24* F(I,3)= 0.27 NS
F(6,6)= 22.165 F(1,6)= 3.58 NS
5.51 NS 6.54 NS
*p<0.05; Cp<0.01; :~p<0.001; NS non-significant. actometry is its lack of a reliable calibration scale. In rats and quails observations of activity were made only during the L period. Bearing this in mind, the discussion will dwell mostly on the results of VCO2 measurements. Circadian rhythmicity of isolated rats and quails has been the object of many studies. Only a few concern circadian respiratory variations. Kayser and Hildwein [6], in isolated rats, reported circadian oxygen consumption and activity variations similar to ours. Boissin [4], in an extensive study, reported circadian motor activity rhythm in isolated Japanese quails which persisted in LL. The activity observations and measurements we performed in grouped rats and quails, and in isolated rats and
quails, corroborate what was reported for isolated animals by the previously mentioned authors. Isolated rats are chiefly active during D, whereas isolated quails are active only during L. Moreover a circadian rhythmicity is maintained in isolated rats and quails submitted for 5-6 days to LL (100 lux) or DD. The measurement of the respiratory exchanges of a group of small vertebrates necessarily multiplies the volume of oxygen consumed and carbon dioxide emitted. So this technique magnifies that which would have been very small for one isolated animal. But the recorded COs concentrations of a group of animals results from the sum of periodic (Fourier's theorem) and aperiodic variations of the CO,, con-
TABLE 4 STATISTICALCORRELATIONSBETWEENRESPIRATORY(VO2and VCO2)AND DISPLACEMENTACTIVITYIN GROUPEDOR ISOLATEDRATS OR QUAILS Species
Strain
Sex
Age days
Animals number
Rats
SpragueDawley
M
40
30
0.585 ~
0.872 ~
0.818
Quails
Japanese
M
38
11
0.462
0.909
0.901
Rats
SpragueDawley
VCO2--activity (inductance) M 337 16 0.733 1:
0.916 :~
0.849 $
Rats
SpragueDawley
Vco2---displacement (animex) M 63 6 0.598 :~
0.506 f
0.715 :~
?p
Correlation coefficient L D L + D
446
STUPFEL ET AL.
centrations of each animal of this group. In addition to this summing up, it should be considered whether grouping has, by itself, a "biological"respiratory effect. Let us first consider the effect of alternating light (L---~D and D--,L) on the respiratory activity, of which VCO.., is an index. In isolated, separated or grouped animals, significantly (p<0.001) greater VCO2 increases to L---~D than to D--~L are observed in rats which are nocturnal rodents. In quails, which are diurnal birds, VCO2 responses are significantly (p<0.001) greater in D---~L than in L---~D. A "biological" group effect is demonstrated in rats where the VCO., response to L---~Dis significantly (p<0.01) greater in grouped than in separated animals. These energy metabolic variations higher in grouped rats than in separated ones provokes a strong LD circadian synchronization. Moreover they could correspond to circadian activity modifications produced in rodents by crowding [1,5]. On the contrary, grouping does not significantly change respiratory variations of quails to D ~ L and L---~D. Secondly let us consider the mean VCO~ level of separated and grouped animals. In LDle:,.,, VCOe is significantly (p<0.001) greater in separated than in grouped rats, while on the contrary it is significantly (0.001
VCO2 curve would result from a desynchronization between different individuals. Societal interaction differences between rats and quails are also demonstrated by observations of the motor activity of isolated individuals belonging to groups. In rats, separated individuals have an activity circadian rhythm synchronized with the other individuals of the groups in LD, LL or DD. Furthermore, as reported above, a long term LL exposure shows that a delay of 13 days is necessary to suppress circadian VCO2 variations in grouped rats. On the contrary, disappearance of circadian variations of motor activity (observation of resting and active individuals) and of respiratory activity (VCO2) is obtained in grouped quails after only 3 days of LL. These interindividual activity and respiratory desynchronizations can be considered as "biological" effects of grouping in quails, for they constrast with the persisting circadian activity rhythmicity observed in LL in isolated quails [4] as well as in LL in isolated birds [2]. A similar interindividual activity desynchronization has been reported by Poppel [7] in a group of 4 men isolated in a bunker and with self-controlled illumination. To conclude, these experiments have tried, by placing rodents and birds of similar volumes in cages of similar dimensions, to reduce the differences which could be mainly related to body size discrepancies which circumscribe displacements in a limited housing. The separation procedure we used suppresses interindividual contacts but leaves visual, acoustical and olfactory communications between separated individuals, placed in separated wire mesh cages and enclosed in the same respiratory chamber. Therefore these energy metabolic differences in grouping effects shown between rats and quails probably result from interindividual contacts which are closer in groups of rats than in groups of quails. This agrees with the observation that, at the temperature of 20-21°C which we used and which was the temperature at which these animals were born and bred, grouped rats (in L) huddle during their resting period, while on the contrary grouped quails (in D) remain isolated. ACKNOWLEDGEMENTS We express all our gratitude to Professor Jtirgen Aschoff of the Max-Planck-Institut for Verhaltensphysiologie, Erling Andechs, West Germany, for suggestions and criticisms. We acknowledge skillful technical assistance of Miss Huguette Thierry and of Mr. Christian Lemercerre. Miss Dani~le Molin of the Acoustical Physiology Laboratory of the French National Institute of Agronomical Research (INRA), Jouy en Josas, 78, has performed the acoustical measurements and Mr. Adrian Pavely has reviewed the manuscript.
REFERENCES
1. Andrews, R. V. The physiology of crowding. Comp. Biochem. Physiol. 63A: 1-6, 1979. 2. Aschoff, J. Circadian rhythms in birds. Proceedings o f the XIV International Ornithology Congress. Oxford and Edinburgh: Blackwell, 1%7, pp. 81-105. 3. Aschoff, J. Desynchronization and resynchronization of human circadian rhythms. Aerospace Med. 40: 844-849, 1969. 4. Boissin, J. Photorrgulation des rythmes circadiens de la fonction corticosurrrnalienne et de l'activit6 grnrrale chez la Caille. Natural Sciences Thesis, Montpellier, 1973. 5. Calhoun, B. Social modification of activity rhythm in rodents. International Society o f Chronobiology, A l l International ConJbrence. Milan: I1 Ponte, 1977, pp. 83-93.
6. Kayser, C. and G. Hildwein. Le rythme circadien de la consommation d'oxyg/me et de l'activit6 locomotrice du rat; ses relations avec les deux formes de sommeil: le sommeil h ondes lentes et le sommeil paradoxal. Archs Sc. Physiol. 28:81-11 l, 1974. 7. Prppel, E. Desynchronisationen circadianer Rhythmen innerhalb einer isoliertien Gruppe. Pfliigers Arch. 299: 364-370, 1%8. 8. Stupfel, M. and G. Bouley. Physiological and biochemical effects on rats and mice exposed to small concentrations of carbon monoxide for long periods. Ann. N. Y. Acad. Sci. 174: 342368, 1970.
GROUPING AND RESPIRATORY BEHAVIOR 9. Stupfel, M. and D. Costagliola. Lifelong variations in heart rates in SPF Sprague Dawley rats of both sexes. Pfliigers Arch. 380: 189-195, 1979. 10. Stupfel, M., A. Perramon, J. M. Gasc, M. Magnier and M. Duriez. Body dimorphism and sex difference of resistance to an acute hypoxic challenge in rodents and birds of different ages. Comp. Biochem. Physiol. 59A: 347-354, 1978.
447 11. Weihe, W. H. The effect of light on animals. In: Control of the Animal House Environment, edited by T. McSheehy. London Laboratory Animals, 1976, pp. 63-76. 12. Wever, R. A. Circadian Systems of Man. Berlin-HeidelbergNew York: Springer, 1979.
Grouping and Respiratory Behavior Induced in Rats and Quails by LD12:12Illumination M A U R I C E S T U P F E L , .1 A R T H U R P E R R A M O N , ¢ P H I L I P P E Mt~RAT,t V I C T O R - H U G O D E M A R I A P E S C E , * Ht~LI~NE MASSIf* A N D V I ~ R O N I Q U E G O U R L E T *
*French National Institute of Health (INSERM) Physiopathological Mechanisms of Environmental Nuisances Research Group, Le Vdsinet, 78, France tFrench National Institute of Agronomical Research (INRA) Factorial Genetic Laboratory, Jouy en Josas, 78, France Received 19 D e c e m b e r 1979 STUPFEL, M., A. PERRAMON, P. MI~RAT, V.-H. DEMARIA PESCE, H. MASSI~ AND V. GOURLET. Grouping and respiratory behavior induced in rats and quails by LD12:12 illumination. PHYSIOL. BEHAV. 25(3) 439--447, 1980.Continuous recordings of the respiratory VCO2 and VOz of isolated, separated or grouped rats (Sprague-Dawley) and quails (Coturnix coturnix japonica) in a LDI~:n (L=100 lux) lighting regimen, under controlled environmental conditions of temperature, humidity and noise, and with food ad lib, show circadian rhythmicity with marked increases in respiratory amplitude at L--~D in rats and at D--*L in quails. Grouping increases these respiratory amplitude variations at L---~Dand D---~L in rats but not in quails. Continuous illumination (LL; 100 lux) provokes respiratory interindividual desynchronization after 13 days in grouped rats and after 3 days in quails. The differences in grouping effects shown between these two species are mostly due to interindividual contacts which, under the above experimental conditions, are closer in rats than in quails. Respiration
Motor activity
Grouping
Rats
THE importance of social synchronization in many human physiological activities is well appreciated. And yet with a few exceptions [3, 7, 12], human chronobiological studies have been performed on isolated individuals. This arises more through difficulties in recruitment, for a sufficiently long time, of a great enough number of volunteers or paid subjects having the same age and genetic origin, than from the task of submitting them to identical synchronizers under controlled environmental parameters. Animal experimentation, on the other hand, enables studies of groups of individuals of the same age, from a genetically defined origin and kept for a long time (considering the short life span of usual laboratory animals) under standardised environmental conditions. Another difficulty in studying groups arises from the necessity of having a great number of apparatus recording simultaneously physiological and biochemical parameters, and from the constraints of calculations involving a large quantity of data recorded for a long time on a large number of iridividuals. Measurement of the variations in respiratory gases emitted by a colony of animals, appears at first sight to be the simplest approach to the simultaneous study of a great number of individuals, since it aims to discover whether the total gas variations measured or recorded correspond to the sum of the respiratory exchanges of each individual. This leads one to investigate whether the resultant concentrations
Quails
Circadian rhythms
Illumination
Darkness
are merely a sum, or a biological addition, in other word whether or not a "group effect" is present. This study considers how respiratory behavior of two species of laboratory vertebrates, diurnal birds and nocturnal rodents, of approximately the same ages and kept under the same environmental conditions (temperature, humidity, ventilation, noise, food and water ad lib) react to alternating L (light) and D (dark). Quails and rats have been compared for, though they have predominant either diurnal or nocturnal activities, they are of similar size and therefore their activity could be compared under the same housing (cages) conditions. A 100 lux lighting and a LDI2:1.2(L=06:00-18:00) regimen has been chosen since it is probably the one most often used in animal houses [11]. METHOD
Animals Rats (Rattus norvegicus) were SPF (specific pathogen free) OFE from the breeding farm of IFFA-CREDO, Les Oncins, 69, France. They were fed with a special sterile commercial food (NoAo 3, UAR, Villemoisson sur Orge, 91, France), the composition of which has been described [9]. The Japanese quails (Coturnix coturnix japonica) were issued from the flock of the Factorial Genetics Laboratory (INRA, Jouy en Josas, 78, France). They were fed with
1Send reprint requests to Dr. Maurice Stupfel, Environmental Nuisances Research Group, INSERM, 44, Chemin de Ronde, 78110, Le
Vrsinet, France.
Copyright © 1980 Brain Research Publications Inc.--0031-9384/80/090439-09502.00/0
S T U P F E L ET AL.
440 INRA quail granules, the composition of which has been previously given [10]. The animals arrived at the I N S E R M Research Group of Le Vrsinet, 5-15 days before physiological measurements were made. On arrival they were placed in air conditioned (temperature 20-21°C; humidity 50-70%) animal rooms, LD,~:,~ (L=06:00-18:00 hr) lighted with 100 lux. All animals were randomly 5-6 grouped, sex separated, in wire mesh cages of either type A (42 cm long, 28 cm wide and 18 cm high), or type B (50 cm long, 40 cm wide and 29 cm high); isolated and separated animals were placed in wire mesh cages of type C (26 cm long, 20 cm wide and 16 cm high). Food and tap water were given ad lib.
Respiratory Measurements Several type A cages, each containing 5-6 grouped animals or several type C cages, each containing one single animal, were enclosed in two translucent lucite chambers (Nos. 1 and 2) (2 m long, 1 m wide and 1 m high) which were placed side by side but separated by blackened opaque faces. The use of these two similar chambers enabled simultaneous measurements and hence comparisons to be made (e.g. in one chamber groups of rats and in the other chamber separate rats, see below). Measurements of the respiratory exchanges of a single animal were made in a translucent lucite chamber (47 cm long, 43 cm wide and 38 cm high). All these "respiratory chambers" were placed in an air conditioned room. Inside them temperature, humidity and atmospheric pressure were continuously recorded (for details see Stupfel and Bouley [8]). The air of each chamber was renewed at the same rate measured with Pitot tubes or flowmeters. The carbon dioxide concentration inside each chamber varied between 0.10 and 0.25% depending on the COs emission of the animal or animals which were enclosed into these "respiratory chambers". Light was provided by fluorescent tubes "blanc (white) industrie" yelding 100 lux at 5 cm above the floor of each of the lucite chambers. Light was either alternating LD,~:,~ (06:00-18:00 hr) or continuously on (LL), or continuously off (DD). Inside the respiratory chambers the temperature range was 20.0-20.5°C and the humidity 60-80%. The level of background noise mainly due to climatiser and analysers was measured with a dB meter, and found to be 76 dB in each chamber. The concentrations of carbon dioxide and oxygen inside the respiratory chambers, sampled 25 cm from the roofs, were measured with two ONERA differential infrared carbon dioxide analysers (0-0.2% CO2) and a Pauling oxygen Beckman F:~ oxygen analyser (20-21% O2). Respiratory measurements were continuously made over periods lasting from 3 to 26 days, gas (O2, COz) concentrations being continuously recorded (MECI recorder) on the same paper. VCO2 and VO2 were expressed STPD, either per animal or per kg body weight.
Activity Measurements An Animex, type DSE activity meter (LKB), was used to measure displacements of isolated rats, placed isolated, in translucent polycarbonate cages (39 cm long, 25 cm wide and 18 cm high) which could be enclosed inside the "respiratory chambers." Numbers of counts for a 1-20 minute time inter-
val were recorded on a paper printer. Measurement of activity of grouped rats was performed by placing an inductance sensor (geophone) on the floor of the large respiratory chamber; vibrations were registered with a Brush recorder. As both types of these activity measurements appear to be not reliable for quails, observation and counts of the number of active and resting (lying down) individuals were performed, from I0:00 to 16:00 at 2-hour intervals, in separated and grouped rats and quails (15 separated and 3 groups of 5 of each species) during 3 days in LD and during 3 days in LL.
Group Effect Determination To compare the effects of interindividual reactions on respiratory activity the following procedure was designed. In one (No. 1) of the 2 identical large respiratory chambers (2 m long, 1 m wide, 1 m high) were placed n wire mesh cages of type A, each containing n' (5--6) rats or quails. In the second (No. 2) respiratory chamber were placed the same number n × n' of rats or quails, but each animal was placed in a separate wire mesh cage of type C. Carbon dioxide and oxygen concentrations were continuously and simultaneously recorded in both chambers. One LKB actometer was placed in each chamber, each recording the displacement activity of a single rat or quail. This procedure enables comparison of the VCO2 and VO2 of both chambers containing the same number of animals but whilst in chamber No. 1 animals have interindividual contacts and so will be called " g r o u p e d " animals, in chamber No. 2 animals have no interindividual contacts and so will be called " s e p a r a t e d " animals. In several experiments " g r o u p e d " animals were placed in chamber No. 2 and " s e p a r a t e d " animals in chamber No. 1, and it was proved that no "chamber effect" was affecting the observed results.
Statistics In expressing the results the arithmetic mean is followed by the standard deviation: mean _+ SD. Assuming that in LD VCOz recordings in L or in D can be considered as straight lines Student's t-test has been used. Furthermore, two-way analysis of variance has been used to compare " g r o u p e d " and " s e p a r a t e d " individuals at different times of LD, L L and DD exposures. Chi-square testing was used to compare active and resting animals during L L and LD exposure. p is the probability, and statistical significance has been assessed for at least p<0.05. RESULTS
Respiratory Activity in Isolated Rats and Quails in LD,2:,2, LL and DD Continuous recordings of VCO2 and of VO2 of an isolated Sprague-Dawley rat and of an isolated Japanese quail were performed on 2-4 individuals of each species placed either in a LD,2:,2, L L or DD regimen. In LD,2:,2 (Fig. 1) t-testing of the mean VCO,., and VO,, taken every 20 minutes on the records, shows that for the rat the respiratory activity is statistically (p<0.001) greater during the dark (D) period than during the light (L) period and that conversely for the quail the respiratory activity is statistically (p<0.001) greater during L than during D. In isolated
GROUPING AND RESPIRATORY BEHAVIOR ~°2
---
3.oi,
441
~,OLATED QUAIL
co 2
!
Q05,I IIIll Ill ,'1 I l l I , I
'"
I
T
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,'
! I I !
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0.04
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1.0HOURS !
l
O0
l
06
I
12
l
18
I
00
I
O6
FIG. 1. Recordings of carbon dioxide emitted by one isolated Sprague-Dawley male rat (solid line) and one isolated Japanese male quail (dotted line) in LD12:,z. In ordinates VCO~ which have been calculated STPD in liter per kg body weight and per hour. Circadian and ultradian VCO2 variations can be observed on the recordings.
17
07
18
19
FIG. 2. Parts of the recording of the carbon dioxide percentages measured in a respiratory chamber containing one male Japanese quail. Peaks A and C are CO.2%increases when light is on at 06:00 (D-~L) and when light is off at 18:00 (L-~D). B and D are CO~% decreases followingpeaks A and C.
Vco2 .... 3.0 1
/ .
individuals of both species sharp VCOz and VO2 increases are noted at 06:00 (D--*L) and 18:00 (L---*D) (portions A and C in gra.phs of Fig. 2). Circadian VCO2 rhythmicity is still observed in an isolated rat or quail maintained for 5 days in an LL or DD exposure. The results of 2 experiments performed with 2 male rats (93 day old) and 2 male quails (167 day old) show changes in r which corroborate data of the literature (i.e. ~'>24 hr in nocturnal rodents in LL and in diurnal birds in DD, and r<24 hr in nocturnal rodents in DD and in diurnal birds in LL).
06
5 GROUPS OF 5 RATS 25 SEPARATED RATS
t%t.-w- ° ; . - " ,,o,,,,,, _:I f n .
2
.
18
( t~ .
, ~
C
O0
06
~'
. n
.$.% I v G L=.s.,.. , ,i w
~
12
18
O0
06
FIG. 3. 36-hour VCO2 variations in Sprague-Dawley male rats, 25 separated individuals (dotted line) and 5 groups of 5 (solid line) in LD,~:12.
Respiratory Activity in Separated and Grouped Rats and Quails in LD12:12 As previously mentioned under methods, an experimental procedure was designed to simultaneously record respiratory activity of grouped and separated animals of the same species. Two such experiments were performed in duplicate, two with separated and grouped rats, two with separated and grouped quails. For rats the first experiment (a) used 5 groups of 5 males (chamber No. 1) compared to 25 isolated males (chamber No. 2); the rats were 36 day old at the beginning of the experiment which lasted 24 days. The second experiment (b) used 3 groups of 5 males compared to 15 isolated males; these rats were 81 day old at the beginning of the experiment which lasted 8 days. For quails the first experiment (c) was done with 1 group of 6 and I group of 5 males compared to 11 separated males; the quails were 37 day old at the beginning of the experiment which lasted 3 days. The second experiment (d) was performed with 3 groups (2 groups of 5 males and 1 group of 6 females) compared to 16 (10 males and 6 females) separated quails; these quails were 58 day old at the beginning of the experiment which lasted 7 days.
Two points will be considered concerning the results of these 4 experiments. (1) In all these experiments VCO2 recordings (Figs. 3 and 4) show the same patterns as found in isolated animals (Fig. 1). Respiratory variations are observed at 06:00 and 18:00 when light is turned on and off. The VCO2 variations in 15 separated and in 15 grouped rats, and in 16 separated and in 16 grouped quails are shown in Table 1. Column 1 shows VCO2 increases (+~VCO2) at 06:00 when light is turned on (D-~L; Fig. 2: part A of the VCO2 recording) and column 5 shows CO2 increases (+AVCO2) at 18:00 when light is turned off (L-~D; Fig. 2 i part C); the duration of these light on and light off induced VCO2 increases are given in columns 2 and 6. The after-effects which follow ~'CO~ increases at the beginning and end of illumination and which appear as VCO,, decreases (-AVCO2; Fig. 2: parts B and D) are shown in columns 3 and 7, and their respective durations in columns 4 and 8. t-testing (statistical results in lines 3 and 6 of Table (1) shows statistically significant differences between separated and grouped rats (at 06:00 for the 2nd VCO2 variation,
442
STUPFEL ET AL. TABLE 1
COMPARISON IN 15 SEPARATED AND IN 15 GROUPED RATS, AND IN 16 SEPARATED AND IN 16 GROUPED QUAILS OF THE FIRST A N D SECOND VCOz VARIATIONS (COLUMNS 1, 3, 5, 7) AND OF THE DURATIONS OF THESE VARIATIONS (COLUMNS 2, 4, 6, 8) AT 06:00 WHEN LIGHT (100 lux) IS TURNED ON (I)-~L) AND AT 18:00 WHEN LIGHT IS TURNED OFF (L--*D); i ± SD (n): MEAN ± STANDARD DEVIATION AND (NUMBER OF EXPERIMENTS)
Species
D--~L (06:00)
(animals number)
Rats (15)
1st variation +A VCO.~ t % min
1st variation +A VCO2 t % min
2nd variation - A VCO~ t % min
5.50 ± 1.52 15.00 ± 0.00 17.83 ± 7.30 34.17 ± 10.21 12.00 ± 2.74 18.00 ± 2.74 2.20 ± 1.10 11.00 ± 2.24
Separated
(6)
Grouped
Quails (16)
L--,D (18:00)
2nd variation - A VCO~ t % min
(6)
(6)
(6)
(5)
(5)
(5)
(5)
5.67 ± 2.16 10.83 ± 2.04 25.55 ± 6.41 37.50 ± II.73 28.80 _ 4.44 25.00 ± 6.12 11.80 ± 3.96 19.00 ± 7.42 (6) (6) (6) (6) (5) (5) (5) (5)
t-test separ-group
NS
"t
Separated
24.20 _+ 5.00 (5)
8.00 _+ 2.74 (5)
Grouped
23.75 _+ 1.09 9.00 _ 2.24
t-test separ-group
*
NS
"~
NS
2.00 _+ 0.71 4.20 _+ 3.49 4.00 ± 2.00 (5) (5) (5)
l-
*
7.00 _+ 2.74 30.60 ± 7.66 42.00 ± 10.37 (5) (5) (5)
2.20 ± 1.10 4.80 ± 3.27 3.20 ± 1.48 6.40 ± 3.51 29.40 ± 3.78 42.00 ± 5.70
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
NS
NS
NS
NS
NS
NS
NS
NS
*p<0.05; ep<0.01; NS non-significant.
p<0.05; at 18:00 for the 1st, p<0.01 and the 2nd, p<0.01 VCO2 variation). In quails no significant differences in ~VCO2% are observed between separated and grouped animals. The duration of these VCO2 variations are significantly shorter for the 1st VCO2 variation (/9<0.01) at 06:00 and significantly longer for the 2nd VCO2 variation (p<0.05) at 18:00 in grouped than in separated rats. N o significant differences of durations of VCO2 variations are observed between grouped and separated quails. (2) Secondly, as in isolated animals, mean respiratory levels of grouped and/or separated animals are significantly different in L and in D.
Vco 2
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06
F o r experiment a, figure 3 shows that the VCO2 levels during L and D are higher in the 25 separated rats than in the 5 batches of 5 grouped rats. This is confirmed by t-tests comparing VCOz (values taken every 20 minutes) during the L and D periods in these separated and grouped rats (p<0.001). For experiment c, Fig. 4 and t-testing show that t h e VCOz levels are higher in the grouped male quails than in the separated male quails (p< 0.001). For experiments b and d, once every 20 minute VCO,, sampling has been performed 72 times during 48 successive hours in 15 separated (347 - 17 g) and 3 groups of 5 (355 _+ 25 g) male rats, 81 day old (experiment b), and in 15 separated (165 _+ 17 g) and 3 groups of 5 (171 - 20 g) male quails, 58 day old (experiment d). This gives the followiflg VCO2 values expressed in ml.kg-Khr -1, during L and D:
1
12
I
18
FIG. 4.36-hour VCO~ variations in Japanese male quails: 1 group of 5 plus 1 group of 6 (solid line), and 11 separated individuals (dotted line) in LD1~:12.
: : : :
L=1177 L = 873 L=2318 L=2962
_+ 92 _+ 93 _+ 185 _ 279
(72) (72) (72) (72)
; D=1507 ; D=1259 ; D=1589 ; D=2267
_+ ± _+ _+
81 (72) 128 (72) 159(72) 194 (72)
t-tests show that separated rats have significantly higher (p<0.001) VCO2 than grouped rats either during L or during D, and that on the other hand separated quails have significantly lower VCO2 than grouped quails either during L (p<0.001) or during D (p<0.05). Moreover in rats (experiment b), both separated and grouped, VCOz is significantly (p<0.001) lower during L than during D. Conversely quails (experiment d), both separated and grouped, have a significantly (p<0.001) higher VCO2 during L than during D. Placing grouped rats or grouped quails in larger wire mesh cages (from cages A to cages B) does not change these differences between separated and grouped rats or quails.
GROUPING A N D RESPIRATORY B E H A V I O R 15/Vco2 • j
~
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FIG. 5.24-hour VCO2 in Sprague-Dawley male rats: 3 groups of 5 (solid line) and 15 separated (dotted line) individuals after 3 days and a half exposure either to LL (left side of figure) or to DD (right side of figure).
Respiratory Activity in Separated and Grouped Rats and Quails in LL and DD Exposure to continuous lighting (LL) or to continuous darkness (DD) suppresses the L--->D and D--~L induced VCO2 increases. L L decreases the mean VCO2 of rats (/9<0.001) and increases the mean VCO2 of quails (0.01
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,
06
FIG. 6.24-hour VCO2 in Japanese quails: 3 groups of 5 (solid line) and 15 separated (dotted line) individuals after 3 days and a half exposure either to LL (left side of figure) or to DD (right side of figure). 7) on the right of Table 2 (second 12 hours of third day and first 12 hours of fourth day of L L or DD) are statistically (p<0.001) different, with the exception of grouped quails in L L for which 2371 - 77 (36) does not statistically differ from 2402 __. 92 (36). A two-way variance analysis of the means has been performed on the 12-hour period (06-18 and 18-06) VCOz data of Table 2. Statistical results of this comparison of mean VCO., sampled either every 12 hours or every 24 hours are shown in Table 3. Significant differences are obtained for rats in all cases, with the exception of the three 18-06 periods during L L where the VCO2 differences between these 3 periods as well as between separated and grouped individuals are not statistically significant. In quails the VCO~ differences between separated and grouped individuals are statistically significant during L L but not significant during DD; moreover in these birds during L L the VCO2 differences between the four 06-18 and the three 18-06 periods are not statistically significant, and during DD the VCO2 differences between the three 06-18 periods are similarly not significant. To assess circadian self-sustainment of respiratory rhythms in grouped animals long term 100 lux L L experiments were performed in preference to long term DD experiments, since constant lighting appears to be more easy and more reliable to achieve than constant darkness. In 5 groups of 5 male rats, 422 day old, placed in respiratory chamber No. 1, disappearance of the whole group (5 x5 individuals) VCO2 circadian variations was observed after 13 days of I00 lux L L exposure. Under the same conditions in 1 group of 8 quails, 45 day old, no more circadian VCO2 variations of the group were obtained after 2 days of 100 lux L L exposure.
Motor Activity Observations and Measurements Concerning motor activity neither agonistic behavior, nor freezing and alarm responses were noted in grouped rats and in grouped quails. Huddling was usually noted in rats during L. Animex activity measurements of rats during 3 days and a half of L L exposure show that rats in a same chamber (whether they are separated or grouped in this chamber) have interindividual parallel records of activity displacement and VCO2, which shows interindividual "syncrhonization." Observations and counts from 10:00 to 16:00 of the number of rats active and resting during 3 days of exposure
S T U P F E L ET A L
444
TABLE 2 TWICE A DAY (06-18 AND 18-06 hr) VCO2, ml-kg-'.hr-' (MEAN -+ SD (n): MEAN LEVEL -+ STANDARD DEVIATION (NUMBER OF DATA)) OF SEPARATED OR GROUPED RATS AND QUAILS DURING 3 DAYS AND A HALF OF EXPOSURE TO CONSTANT 100 LUX ILLUMINATION (LL) OR CONSTANT DARKNESS (DD) 1st day
2nd day
06-18
18-06
Separated
837 ± 51 (36)
1052 ± 56 (33)
Grouped
766 ± 56 (36)
991 ± 92 (33)
t-test separ-grouped
:~
?
06-18
18-06
4th day 06-18
879± 166 1080± 56 (33) (36)
886± 64 (36)
1007 ± 81 (36)
907 ± 85 (36)
825 ± 89 (33)
982 ± 88 (36)
825 ± 49 (36)
988 ± 85 (36)
845 ± 88 (36)
~
$
06-18
3rd day 18-06 LL
Rats
Quails
NS
NS
?
Separated
2498 ± 137 2216 ± 295 (36) (36)
2260 ± 105 2054 ± 94 2188 ± 109 2056± 106 2256 ± 105 (32) (36) (29) (36) (36)
Grouped
2850 ± 154 2568 ± 180 2639 ± 154 2507 ± 153 2555 ± 128 2371 ± 77 2402 ± 92 (32) (36) (29) (36) (36) 36) (36)
t-test separ-group
:~
$
Separated
1353 ± 72 (36)
1158 ± 72 (36)
1448_+ 63 (36)
1185_+ 104 (36)
1434_+ 56 (36)
1186+_ 69 (36)
1452_+ 44 (36)
Grouped
1234 ± 78 (36)
987 ± 81 (36)
1329_+ 77 (36)
1007_+ 122 (36)
1329±- 68 (36)
1015 ± 102 (36)
1362 ± 68 (36)
t-test separ-group
:~
?
Separated
1561 ± 125 (36)
1901 ± 170 (36)
1764_+ 51 (36)
1970_+ 98 (36)
1830_+ 63 (36)
1994_+ 80 (36)
1826-+ 68 (36)
Grouped
1579 ± 101 (36)
1938 ± 127 (36)
1710_+ 64 2066_+ 277 (36) (36)
1909_+ 75 2170_+ 141 (36) (36)
1840-- 41 (36)
t-test separ-group
NS
NS
DD Rats
Quails
NS
*
NS
*
NS
*p<0.05; Cp<0.01; ~p<0.001; NS non-significant.
to L L and 3 days to L D corroborate the findings of no significant differences b e t w e e n separated and grouped rats (percentages of resting individuals 84--85%, n u m b e r of observations n=240-300). Concerning quails, percentages of resting individuals are: in in in in
LD LD LL LL
: : : :
53.33% 50.00% 66.11% 75.00%
(n=240) (n=240) (n= 180) (n= 180)
in in in in
separated individuals, grouped individuals, separated individuals, grouped individuals.
Chi-square testing shows greater (0.001
Correlations Between VCO.2 and (/0.2, (1C02 and Motor Activity Table 4 shows correlation coefficients in rats and quails b e t w e e n VCO2 and VOw, and b e t w e e n VCO2 and activity (either inductance or A n i m e x measurements) of rats measured e v e r y 20 minutes. The last 3 columns on the left of Table 4 give the correlation coefficients for the light (L), or dark (D) period, or for the whole circadian ( L + D ) period. All results shown are statistically significant (0.001
GROUPING AND RESPIRATORY BEHAVIOR
445
TABLE 3 TWO-WAYVARIANCEANALYSISOF SEPARATEDAND GROUPEDRATSAND QUAILSMEANVCO~ FROM TABLE 2. MEAN VCO~HAVE BEEN SAMPLEDEITHER EVERY24 hr, OR EVERY 12 hr DURING 3 DAYSAND A HALF OF EXPOSURETO CONSTANT100 LUX ILLUMINATION(LL) OR CONSTANTDARKNESS(DD); (n=NUMBEROF EXPERIMENTS) Mean VCO~ sampled every 12 hr
Mean VCO2 sampled every 24 hr
LL 06-18 (n=4)
18-06 (n=3)
06-06 (n=7)
Rats
between periods separ-group
F(3,3)= 82.36f F(1,3)= 315.95:~
F(2,2)= 0.76 NS F(I,2)= 6.76 NS
F(6,6)= 65.055 F(I,6)= 47.385
Quails
between periods separ-group
F(3,3)= 7.73 NS F(1,3)= 3 1 . 6 5 "
F(2,2)= 6.37 NS F(1,2)= 81.95'
F(6,6)= 9.819 F(1,6)= 89.48:~
DD 06--18 (n=3)
18-06 (n=4)
06-06 (n=7)
Rats
between periods separ-group
F(2,2)= 55.37* F(1,2)= 5518.36~:
F(3,3)= 5 3 . 4 1 7 F(1,3)= 244.66~
F(6,6)= 71.46:~ F(1,6)= 98.93:~
Quails
between periods separ-group
F(2,2)= F(1,2)=
F(3,3)= 24.24* F(I,3)= 0.27 NS
F(6,6)= 22.165 F(1,6)= 3.58 NS
5.51 NS 6.54 NS
*p<0.05; Cp<0.01; :~p<0.001; NS non-significant. actometry is its lack of a reliable calibration scale. In rats and quails observations of activity were made only during the L period. Bearing this in mind, the discussion will dwell mostly on the results of VCO2 measurements. Circadian rhythmicity of isolated rats and quails has been the object of many studies. Only a few concern circadian respiratory variations. Kayser and Hildwein [6], in isolated rats, reported circadian oxygen consumption and activity variations similar to ours. Boissin [4], in an extensive study, reported circadian motor activity rhythm in isolated Japanese quails which persisted in LL. The activity observations and measurements we performed in grouped rats and quails, and in isolated rats and
quails, corroborate what was reported for isolated animals by the previously mentioned authors. Isolated rats are chiefly active during D, whereas isolated quails are active only during L. Moreover a circadian rhythmicity is maintained in isolated rats and quails submitted for 5-6 days to LL (100 lux) or DD. The measurement of the respiratory exchanges of a group of small vertebrates necessarily multiplies the volume of oxygen consumed and carbon dioxide emitted. So this technique magnifies that which would have been very small for one isolated animal. But the recorded COs concentrations of a group of animals results from the sum of periodic (Fourier's theorem) and aperiodic variations of the CO,, con-
TABLE 4 STATISTICALCORRELATIONSBETWEENRESPIRATORY(VO2and VCO2)AND DISPLACEMENTACTIVITYIN GROUPEDOR ISOLATEDRATS OR QUAILS Species
Strain
Sex
Age days
Animals number
Rats
SpragueDawley
M
40
30
0.585 ~
0.872 ~
0.818
Quails
Japanese
M
38
11
0.462
0.909
0.901
Rats
SpragueDawley
VCO2--activity (inductance) M 337 16 0.733 1:
0.916 :~
0.849 $
Rats
SpragueDawley
Vco2---displacement (animex) M 63 6 0.598 :~
0.506 f
0.715 :~
?p
Correlation coefficient L D L + D
446
STUPFEL ET AL.
centrations of each animal of this group. In addition to this summing up, it should be considered whether grouping has, by itself, a "biological"respiratory effect. Let us first consider the effect of alternating light (L---~D and D--,L) on the respiratory activity, of which VCO.., is an index. In isolated, separated or grouped animals, significantly (p<0.001) greater VCO2 increases to L---~D than to D--~L are observed in rats which are nocturnal rodents. In quails, which are diurnal birds, VCO2 responses are significantly (p<0.001) greater in D---~L than in L---~D. A "biological" group effect is demonstrated in rats where the VCO., response to L---~Dis significantly (p<0.01) greater in grouped than in separated animals. These energy metabolic variations higher in grouped rats than in separated ones provokes a strong LD circadian synchronization. Moreover they could correspond to circadian activity modifications produced in rodents by crowding [1,5]. On the contrary, grouping does not significantly change respiratory variations of quails to D ~ L and L---~D. Secondly let us consider the mean VCO~ level of separated and grouped animals. In LDle:,.,, VCOe is significantly (p<0.001) greater in separated than in grouped rats, while on the contrary it is significantly (0.001
VCO2 curve would result from a desynchronization between different individuals. Societal interaction differences between rats and quails are also demonstrated by observations of the motor activity of isolated individuals belonging to groups. In rats, separated individuals have an activity circadian rhythm synchronized with the other individuals of the groups in LD, LL or DD. Furthermore, as reported above, a long term LL exposure shows that a delay of 13 days is necessary to suppress circadian VCO2 variations in grouped rats. On the contrary, disappearance of circadian variations of motor activity (observation of resting and active individuals) and of respiratory activity (VCO2) is obtained in grouped quails after only 3 days of LL. These interindividual activity and respiratory desynchronizations can be considered as "biological" effects of grouping in quails, for they constrast with the persisting circadian activity rhythmicity observed in LL in isolated quails [4] as well as in LL in isolated birds [2]. A similar interindividual activity desynchronization has been reported by Poppel [7] in a group of 4 men isolated in a bunker and with self-controlled illumination. To conclude, these experiments have tried, by placing rodents and birds of similar volumes in cages of similar dimensions, to reduce the differences which could be mainly related to body size discrepancies which circumscribe displacements in a limited housing. The separation procedure we used suppresses interindividual contacts but leaves visual, acoustical and olfactory communications between separated individuals, placed in separated wire mesh cages and enclosed in the same respiratory chamber. Therefore these energy metabolic differences in grouping effects shown between rats and quails probably result from interindividual contacts which are closer in groups of rats than in groups of quails. This agrees with the observation that, at the temperature of 20-21°C which we used and which was the temperature at which these animals were born and bred, grouped rats (in L) huddle during their resting period, while on the contrary grouped quails (in D) remain isolated. ACKNOWLEDGEMENTS We express all our gratitude to Professor Jtirgen Aschoff of the Max-Planck-Institut for Verhaltensphysiologie, Erling Andechs, West Germany, for suggestions and criticisms. We acknowledge skillful technical assistance of Miss Huguette Thierry and of Mr. Christian Lemercerre. Miss Dani~le Molin of the Acoustical Physiology Laboratory of the French National Institute of Agronomical Research (INRA), Jouy en Josas, 78, has performed the acoustical measurements and Mr. Adrian Pavely has reviewed the manuscript.
REFERENCES
1. Andrews, R. V. The physiology of crowding. Comp. Biochem. Physiol. 63A: 1-6, 1979. 2. Aschoff, J. Circadian rhythms in birds. Proceedings o f the XIV International Ornithology Congress. Oxford and Edinburgh: Blackwell, 1%7, pp. 81-105. 3. Aschoff, J. Desynchronization and resynchronization of human circadian rhythms. Aerospace Med. 40: 844-849, 1969. 4. Boissin, J. Photorrgulation des rythmes circadiens de la fonction corticosurrrnalienne et de l'activit6 grnrrale chez la Caille. Natural Sciences Thesis, Montpellier, 1973. 5. Calhoun, B. Social modification of activity rhythm in rodents. International Society o f Chronobiology, A l l International ConJbrence. Milan: I1 Ponte, 1977, pp. 83-93.
6. Kayser, C. and G. Hildwein. Le rythme circadien de la consommation d'oxyg/me et de l'activit6 locomotrice du rat; ses relations avec les deux formes de sommeil: le sommeil h ondes lentes et le sommeil paradoxal. Archs Sc. Physiol. 28:81-11 l, 1974. 7. Prppel, E. Desynchronisationen circadianer Rhythmen innerhalb einer isoliertien Gruppe. Pfliigers Arch. 299: 364-370, 1%8. 8. Stupfel, M. and G. Bouley. Physiological and biochemical effects on rats and mice exposed to small concentrations of carbon monoxide for long periods. Ann. N. Y. Acad. Sci. 174: 342368, 1970.
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447 11. Weihe, W. H. The effect of light on animals. In: Control of the Animal House Environment, edited by T. McSheehy. London Laboratory Animals, 1976, pp. 63-76. 12. Wever, R. A. Circadian Systems of Man. Berlin-HeidelbergNew York: Springer, 1979.