Physiology & Behavior, Vol. 17, pp. 735-741. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Environmental Enrichment and Crowding: Behavioral and Hormonal Effects 1 ELAINE M. HULL, CHRIS KASTANIOTIS, GEORGIA L'HOMMEDIEU AND JONATHAN FRA N Z
Department o f Psychology, S. U. N. Y. at Buffalo, 4230 Ridge Lea Road, Buffalo N Y 14226 (Received 22 July 1975) HULL, E. M., C. KASTANIOTIS, G. L'HOMMEDIEU AND J. R. FRANZ. Environmental enrichment and crowding: behavioral and hormonal effects. PHYSIOL. BEHAV. 17(5) 735-741, 1976. - - Male and female gerbils lived from weaning to adulthood in Enriched Crowded, Plain Crowded, Dense Crowded, or Paired conditions. Daily observations, social interaction tests with one animal from each of the 4 groups, and anatomical measures were made, and levels of cortisol and testosterone were determined by radioimmunoassay. In the social interaction tests Paired gerbils marked and fought the most. Home cage observations estabished that Enriched animals failed to pair bond or establish nests in the small cubicles provided. They also reproduced no more successfully than did other crowded groups, while all crowded groups reproduced less than Paired animals. All groups of crowded animals had lighter testes and adrenal glands than Paired animals, although their cortisol levels were consisderably higher than Paired animals'. This inverse relationship of cortisol and adrenal weight was interpreted as a sign of extreme adrenal activity on the part of crowded animals. Enriched animals had cortisol levels intermediate between those of Paired and of Dense and Plain crowded animals, and their testosterone levels were as high as those of Paired males, indicating some diminution of hormonal effects of crowding. There was, however, little evidence of diminished behavioral and reproductive effects of crowding by environmental enrichment. Environmental crowding
Hormonal effects
Aggression
Reproduction
of crowding but rather appeared to exacerbate them. Most frequently, animals which have been crowded for a relatively long period (at least 2 weeks) exhibit decreased aggressiveness. However, Intermittent Isolation animals in this previous experiment were more aggressive, and this factor may have accounted for their reproduction being poorer than even the Plain-Crowded group. Since isolation is known to increase aggressiveness, and since in the previous experiment much of the aggression was observed immediately after regrouping, the present experiment was designed to provide free access to refuge, rather than enforced isolation. Thus, decreased intensity of social stimulation could be available whenever an animal sought it without the disruptive effects of daily handling, isolation and regrouping. One group of animals lived from weaning to adulthood in a physically enriched environment with access to small tunnels and cubicles leading off from the central area. A second group was housed in a plain square enclosure, with' an equal amount of space per animal. A third group lived in a much smaller space. The fourth group lived in male-female pairs, with the same amount of space per animal as the first two groups. Daily observations were made of the animals' behavior in the home cages. At adulthood controlled social interaction tests were made. At the termination of the experiment, animals were sacrificed, organ weights recorded, and blood obtained for radioimmunoassay of cortisol and testosterone levels.
IT has been frequently observed that crowding animals leads to decreased reproduction, increased adrenal activity, and behavioral changes, including decreased aggression in social interaction tests and in the home cage if a sufficient period of adjustment is allowed [2, 5, 7, 12, 13, 16, 18, 27]. However, several investigators have reported increased aggressiveness among crowded animals [9, 15, 28], and/or no adrenal hypertrophy [4, 7, 17, 29, 33 ]. In gerbils we have observed depressed ventral gland marking, aggression, and nonaggressive contacts, as well as diminished reproductive success, resulting from crowding normal animals [17,18]. We have also seen smaller ventral glands (used by gerbils to deposit a sebaceous substance on low-lying objects in the environment) and in some experiments smaller testes and/or enlarged adrenal glands in crowded animals [ 17,18]. In a previous experiment in this lab [ 17], one group of crowded gerbils was placed into individual, physically enriched boxes for 2 hr per day for 10 weeks in order to determine whether this intermittent isolation would diminish the physiological and/or behavioral effects of crowding. However, the crowded animals subjected to intermittent isolation became more aggressive both in the home cage and in social interaction tests, and reproduced less than either paired or continuously crowded animals. There was no indication of adrenal hyperactivity, although testes weights were lower in all crowded males. Thus, the enforced periods of isolation did not ameliorate the effects
~We wish to thank Dr. W. Roy Slaunwhite and Dr. N. Kundu for their help and use of facilities. This work was partially supported by grant No. 1 RO3 MH 25782-01 from NIMH and by Institutional Funds from a Biomedical Support Grant to SUNY/Buffalo. 735
H U L L ET AL.
736 METHOD
Animals Sixty-four weanling gerbils (32 male, 32 female) were obtained from T u m b l e b r o o k Farms (West Brookfield, Mass.) and individually earmarked. Animals of each sex were r a n d o m l y assigned to the following four conditions: 16/large enriched cage (Enriched, or E), 16/large plain cage (Plain, or P1), 16/small cage (Dense, or D), or 2/small cage (Paired, or Pr). Food, water, and w o o d e n blocks for gnawing were freely available; paper for shredding was supplied daily. E, P1, and Pr animals were allowed 64 cm: per animal; D animals were allowed 8 cm ~ per animal. All cages were made out of clear Plexiglas. The enriched e n v i r o n m e n t consisted of a large central octagonal space (34 cm/side, 42 cm high), with a small tunnel connecting to a red Plexiglas cubicle emerging from each of the eight sides. Rocks, sticks, a hollow cinderblock, and a food and water dispenser were in the central area. The plain e n v i r o n m e n t was a square (81 × 81 x 42 cm) enclosure with a f o o d and water dispenser in the center. Pr and D animals were housed in standard (10.5 x 40.5 × 16 cm) laboratory cages with overhead f o o d and water dispensers.
Apparatus Social interaction tests were c o n d u c t e d in a 1 m 2 open field apparatus, painted gray and lined off into 16 squares of equal size. Nine pegs made of clear plastic dowels, 2 cm high, were inserted into holes at the corners of each square except along the b o u n d a r y of the field. The sides were 48 cm high. A sheet of Plexiglas, with holes into which the pegs fit, covered the floor for easy cleaning. A manual keyboard connected to electromechanical counters was used to record each of the social behaviors.
Procedure Daily 1 hr observations were made of animals in their h o m e cages. Three observations were made at night. Location and quality of nests, animals most active, instances of aggression, other social interactions, ventral gland marking, and presence of pups were recorded. At 120 days of age social interaction (SI) tests were c o n d u c t e d in the open field apparatus. Each test lasted 10 min., and the number of ventral gland rubs, aggressive encounters, sniff or nonaggressive contacts, and escape a t t e m p t s initiated by each animal were recorded. Each animal was tested against one other animal of the same sex f r o m each of the 4 environments (4 tests per animal), in counter balanced order. When behavioral measures were completed, animals were sacrificed by exsanguination under ether anesthesia, and body, ventral gland, adrenal gland, and testes weights were recorded. Blood was collected in heparinized tubes, and plasma levels of cortisol (for all animals) and testosterone (for males) were determined by radioimmunoassay using commercially prepared a n t i b o d y and standard and tritiated h o r m o n e s (New England Nuclear). Steroids were extracted from 0.1 ml samples of plasma with m e t h y l e n e chloride. Extraction of relevant steroids was determined to be at least 80% of the total. Blank, zero, standard and sample tubes were run in duplicate. Following an incubation period to allow labeled-unlabeled antigen c o m p e t i t i o n to reach
equilibrium, an activiated charcoal suspension was added to appropriate tubes to adsorb u n b o u n d antigen. After centrifugation, the supernatant containing the bound antigen was transferred to scintillation vials for counting under conditions optimized for tritium. The percent b o u n d for each sample was used to interpolate the q u a n t i t y of h o r m o n e in nanograms from the standard curve. Calculation of the u n k n o w n h o r m o n e c o n c e n t r a t i o n in ug% was then made, taking into account dilutions, v o l u m e extracted, and fractional recovery.
RESULTS
Home Environment Observations One litter was produced in the E environment; 4, in the P1; 3, in the D; and 7, in the Pr cages. Parturient females exhibited p o s t p a r t u m estrus, and were frequently chased by numerous males. A l t h o u g h the mothers a t t e m p t e d to retrieve the pups, all 28 offspring in the 3 crowded cages were trampled to death within a few hours. All 37 pups born to the p a i r e d a n i m a l s survived until weaning. In each group s o m e fairly consistent dominance/submissiveness relationships could be discerned, in which a given d o m i n a n t animal regularly chased a n d / o r attacked one or more other animals. However, a gerbil attacked by one d o m i n a n t animal was also f r e q u e n t l y attacked by others as well. F o r example, the 4 animals in the P1 condition which were eventually killed were repeatedly attacked by n u m e r o u s cagemates. In the 3 crowded dages there were somewhat more instances of aggression by females than by males, and most of the aggression by b o t h males and females was directed toward females. A m o n g Pr animals, males and females were equally aggressive, though, of course, same-sex aggression was precluded by the housing conditions. Positive correlations for all animals were found between activity and grooming another animal (r = .50, d f = 57, p < 0 . 0 1 ) . Within-group correlations are given in Table 1. However, correlations between daily observation scores and social interaction scores were near zero. TABLE1 WITHIN-GROUP CORRELATIONS AMONG DAILY OBSERVATION MEASURES*
Activity Aggression
Groom
r = r = r = r = r = r = r =
0.6391 for PI 0.9224 for En 0.7903 for PI 0.8391 for PI 0.7434 for Pr 0.6379 for P1 0.5744 for D
Aggression d (2 ~? 9 9 9
r = 0.6691 for PI r = 0.8054 for Pr c~ r = 0.7666 for PI 9
*For all correlations df = 8, rt0.05)= 0.6319, rt0.011= 0.7646 In all three crowded conditions, at least half the animals were observed on any occasion to be huddled together. F r e q u e n t l y in the E condition one or two cubicles were literally stuffed with gerbils; the particular cubicles chosen varied from time to time. Also, one or two cubicles were frequently stuffed with wood shavings and shredded paper; the location of these also varied. Neither pair-bonding nor
ENVIRONMENTAL ENRICHMENT AND CROWDING
737
t e r r i t o r i a l defense was o b s e r v e d . No n e s t s were e s t a b l i s h e d in E cubicles, a n d t h e o n e litter p r o d u c e d in this env i r o n m e n t was f o u n d dead in t h e c e n t r a l o p e n area. Most a n i m a l s w h i c h were active in t h e c r o w d e d e n v i r o n m e n t s o n any one day returned periodically to the group huddle. On a n o t h e r d a y t h e y m i g h t n o t leave t h e g r o u p d u r i n g t h e p e r i o d of o b s e r v a t i o n . T h u s , t h e g r o u p h u d d l e s e e m e d to b e t h e focal p o i n t f r o m w h i c h s o m e a n i m a l s e m e r g e d to b e active for a t i m e a n d t o w h i c h t h e y r e t u r n e d at t h e end of t h a t time. T h e r e were, h o w e v e r , s o m e a n i m a l s (2 P1 males, 2 P1 females, a n d 1 D m a l e ) w h i c h were m o r e or less p e r m a n e n t o u t c a s t s . T h e y were f r e q u e n t l y a t t a c k e d and all 5 were e v e n t u a l l y killed ( a f t e r social i n t e r a c t i o n tests were c o m p l e t e d ) . T h e y were n o t replaced. T w o Pr f e m a l e s e i t h e r died or were killed b y t h e i r c a g e m a t e s w i t h i n t h e first 30 days; t h e t w o were r e p l a c e d b y a n i m a l s of t h e same age a n d sex f r o m o u r c o l o n y , and o n e r e p l a c e m e n t later p r o d u c e d a litter.
Social Interaction Tests As can be seen in T a b l e 2, paired males engaged in m o r e v e n t r a l m a r k i n g , F ( 3 , 3 0 ) = 5.01, p < 0 . 0 1 , a n d aggression, F ( 3 , 3 0 ) = 4.29, p < 0 . 0 5 , t h a n a n y o t h e r g r o u p of males. Paired females also m a r k e d m o r e t h a n a n y o t h e r f e m a l e
group, F ( 3 , 3 0 ) = 6.65, p < 0 . 0 1 , a l t h o u g h t h e i r increased aggressiveness did n o t r e a c h statistical significance. O t h e r g r o u p s did n o t differ significantly a c c o r d i n g to N e w m a n Keuls c o m p a r i s o n s for e i t h e r sex. Males m a r k e d m o r e f r e q u e n t l y t h a n did females (t = 1.67, df = 62, p < 0 . 0 5 ) . T h e r e were n o significant main effects in nonaggressive sniffs a n d c o n t a c t s . Aggression was c o r r e l a t e d w i t h v e n t r a l m a r k i n g (r = .58, df = 57, p < 0 . 0 1 ) a n d w i t h nonaggressive sniffs a n d c o n t a c t s (r = 42, d f = 57, p < 0 . 0 1 ) . H o u s i n g c o n d i t i o n o f o p p o n e n t p r o d u c e d n o c o n s i s t e n t effect o n social i n t e r a c t i o n scores (Table 3).
Physiological Measures As can be seen in Table 4, Pr males h a d t h e heaviest testes, F ( 3 , 2 6 ) = 3.07, p < 0 . 0 5 , while o t h e r groups did n o t differ f r o m each o t h e r o n N e w m a n - K e u l s c o m p a r i s o n s . V e n t r a l gland weights of t h e Pr and P1 g r o u p s were h i g h e r t h a n t h o s e o f t h e o t h e r t w o groups, F ( 3 , 5 0 ) = 8.34, p < 0 . 0 1 , a n d males h a d heavier v e n t r a l glands t h a n did females, F ( 1 , 5 0 ) = 138.5, p < 0 . 0 0 0 1 . Males also h a d heavier a d r e n a l glands t h a n did females, F ( 1 , 5 2 ) = 21.75, p < 0 . 0 1 . T h e Pr g r o u p h a d heaviest adrenals, f o l l o w e d by E, P1, a n d D, respectively, F ( 3 , 5 2 ) = 4.08, p < 0 . 0 5 , t h o u g h t h e r e was a significant sex x e n v i r o n m e n t i n t e r a c t i o n with Pr females
TABLE 2 SOCIAL INTERACTION TEST SCORES (MEANS PER INTERACTION FOR EACH GROUP) Enriched Mean S.D.
Plain Mean S.D
Marking 1.31 Aggression 4.69 Sniff Contact 57.25
1.82 5.70
0.41 2.31
3.70
58.50
Marking 0.91 Aggression 6.66 Sniff Contact 70.34
1.02 5.48
0.25 3.66
12.07
53.19
Dense Mean S.D.
Males 0.68 3.42 7.04 Females .08 2.57 8.08
Paired Mean S.D.
2.69 1.81
4.56 1.64
20.13 11.31
24.17 9.90
59.72
15.86
70.44
13.90
0.06 5.38
0.18 5.12
5.97 12.69
5.59 14.81
60.16
20.75
63.94
17.61
TABLE 3 WITHIN-GROUP CORRELATIONS AMONG SOCIAL INTERACTION MEASURES Sniff & Contact Marking Aggression
Grooming Escape
r r r r r
= = = = =
0.8226 0.8042 0.7807 0.7749 0.6917
for for for for for
Pr D 9 D o~ D 9 Pr 9
Marking
Aggression
r = 0.7945 for Pr 0-> r = 0.8098 for D 9 r = 0.6338 for D o->
*For all correlations df = 8, qo.os) = 0.6319, rto.ol)= 0.7646.
r = 0.8498 for En o~ r = 0.6592 for En 9
HULL E T A L
738
TABLE 4 PHYSIOLOGICAL MEASURES
Enriched Mean S.D.
Plain Mean
Dense S.D.
Paired
Mean
S.D.
Mean
S.D.
Males Body weight (g) Testes (mg) Ventral gland (mg) Adrenal glands (mg) Relative testes (testes in mg) (body in g) Relative Adrenal (adrenals in mg) (body in g) Relative ventral gland (ventral gland in mg) (body in g) Cortisol ( ~zg%) Testosterone ( ~ g%)
72.62
5.40
68.38
2.01
75.29
10.43
70.38
7.23
958.37
83.02
968.75
52.51
944.38
171.35
1095.38
83.93
104.75
26.68
120.30
25.24
129.06
63.23
140.84
22.70
31.84
6.13
32.61
3.20
30.84
9.83
31.21
5.50
13.38
1.41
14.62
1.30
12.75
2.19
15.00
1.31
.438
.118
.477
.054
1.442
0.352
1.756
0.353
60.52
56.10
88.07
25.91
4.92
5.60
2.23
1.07
.410
.085
.443
.083
1.67
0.59
2.01
0.343
101.74
24.41
45.14
22.56
3.61
2.40
4.82
4.46
Females Body weight (g) Testes (mg) Ventral gland (mg) Adrenal glands (mg) Relative testes wt. (testes in mg) (body in g) Relative adrenal wt. (adrenals in mg) (body in g) Relative ventral gland (ventral gland in mg) (body in g) Cortisol (~,g%) Testosterone ( t~ g%)
62.06
5.82
--
--
66.21
4.05
61.58
7.35
74.53
7.99
25.51
17.82
78.60
28.96
36.29
25.51
66.92
16.77
27.78
5.60
25.88
2.50
21.36
4.80
31.35
2.53
--
--
.447
.096
.390
.050
.347
.090
.421
.037
.411
.286
1.187
.774
.558
.335
.890
.159
39.92
26.82
51.12
30.36
58.24
36.35
24.55
25.56
ENVIRONMENTAL ENRICHMENT AND CROWDING and P1 males having heaviest adrenals and D animals of both sexes possessing lightest adrenal glands, F(3,52) = 14.92, p
Environmental Enrichment and Crowding: Behavioral and Hormonal Effects 1 ELAINE M. HULL, CHRIS KASTANIOTIS, GEORGIA L'HOMMEDIEU AND JONATHAN FRA N Z
Department o f Psychology, S. U. N. Y. at Buffalo, 4230 Ridge Lea Road, Buffalo N Y 14226 (Received 22 July 1975) HULL, E. M., C. KASTANIOTIS, G. L'HOMMEDIEU AND J. R. FRANZ. Environmental enrichment and crowding: behavioral and hormonal effects. PHYSIOL. BEHAV. 17(5) 735-741, 1976. - - Male and female gerbils lived from weaning to adulthood in Enriched Crowded, Plain Crowded, Dense Crowded, or Paired conditions. Daily observations, social interaction tests with one animal from each of the 4 groups, and anatomical measures were made, and levels of cortisol and testosterone were determined by radioimmunoassay. In the social interaction tests Paired gerbils marked and fought the most. Home cage observations estabished that Enriched animals failed to pair bond or establish nests in the small cubicles provided. They also reproduced no more successfully than did other crowded groups, while all crowded groups reproduced less than Paired animals. All groups of crowded animals had lighter testes and adrenal glands than Paired animals, although their cortisol levels were consisderably higher than Paired animals'. This inverse relationship of cortisol and adrenal weight was interpreted as a sign of extreme adrenal activity on the part of crowded animals. Enriched animals had cortisol levels intermediate between those of Paired and of Dense and Plain crowded animals, and their testosterone levels were as high as those of Paired males, indicating some diminution of hormonal effects of crowding. There was, however, little evidence of diminished behavioral and reproductive effects of crowding by environmental enrichment. Environmental crowding
Hormonal effects
Aggression
Reproduction
of crowding but rather appeared to exacerbate them. Most frequently, animals which have been crowded for a relatively long period (at least 2 weeks) exhibit decreased aggressiveness. However, Intermittent Isolation animals in this previous experiment were more aggressive, and this factor may have accounted for their reproduction being poorer than even the Plain-Crowded group. Since isolation is known to increase aggressiveness, and since in the previous experiment much of the aggression was observed immediately after regrouping, the present experiment was designed to provide free access to refuge, rather than enforced isolation. Thus, decreased intensity of social stimulation could be available whenever an animal sought it without the disruptive effects of daily handling, isolation and regrouping. One group of animals lived from weaning to adulthood in a physically enriched environment with access to small tunnels and cubicles leading off from the central area. A second group was housed in a plain square enclosure, with' an equal amount of space per animal. A third group lived in a much smaller space. The fourth group lived in male-female pairs, with the same amount of space per animal as the first two groups. Daily observations were made of the animals' behavior in the home cages. At adulthood controlled social interaction tests were made. At the termination of the experiment, animals were sacrificed, organ weights recorded, and blood obtained for radioimmunoassay of cortisol and testosterone levels.
IT has been frequently observed that crowding animals leads to decreased reproduction, increased adrenal activity, and behavioral changes, including decreased aggression in social interaction tests and in the home cage if a sufficient period of adjustment is allowed [2, 5, 7, 12, 13, 16, 18, 27]. However, several investigators have reported increased aggressiveness among crowded animals [9, 15, 28], and/or no adrenal hypertrophy [4, 7, 17, 29, 33 ]. In gerbils we have observed depressed ventral gland marking, aggression, and nonaggressive contacts, as well as diminished reproductive success, resulting from crowding normal animals [17,18]. We have also seen smaller ventral glands (used by gerbils to deposit a sebaceous substance on low-lying objects in the environment) and in some experiments smaller testes and/or enlarged adrenal glands in crowded animals [ 17,18]. In a previous experiment in this lab [ 17], one group of crowded gerbils was placed into individual, physically enriched boxes for 2 hr per day for 10 weeks in order to determine whether this intermittent isolation would diminish the physiological and/or behavioral effects of crowding. However, the crowded animals subjected to intermittent isolation became more aggressive both in the home cage and in social interaction tests, and reproduced less than either paired or continuously crowded animals. There was no indication of adrenal hyperactivity, although testes weights were lower in all crowded males. Thus, the enforced periods of isolation did not ameliorate the effects
~We wish to thank Dr. W. Roy Slaunwhite and Dr. N. Kundu for their help and use of facilities. This work was partially supported by grant No. 1 RO3 MH 25782-01 from NIMH and by Institutional Funds from a Biomedical Support Grant to SUNY/Buffalo. 735
H U L L ET AL.
736 METHOD
Animals Sixty-four weanling gerbils (32 male, 32 female) were obtained from T u m b l e b r o o k Farms (West Brookfield, Mass.) and individually earmarked. Animals of each sex were r a n d o m l y assigned to the following four conditions: 16/large enriched cage (Enriched, or E), 16/large plain cage (Plain, or P1), 16/small cage (Dense, or D), or 2/small cage (Paired, or Pr). Food, water, and w o o d e n blocks for gnawing were freely available; paper for shredding was supplied daily. E, P1, and Pr animals were allowed 64 cm: per animal; D animals were allowed 8 cm ~ per animal. All cages were made out of clear Plexiglas. The enriched e n v i r o n m e n t consisted of a large central octagonal space (34 cm/side, 42 cm high), with a small tunnel connecting to a red Plexiglas cubicle emerging from each of the eight sides. Rocks, sticks, a hollow cinderblock, and a food and water dispenser were in the central area. The plain e n v i r o n m e n t was a square (81 × 81 x 42 cm) enclosure with a f o o d and water dispenser in the center. Pr and D animals were housed in standard (10.5 x 40.5 × 16 cm) laboratory cages with overhead f o o d and water dispensers.
Apparatus Social interaction tests were c o n d u c t e d in a 1 m 2 open field apparatus, painted gray and lined off into 16 squares of equal size. Nine pegs made of clear plastic dowels, 2 cm high, were inserted into holes at the corners of each square except along the b o u n d a r y of the field. The sides were 48 cm high. A sheet of Plexiglas, with holes into which the pegs fit, covered the floor for easy cleaning. A manual keyboard connected to electromechanical counters was used to record each of the social behaviors.
Procedure Daily 1 hr observations were made of animals in their h o m e cages. Three observations were made at night. Location and quality of nests, animals most active, instances of aggression, other social interactions, ventral gland marking, and presence of pups were recorded. At 120 days of age social interaction (SI) tests were c o n d u c t e d in the open field apparatus. Each test lasted 10 min., and the number of ventral gland rubs, aggressive encounters, sniff or nonaggressive contacts, and escape a t t e m p t s initiated by each animal were recorded. Each animal was tested against one other animal of the same sex f r o m each of the 4 environments (4 tests per animal), in counter balanced order. When behavioral measures were completed, animals were sacrificed by exsanguination under ether anesthesia, and body, ventral gland, adrenal gland, and testes weights were recorded. Blood was collected in heparinized tubes, and plasma levels of cortisol (for all animals) and testosterone (for males) were determined by radioimmunoassay using commercially prepared a n t i b o d y and standard and tritiated h o r m o n e s (New England Nuclear). Steroids were extracted from 0.1 ml samples of plasma with m e t h y l e n e chloride. Extraction of relevant steroids was determined to be at least 80% of the total. Blank, zero, standard and sample tubes were run in duplicate. Following an incubation period to allow labeled-unlabeled antigen c o m p e t i t i o n to reach
equilibrium, an activiated charcoal suspension was added to appropriate tubes to adsorb u n b o u n d antigen. After centrifugation, the supernatant containing the bound antigen was transferred to scintillation vials for counting under conditions optimized for tritium. The percent b o u n d for each sample was used to interpolate the q u a n t i t y of h o r m o n e in nanograms from the standard curve. Calculation of the u n k n o w n h o r m o n e c o n c e n t r a t i o n in ug% was then made, taking into account dilutions, v o l u m e extracted, and fractional recovery.
RESULTS
Home Environment Observations One litter was produced in the E environment; 4, in the P1; 3, in the D; and 7, in the Pr cages. Parturient females exhibited p o s t p a r t u m estrus, and were frequently chased by numerous males. A l t h o u g h the mothers a t t e m p t e d to retrieve the pups, all 28 offspring in the 3 crowded cages were trampled to death within a few hours. All 37 pups born to the p a i r e d a n i m a l s survived until weaning. In each group s o m e fairly consistent dominance/submissiveness relationships could be discerned, in which a given d o m i n a n t animal regularly chased a n d / o r attacked one or more other animals. However, a gerbil attacked by one d o m i n a n t animal was also f r e q u e n t l y attacked by others as well. F o r example, the 4 animals in the P1 condition which were eventually killed were repeatedly attacked by n u m e r o u s cagemates. In the 3 crowded dages there were somewhat more instances of aggression by females than by males, and most of the aggression by b o t h males and females was directed toward females. A m o n g Pr animals, males and females were equally aggressive, though, of course, same-sex aggression was precluded by the housing conditions. Positive correlations for all animals were found between activity and grooming another animal (r = .50, d f = 57, p < 0 . 0 1 ) . Within-group correlations are given in Table 1. However, correlations between daily observation scores and social interaction scores were near zero. TABLE1 WITHIN-GROUP CORRELATIONS AMONG DAILY OBSERVATION MEASURES*
Activity Aggression
Groom
r = r = r = r = r = r = r =
0.6391 for PI 0.9224 for En 0.7903 for PI 0.8391 for PI 0.7434 for Pr 0.6379 for P1 0.5744 for D
Aggression d (2 ~? 9 9 9
r = 0.6691 for PI r = 0.8054 for Pr c~ r = 0.7666 for PI 9
*For all correlations df = 8, rt0.05)= 0.6319, rt0.011= 0.7646 In all three crowded conditions, at least half the animals were observed on any occasion to be huddled together. F r e q u e n t l y in the E condition one or two cubicles were literally stuffed with gerbils; the particular cubicles chosen varied from time to time. Also, one or two cubicles were frequently stuffed with wood shavings and shredded paper; the location of these also varied. Neither pair-bonding nor
ENVIRONMENTAL ENRICHMENT AND CROWDING
737
t e r r i t o r i a l defense was o b s e r v e d . No n e s t s were e s t a b l i s h e d in E cubicles, a n d t h e o n e litter p r o d u c e d in this env i r o n m e n t was f o u n d dead in t h e c e n t r a l o p e n area. Most a n i m a l s w h i c h were active in t h e c r o w d e d e n v i r o n m e n t s o n any one day returned periodically to the group huddle. On a n o t h e r d a y t h e y m i g h t n o t leave t h e g r o u p d u r i n g t h e p e r i o d of o b s e r v a t i o n . T h u s , t h e g r o u p h u d d l e s e e m e d to b e t h e focal p o i n t f r o m w h i c h s o m e a n i m a l s e m e r g e d to b e active for a t i m e a n d t o w h i c h t h e y r e t u r n e d at t h e end of t h a t time. T h e r e were, h o w e v e r , s o m e a n i m a l s (2 P1 males, 2 P1 females, a n d 1 D m a l e ) w h i c h were m o r e or less p e r m a n e n t o u t c a s t s . T h e y were f r e q u e n t l y a t t a c k e d and all 5 were e v e n t u a l l y killed ( a f t e r social i n t e r a c t i o n tests were c o m p l e t e d ) . T h e y were n o t replaced. T w o Pr f e m a l e s e i t h e r died or were killed b y t h e i r c a g e m a t e s w i t h i n t h e first 30 days; t h e t w o were r e p l a c e d b y a n i m a l s of t h e same age a n d sex f r o m o u r c o l o n y , and o n e r e p l a c e m e n t later p r o d u c e d a litter.
Social Interaction Tests As can be seen in T a b l e 2, paired males engaged in m o r e v e n t r a l m a r k i n g , F ( 3 , 3 0 ) = 5.01, p < 0 . 0 1 , a n d aggression, F ( 3 , 3 0 ) = 4.29, p < 0 . 0 5 , t h a n a n y o t h e r g r o u p of males. Paired females also m a r k e d m o r e t h a n a n y o t h e r f e m a l e
group, F ( 3 , 3 0 ) = 6.65, p < 0 . 0 1 , a l t h o u g h t h e i r increased aggressiveness did n o t r e a c h statistical significance. O t h e r g r o u p s did n o t differ significantly a c c o r d i n g to N e w m a n Keuls c o m p a r i s o n s for e i t h e r sex. Males m a r k e d m o r e f r e q u e n t l y t h a n did females (t = 1.67, df = 62, p < 0 . 0 5 ) . T h e r e were n o significant main effects in nonaggressive sniffs a n d c o n t a c t s . Aggression was c o r r e l a t e d w i t h v e n t r a l m a r k i n g (r = .58, df = 57, p < 0 . 0 1 ) a n d w i t h nonaggressive sniffs a n d c o n t a c t s (r = 42, d f = 57, p < 0 . 0 1 ) . H o u s i n g c o n d i t i o n o f o p p o n e n t p r o d u c e d n o c o n s i s t e n t effect o n social i n t e r a c t i o n scores (Table 3).
Physiological Measures As can be seen in Table 4, Pr males h a d t h e heaviest testes, F ( 3 , 2 6 ) = 3.07, p < 0 . 0 5 , while o t h e r groups did n o t differ f r o m each o t h e r o n N e w m a n - K e u l s c o m p a r i s o n s . V e n t r a l gland weights of t h e Pr and P1 g r o u p s were h i g h e r t h a n t h o s e o f t h e o t h e r t w o groups, F ( 3 , 5 0 ) = 8.34, p < 0 . 0 1 , a n d males h a d heavier v e n t r a l glands t h a n did females, F ( 1 , 5 0 ) = 138.5, p < 0 . 0 0 0 1 . Males also h a d heavier a d r e n a l glands t h a n did females, F ( 1 , 5 2 ) = 21.75, p < 0 . 0 1 . T h e Pr g r o u p h a d heaviest adrenals, f o l l o w e d by E, P1, a n d D, respectively, F ( 3 , 5 2 ) = 4.08, p < 0 . 0 5 , t h o u g h t h e r e was a significant sex x e n v i r o n m e n t i n t e r a c t i o n with Pr females
TABLE 2 SOCIAL INTERACTION TEST SCORES (MEANS PER INTERACTION FOR EACH GROUP) Enriched Mean S.D.
Plain Mean S.D
Marking 1.31 Aggression 4.69 Sniff Contact 57.25
1.82 5.70
0.41 2.31
3.70
58.50
Marking 0.91 Aggression 6.66 Sniff Contact 70.34
1.02 5.48
0.25 3.66
12.07
53.19
Dense Mean S.D.
Males 0.68 3.42 7.04 Females .08 2.57 8.08
Paired Mean S.D.
2.69 1.81
4.56 1.64
20.13 11.31
24.17 9.90
59.72
15.86
70.44
13.90
0.06 5.38
0.18 5.12
5.97 12.69
5.59 14.81
60.16
20.75
63.94
17.61
TABLE 3 WITHIN-GROUP CORRELATIONS AMONG SOCIAL INTERACTION MEASURES Sniff & Contact Marking Aggression
Grooming Escape
r r r r r
= = = = =
0.8226 0.8042 0.7807 0.7749 0.6917
for for for for for
Pr D 9 D o~ D 9 Pr 9
Marking
Aggression
r = 0.7945 for Pr 0-> r = 0.8098 for D 9 r = 0.6338 for D o->
*For all correlations df = 8, qo.os) = 0.6319, rto.ol)= 0.7646.
r = 0.8498 for En o~ r = 0.6592 for En 9
HULL E T A L
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TABLE 4 PHYSIOLOGICAL MEASURES
Enriched Mean S.D.
Plain Mean
Dense S.D.
Paired
Mean
S.D.
Mean
S.D.
Males Body weight (g) Testes (mg) Ventral gland (mg) Adrenal glands (mg) Relative testes (testes in mg) (body in g) Relative Adrenal (adrenals in mg) (body in g) Relative ventral gland (ventral gland in mg) (body in g) Cortisol ( ~zg%) Testosterone ( ~ g%)
72.62
5.40
68.38
2.01
75.29
10.43
70.38
7.23
958.37
83.02
968.75
52.51
944.38
171.35
1095.38
83.93
104.75
26.68
120.30
25.24
129.06
63.23
140.84
22.70
31.84
6.13
32.61
3.20
30.84
9.83
31.21
5.50
13.38
1.41
14.62
1.30
12.75
2.19
15.00
1.31
.438
.118
.477
.054
1.442
0.352
1.756
0.353
60.52
56.10
88.07
25.91
4.92
5.60
2.23
1.07
.410
.085
.443
.083
1.67
0.59
2.01
0.343
101.74
24.41
45.14
22.56
3.61
2.40
4.82
4.46
Females Body weight (g) Testes (mg) Ventral gland (mg) Adrenal glands (mg) Relative testes wt. (testes in mg) (body in g) Relative adrenal wt. (adrenals in mg) (body in g) Relative ventral gland (ventral gland in mg) (body in g) Cortisol (~,g%) Testosterone ( t~ g%)
62.06
5.82
--
--
66.21
4.05
61.58
7.35
74.53
7.99
25.51
17.82
78.60
28.96
36.29
25.51
66.92
16.77
27.78
5.60
25.88
2.50
21.36
4.80
31.35
2.53
--
--
.447
.096
.390
.050
.347
.090
.421
.037
.411
.286
1.187
.774
.558
.335
.890
.159
39.92
26.82
51.12
30.36
58.24
36.35
24.55
25.56
ENVIRONMENTAL ENRICHMENT AND CROWDING and P1 males having heaviest adrenals and D animals of both sexes possessing lightest adrenal glands, F(3,52) = 14.92, p
