Interstrain fighting in male mice

INTERSTRAIN FIGHTING IN MALE MICE* LOUIS LEVINE, CAROL ANNE DIAKOW AND GAIL ELKIN BARSEL Biology Department of The City College, of the City University of New York .

BY

Introduction Fighting behaviour in mice has been studied by many different investigators (for general review see Scott, 1958) . Most of these studies have been on problems of conditioning (Ginsburg & Allee, 1942), competition for food (Fredericson & Birnbaum, 1954), effects of early social experience (Kahn, 1954 ; King, 1957), effects of hormones (Beeman, 1947), or the effects of learned fighting on subsequent sexual behaviour (Kahn, 1961) . Although the relationship between social dominance and sexual activity has been studied in chickens (Fennell, 1945 ; Guhl, 1953), there have not been any studies which examine the correlation between fighting ability and mating effectiveness in male mice taken from different inbred strains . The importance of this type of experiment lies in the fact that any behaviour which results in a reproductive superiority of one type of male over his competitor will lead, over many generations, to evolutionary changes in a population . The use of inbred strains permits a determination of the extent of genetic control over the behaviour since, under identical environments, any behavioural differences of the inbred strains must be due to their different hereditary backgrounds . A proper analysis of the relation of hereditary background, behaviour and reproductive effectiveness is necessary if we are to achieve an understanding of the origin and development of the complex behavioural patterns that exist in organisms . The present experiments are outgrowths of a study by one of us (Levine, 1958) on mating competition between males from two inbred strains of mice . In that study it was found that when a pigmented, black-agouti, male (CBA/J),

an albino male (ST/J), and an albino female (ST/J) are housed together, the albino male sires 83 per cent . of the offspring . A number of hypotheses were proposed as possible explanations of the reproductive superiority of the albino male. One of these hypotheses was that there may have been a male-male interaction in which social dominance, as achieved by fighting, led to reproductive dominance . In support of this hypothesis, it was reported that in chance observations of 27 fights which had occurred in the experimental cages, the albino males were the winners in 25, while the pigmented males were the victors in only 2 . The experiments reported here are parts of a continuing study of the relationship between mating success and different behaviour patterns in these two strains of mice . This paper is a comparison of the fighting abilities of males of two strains of mice under three different sets of laboratory conditions . Future papers will extend our analysis of the relation between fighting behaviour and mating success . Materials and Methods The males used in the first two experiments were born and raised in this laboratory and were the offspring of mice obtained from The Jackson Laboratory. Those used in the third experiment were born at The Jackson Laboratory . The mice were fed a diet of Rockland Mouse Pellets and Rockland Rabbit Ration . Food and water were available at all times. Wood shavings were used as bedding material . The animals were housed in cages that were I1 inches by 7 inches, by 5 inches deep . Once a week, when their cages were cleaned, the males were bathed in a 2 . 4 per cent . Kreso Dip solution, as a protection against ectoparasites . In all three experiments, the males were paired for interstrain fighting tests, every male of one strain meeting each male of the opposite strain but once (Ginsburg & Allee, 1942) . For the contests, the males were placed in a cage which was foreign to both of them . The fighting cage was of the same size as the cages in which the males were housed . There was a removable partition that extended diagonally

*The research in this report was supported by Grants GB-312 and GB-1390 from the National Science Foundation, and by grants from the Society of the Sigma Xi and Resa Research Fund and The City College Fund . The authors wish to thank Miss Gail Gelband, Miss Jean Hapliznik and Mrs . Nimia Patran for their efforts in caring for the mouse colony at The City College . The authors also thank Dr. Ethel Tobach for statistical advice . During the course of this work, one of us (G .E.B .) was the recipient of a Sergei Zlinkoff Scholarship from The City College of the City University of New York. 52

LEVINE el a/. : INTERSTRAIN FIGHTING IN MALE MICE

across the fighting cage, separating it into two compartments . A fluorescent light was placed 14-} inches above the floor of the pen . The males were left, each in his own compartment, for three minutes before the partition was removed . No food or water was available to the males during the time they were in the fighting pen . The number of contests resulting in fights were recorded . Contests were classified as no fight, draw, CBA victory, or ST victory . Contests were further classified as ST, CBA, or mutual initial attack . The latency of initial attack was also noted . (This is the time from lifting of the partition to the first attack .) The number of attacks initiated by each male was recorded in each contest as were mutual attacks (simultaneous attacks by both males on one another) . A male was declared to be a victor if he was the first to elicit submissive behaviour from his opponent after an attack . We have defined an attack as the biting or attempted biting of one male by another . Our criterion for submission was the appearance of one or more of the following : (1) running, (2) squealing, (3) leaping (a jump across the cage), (4) freezing (attacked male just stands still, often sitting on his genital region) . Except where otherwise indicated, the MannWhitney U two-tailed test was used for statistical analysis of the data (Siegel, 1956) . Experiment I Eleven albino (ST) males and 11 pigmented (CBA) males, isolated since weaning, were paired in round-robin fashion once each week and left together until one male attacked the other and elicited submission behaviour from him, or until 30 minutes had passed without a submission . The contests were begun when the median age of both CBA and ST males was 101 days, with a range of 92 to 122 days for the CBA males and a range of 93 to 115 days for the ST males . Fights were conducted between 8 .40 a .m . and 3 .30 p .m. The mean temperature of the room was 78 ° F, varying between 75 ° and 81'F . In the 3 minutes allowed the isolated males in the fighting pen, the CBA males were more active, in exploring the cage than the ST males . When the partition was lifted, it was almost always the CBA who approached the ST rather than vice-versa . This might have been a natural result of the greater activity of the CBA, since after the partition was lifted he continued to explore more than the ST . Very

53

often the approach of a male to his opponent seemed to be an accidental result of his cage explorations . There were no evident strain differences in the methods of attack and submission of the males . During the 11 weeks of the experiment, 121 contests were observed . The results of the contests are shown in Table I, column a . The data clearly illustrate the superiority of the CBA mares . They won more contests, were more often the first to attack, and had a greater number of total attacks than the ST males (p<0 .002 for each of the above) . The CBA attack latency was lower than that of the ST (p<0 . 05) . As shown in Table II, a distinct pattern of victories emerged during the 11 weeks of the contests . In the first week, the ST males won 5 contests while the CBA males won only 3 . Thereafter, the CBA males won no less than 9 of the I1 contests each week, and eventually won all 11 fights each week . The average attack latency per week is shown in Table III . It was greatest in the first week (11 . 9 minutes), falling thereafter to a fairly constant level between 2 . 0 and 4 . 0 minutes . After the first week, the average attack latency closely resembles the CBA average attack latency . This is due to the fact that after the first week, the CBA males were most often the first to attack in any contest . Experiment II The methods and materials used in this experiment closely paralleled those of Experiment I, with one major change . The length of time of the contests was extended to 30 minutes . As in the first experiment, 11 albino (ST) and 11 pigmented (CBA) males were used . At the start of this series of contests, the median age of the CBA males was 108 days, with a range from 108 to 115 days and the median age of the ST males was 109 days, with a range from 95 to 113 days . The apparatus used for observations was modified somewhat from that used in Experiment I . High glass walls were placed around the regular fighting pen, increasing in depth from 5 to 12 inches . This was done to prevent the attacked male from jumping out of the cage, as had been observed to occur in an earlier trial series of 30minute fights . The temperature of the room averaged 77°F, varying between 70° and 80°F . Observations were conducted between 7 .45 a .m . and 7 .45 p .m . The initial behaviour of the mice in this series of 121 contests was similar to that in Experiment I . The CBA males were more active than



54

ANIMAL BEHAVIOUR, XIII, I Table I. Comparison of the Results of Experiments I, II and III . a

b

c

Experiment I

Experiment If

Experiment III

1st victory isolated d8'

30 minutes isolated dd

1st victory social experience

Number of victories ST

13 (10 . 7 %)

6 (5 . 0 %)

103(85-2%)

106(87-6%)

87 (71 . 9 %)

Number of draws

4(3-3%)

1(, 0 . 8%)

7 (5 .8 %)

Number of no-fights

1 (0 . 8 %)

8 (6 . 6 %)

27 (22 . 3 %)

CBA

0

I

Average Attack Latency (minutes) ST

9 . 7±2 . 7

9 . 3±0 . 7

2 .0-*-

CBA

3 . 0±0 .4

3 . 1±0 . 7

4 . 5±0 . 6

Mutual

1 . 0---

5 . 2±3 .5

4 . 3±2 . 4

ST

19(15 .8%)

6(5 . 3%)

1 ( :1 . 1%)

CBA

99(82-5%)

102(90-3%)

89(94-7%)

Mutual

2(1 .7%)

5(4-4%)

4(4-2%)

ST

48(25 . 5%)

35(0 . 7%)

10(6 . 2%)

137(72-9%)

5283 (98-9%)

130(81-3%)

3(l .6%)

23(0 . 4%)

20(12-5%)

Number of times first to attack

Number of attacks

CBA Mutual

*Based on only one case .

the ST males both before and after the partition was removed, and continued to be so throughout the 30 minutes of each of the contests . The results of the contests are shown in Table I, column b . Again the data clearly indicate the superiority of the CBA males . To facilitate a comparison of Experiment I and II, a male was considered to be a winner of a contest if he was the first to elicit submissive behaviour from his opponent . In comparing the performances of CBA and ST males in Experiment H, it is found that the CBA males were victors more times, they were first to attack more often, their overall number of attacks was greater, and their attack latencies were less than those of the ST males (p < •002 for each of the above) . Again, as in Experiment I, a distinct pattern of victories emerged during the 11 weeks (Table II) . In

the first week, the ST males won 6 contests while the CBA males won only 3 . On all subsequent weeks the ST males failed to win any of the contests and the CBA males were victorious in no less than 9 . Here too, the CBA males eventually won all 11 contests each week . The amount and ferocity of the fighting increased during the 11 weeks of observation . Attacks were not just single events . Often one attack quickly followed another .. A CBA male would attack a number of times, stop for a while, then begin another series of attacks . This pattern was evident all though the contests . Many injuries were received by the ST males, though none severe enough to cripple them . Most injuries were received on the tail, posterior end of the body (back and sides), hind legs and feet . Since the ST males rarely fought hack, the



55

LEVINE et al. ; INTERSTRAIN FIGHTING IN MALE MICE Table II . Number of Initial Victories . Experiment I (Observations terminated after 1st victory-isolated d' d') Week

1

2

3

4

5

6

7

8

9

ST

5

2

1

2

1

2

0

0

0

CBA

3

9

9

9

9

9

11

11

11

Draw

3

0

1

0

0

0

0

0

0

No-fight

0

0

1

0

0

I I

10

11

Total

0

0

13

11

11

103

0

0

4

0

1

Total

0

1 Experiment II (Observations terminated after 30 minutes-isolated (I8) Week

1

2

3

4

5 i

6

ST

6

0 ~ 0

0

0

0

CBA

3

11

10

9

10

10

Draw

1

0

0

0

0

0

No-fight

1

0

1

1

7

8

9

10

11

0'

0

0

0

0

6

10

11

10

11

11

106

0

0

0

0

0

1

1

1

0

8 I

Experiment III (Observations terminated after 1st victory-a f with social experience) Week

1

2

3

4

ST

0

0

0

0

CBA

3

5

5

8

7

Draw

2

2

2

0

1

No-fight

6

4

3

Table ID. Average Attack Latencies for Each Week.

Week Experiment I

Experiment II

Experiment III

I

119±2 •i

6 . 1±1 . 6

9 . 8+2 . 7

2

4 . 0±0 . 9

3 . 2±0 . 6

6 .2±1 . 6

3

4 . 0±1 . 4

4 . 2±1 . 0

4 .8±1 . 3

4

2 . 0±0 . 4

3 . 8+1 . 1

9 .0±2 . 8

5

2 . 0±0 . 3

2 . 0±0 . 4

3 .4+0 . 8

6

2 . 640-5

2 . 94-0 . 7

2 . 6+0 . 8

7

2 . 91-0 .9

2 . 010 .4

3 . 8-f-0 . 8

8

3 .0 i-0 .4

2 . 2+0 .4

3 . 1-4-1 . 0

9

2 . 5+0 . 7

1 . 4±0 .4

2-6-L-1-1

10

2 .0±0 . 6

3 .9±1 . 8

3 . 4±1 .6

ll

2 . 2±0 . 6

1 . 6±0 . 3

2 . 0±0 . 7

5

6

7

8

9

0

0

0

10 0

0

1

10

11

Total

0

0

0

0

9

11

9

11

87

0

0

0

_ 0

7

0

27

ki

actual encounters of the mice were a constant series of attacks of submissions . It was found that 51 per cent . of all attacks occurred within the first 10 minutes of the 30-minute observation period . The attack latency pattern follows very closely that of Experiment 1 . The over-all attack latency for the first week was 6 . 1 minutes and then dropped sharply to a level between 1 . 4 and 4 . 2 minutes (Table Ill). Experiment III The procedure followed in this experiment was similar to that of Experiment I in that the contests were discontinued after the first victory, or after 30 minutes if there was no submission . However, in Experiment l, the males were isolated from time of weaning, whereas in this experiment the males were housed with males of their own strain until one week before the experiment began . The mice used were received from the Jackson Laboratory, already weaned



56

ANIMAL

BEHAVIOUR,

and housed with others of their own strain and sex . At the time they were received, the CBA males were 49 days old while the ST males were between 63 and 69 days old . Four cages (1I inches by 7 inches by 5 inches deep) were set up in such a way that 2 cages contained 6 CBA males each and 2 cages held 6 ST males each. One week before the start of the contests, 11 males of each strain were put into separate cages of the same size as above . At the start of this series of contests, all the CBA males were 90 days old, six of the ST males were 104 days old and five were 110 days old . The observations were made between 7 .40 a .m. and 5 .00 p .m . The room temperature averaged 71'F, varying between 66 ° and 78 ° F . The results are shown in Table I, column c . Here, too, the superiority of the CBA males was evident . The CBA males were victors more often, they were the first to attack more frequently and their total number of attacks was greater than the ST males (p<0 . 002 for each of the above) . The week by week analysis of victories is shown in Table II . In the first week, there were no ST victories and 3 CBA victories . Although the lack of ST victories persisted in subsequent weeks, there was no dramatic jump in the number of CBA victories . It was not until the sixth week that the CBA males achieved 9 victories, and although in some weeks the CBA males won every contest, this did not develop into a consistent pattern . In examining the week by week attack latency (Table III), it will be seen that it dropped sharply after the fourth week, and although it was 9 .0 minutes that week, it never again rose above 3 . 8 minutes . In this experiment one other type of behaviour was observed that had not appeared in the other experiments . In 8 of the 121 contests, homosexual behaviour was observed . In 3 cases, ST males mounted CBA males or both mounted and • thrust . In 5 cases, CBA males mounted ST males but did not exhibit thrusting . Discussion The results of Experiment I (contests concluded after first submission) and Experiment II (contests concluded after 30 minutes) are essentially the same . There is no significant difference in the number of ST victories, CBA victories, and draws . There is a difference in the number of no-fights for ST males in the two experiments (p<0 . 02), but not for the CBA males . The reason is that one CBA male accounts for 5 of the 8 no-fights in Experiment II (p <0 .001 by Cochran

XIII,

I

Q test substantiates the fact that the probability of a no-fight is not the same for all CBA males . The probability is the same for the ST males) . The patterns of victories (Table 11) in both experiments are also very similar . In the first week of both experiments, the ST males won the majority of the contests . Thereafter in both experiments, the CBA males won no less than 9 contests per week and eventually won all 11 contests each week . These results are puzzling . It is normally assumed that a victory conditions the mouse for further victories and a defeat conditions the mouse for further defeats (Ginsburg & Allee, 1942) . In both of our experiments, the CBA males who had been defeated at first were overwhelmingly victorious thereafter, while the ST males who had been victors, lost their superiority . Although the results of Experiment I and II are essentially the same they do differ in two respects . First, the ST males were the first to attack a significantly greater number of times in Experiment I (p=0 .04) . It appears that the ST males were conditioned to defeat faster in Experiment II than in Experiment I, because they experienced a more concentrated show of CBA aggressiveness during the extended contest period . The second difference in the two experiments is in the attack latencies . Those of the first weeks of Experiment I and II are significantly different (p<0 . 01) by chi-square test) . This discrepancy cannot be explained at this time . The experimental procedures of Experiments I and III were similar in that the mice were separated immediately after a victory . They differed in that the males of Experiment I were isolated since weaning at thirty days of age, while the males of Experiment III had social experience from weaning until one week before the first contest . The fighting ability of the CBA males seems to be the same in both experiments with respect to number of victories, number of times they were first to attack, and total number of attacks . The only apparent effect of social experience on the fighting ability of the CBA males was the increase of their average attack latency from 3 . 0 to 4 . 5 minutes (p<0 . 05) . On the other hand, pre-fight social experience appears to have a radical effect on the fighting ability of the ST males . In comparing the two experiments, it is seen that in Experiment III, the ST males were never victorious (p<0 .02), they were the first to attack in only one case (p=0 .004), and their total number of attacks dropped to 10 (p=0 .003)

LEVINE

etl al. :

INTERSTRATN FIGHTING IN MALE MICE

the two experiments concerns the number of no-fights which increased from I in Experiment 1 to 27 in Experiment 111 . It seems that the increased number of no-fights is due to the decreased aggressiveness of the ST males . There appears to be an inverse relationship between the number of no-fights and the number of times the ST males were the first to attack . It can be concluded that in inter-strain fighting between CBA and ST males, the effect of social experience before fighting is to drastically reduce the aggressiveness of the ST males but to effect the CBA males only in increasing their attack latency . This finding indicates that the different hereditary backgrounds of the two types of mice produce different responses to social experience . It illustrates the need to consider genetic factors in assessing the effect of early experience on adult behaviour . The results of other investigators, with respect to the effect of social experience on aggressiveness, have been variable . Kahn (1954), using response to training as fighters as a criterion, found that males isolated after weaning were more aggressive than males raised in social groups, while King (1957), using fighting response latency as a criterion, found that males isolated after weaning were less aggressive than males raised in social groups . Both of the above investigators used the same inbred strain of mice (C57BL/10) . Such seemingly contradictory results can be understood when it is appreciated that the investigators used different experimental procedures and criteria in their measurement of aggressiveness . This dependency of animal behaviour upon the situation in which the behaviour is elicited has been noted by others . Fredericson & Birnbaum (1954) compared the aggressive behaviour of two strains (BALB/c and C57BL/10) of mice under intrastrain competition for food and under insterstrain housing together . Under the first condition, the BALB/c strain was found to be less aggressive while under the latter condition, it was found to be more aggressive . Our own results also show that the fighting ability of inbred strains of mice is a function of the experimental procedure used to test this ability . With respect to this point, the results obtained when ST and CBA males are housed together permanently with ST females should be considered . In chance observations of 27 fights, it was found that the ST males won 25, while the CBA males won 2. This ST superiority is in sharp contrast to the results obtained

57

in the present experiments . When CBA and ST males are housed together permanently with ST females, the ST males are superior in fighting. However, if they are separated after each fighting contest and housed without females, any original ST superiority disappears and the CBA males achieve overwhelming fighting success . Summary lnterstrain fighting between males of the ST/J (albino) and the CBA/J (black-agouti) strains was studied under three different sets of conditions : (a) contests terminated after first victory using males isolated since weaning ; (b) 30minute contests with males isolated since weaning ; (c) contests terminated after the first victory using socially experienced males . It was found that : (1) Under all three sets of conditions, the CBA males achieved fighting superiority . (2) The CBA superiority in fighting was not evident in the first contests but developed rapidly thereafter . (3) When the contests are conducted for a 30-minute period, rather than until first submission, the conditioning of the ST males to defeat occurs more rapidly . (4) Social experience before fighting has different effects on the two types of males . It drastically reduces all aspects of aggressiveness of the ST males . Its only effects on the CBA males are to increase their attack latency and to extend the number of contests required to achieve their superiority in fighting . The above results are compared with those obtained elsewhere . The dependency of fighting ability upon the situation in which the behaviour is elicited is discussed . REFERENCES Beeman, E . A . (1947) . The effect of male hormones on aggressive behavior in mice . Physiol . Zool ., 20, 373-405 . Fennell, R . A . (1945) . The relation between heredity, sexual activity and training to dominancesubordination in game cocks . Amer . Nat ., 79, 142-151 . Fredericson, E. & Birnbaum, E . A . (1954). Competitive fighting between mice with different hereditary backgrounds . J. genet . Psychol ., 85, 271-280 . Ginsburg, B . & Allee, W . C. (1942) . Effects of conditioning on social dominance and subordination in inbred strains of mice . Physiol. Zool., 14, 485-506 . Guhl, A. M . (1953) . Social behavior of the domestic fowl . Kansas State College ; Agricultural Experiment Station Tech . Bull . No . 73 .



58

ANIMAL BEHAVIOUR, XIII, I

Kahn, M . W . (1954). Infantile experience and mature aggressive behavior of mice : some maternal influences . J. genet. Psychol., 84, 65-76. Kahn, M . W. (1%1). The effect of socially learned aggression or submission mating behavior of C57 mice . J. genet . Psychol., 98, 211-217. King, J . A. (1957) . Relationships between early social experience and adult aggressive behavior in inbred mice. J. genet . Psychol., 90, 151-166.

Levine, L . (1956) . Studies on sexual selection in mice . I. Reproductive competition between albino and black agouti males . Amer. Nat., 92, 21-26 . Scott, J . P. (1958) . Aggression. Chicago : The University of Chicago Press, xi, p. 149. Siegel, S . (1956) . Nonparametric Statistics for the Behavioral Sciences. Mc-Graw Hill . (Accepted for publication 28th September, 1964 ; Ms. number : 438) .