Relation between the seasonal changes in aggression, plasma testosterone and the photoperiod in male rhesus monkeys

Ps.vchoneurorndocrinolog.v, Printed in Great Britain.

Vol.

6. No.

2. pp.

145-

158.

1981

03C+-4530/81/02014S-14$02,00/O @ 1981 Pergamon Press Ltd.

RELATION BETWEEN THE SEASONAL CHANGES IN AGGRESSION, PLASMA TESTOSTERONE AND THE PHOTOPERIOD IN MALE RHESUS MONKEYS RICHARD P. MICHAEL and DORIS ZUMPE Department of Psychiatry, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. and The Georgia Mental Health Institute, Atlanta, GA 30306, U.S.A. (Received 28 July 1980) SUMMARY Changes in direct and redirected aggression and in plasma testosterone levels were studied during a 39-month period in eight male rhesus monkeys maintained under laboratory conditions in a constant photoperiod. Males were tested with the same four ovariectomized, estrogen-treated females (32 pairs, 6357 one-hour tests), and plasma samples were obtained weekly at 0800 and 1600 hr and every two weeks at 2200 hr (2630 samples). Four males remained intact throughout and four males were castrated and received testosterone implants subcutaneously at the start of the second year. Annual changes in male aggression continued to occur at the expected time in the autumn in both intact and castrated males which indicated a degree of independence of the behavior from the photoperiod and from changes in plasma testosterone. There was a highly significant temporal association between 0800 hr testosterone and direct aggression occurring 3 - 4 weeks later during the first year, but this association was lost in subsequent years. Although the annual rhythms in plasma restosterone persisted for three years in the intact group, the maxima for the hormonal and the behavioral changes drifted apart in the constant photoperiod. The view is propounded that essentially independent long-term endocrine and behavioral rhythms are normally entrained by exteroceptive factors. INTRODUCTION SEXUAL activity and aggressive tendencies are intricately related in many vertebrates including fishes, reptiles, birds and mammals. This association is almost the rule for those territorial species in which a prospective mate intrudes into the territory of a future partner and where this is vigorously defended, at least initially, against all other conspecifics. Aggression in a sexual context also occurs in many social species where the males and females do not associate very intimately except during the mating season when temporary or more permanent pair bonds are established between individuals within the social group. The agonistic tension existing between prospective mates is diffused and dissipated primarily by the process of redirection (Bastock, Morris & Moynihan, 1954). The aggression continues to be expressed as courtship proceeds, but instead of directing threats at their prospective mates, both members of the bonding pair now redirect their threats away from each other. Such threats may be redirected at another conspecific, such as a territory neighbour or troop member, or at inanimate objects in the environment, or even at nothing in particular (vacuum activity). Many courtship displays have been shown to be ritualizations of such redirected aggression and, unless redirection occurs successfully, attacks may become too intense and courtship is prevented from proceeding (Lorenz, 1941; Baerends & Baerends-Van Roon, 1950; Andrew, 1957; Tinbergen, 1959). 145

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RICHARDP. MICH~,et.and ER)RJsZUMPE

The whole process serves to strengthen and intensify pair bonds, and helps to isolate the sexually interacting pair from interference by other conspecifics. Like many infraprimate species, rhesus monkeys (Macaca mulatta) are seasonal breeders and have an autumn mating season both in the wild in India (Southwick, Beg & Siddiqi, 1965; Lindburg, 1971) and under free-ranging conditions in the Caribbean (Koford, 1965; Drickamer, 1974). Autumn mating also occurs in captivity in outdoor enclosures (Gordon & Bernstein, 1973) and under laboratory conditions (Michael & Keverne, 1971). The start of the mating season is associated with increased testicular activity (Conaway & Sade, 1965), increased plasma androgen levels (Plant, Zumpe, Sauis & Michael, 1974; Robinson, Scheffler, Eisele & Goy, 1975; Gordon, Bernstein & Rose, 1978; M. radiata: Glick, 1979) and increased aggression, more particularly by males towards other males (Wilson & Boelkins, 1970; Gordon, Rose & Bernstein, 1976; M. fuscata: Eaton, 1978; Cercopithecus aethiops: Henzi & Lucas, 1980), but also towards their female partners (Michael & Zumpe, 1978). Both under free-ranging conditions (Altmann, 1962) and in the laboratory (Zumpe & Michael, 1970), sexual partners may redirect threats away from their mates onto nothing in particular. When oppositely-sexed pairs of rhesus monkeys are tested in isolation from conspecifics under standardized conditions, any threats not directed towards the sexual partner can readily be identified as redirected aggression since there is no possibility of their being evoked by another conspecific. Under these conditions, if the sexual interest of male rhesus monkeys in their female partners is changed by giving them estrogen, there is a corresponding change from direct to redirected aggression in these males (Zumpe & Michael, 1979); the negative association between these two forms of aggression is quite similar to that seen in the courtship displays of many infraprimate species. In seasonal breeders, the timing of the mating season is determined by exteroceptive factors, notably by the photoperiod, although the mechanisms may be more complex than was once thought (Turek & Campbell, 1979). Primate breeding seasonality is also influenced by the photoperiod (Van Horn, 1975, 1980; Barsotti, Abrahamson, Marlar & Allen, 1980), although in the rhesus monkey, seasonal factors may partially be overridden by social factors such as the presence or absence of sexually active partners (Vandenbergh & Drickamer, 1974; Gordon & Bernstein, 1973). The sequence of events at the start of the mating season, namely, gonadal recrudescence followed by increased aggression and then sexual activity, together with the long recognized facts that in many mammals castration reduces both aggressiveness and sexual activity while testosterone replacement restores them, has led to the plausible and widely held view that the hormonal changes are primarily responsible for the changes in behavior. However, in macaques it has been quite difficult to demonstrate any direct relation between androgens and aggression (Wilson & Vessey, 1968; Phoenix, Slob & Goy, 1973; Eaton & Resko, 1974; Cochran & Perachio, 1977; Gordon, Rose, Grady & Bernstein, 1979), and an alternative possibility would be that the photoperiod simply serves to entrain several intrinsic, endogenous circannual rhythms (Gwinner & Dorka, 1976). In rhesus monkeys, for example, it has been shown that whereas the annual rhythm in ejaculatory activity was lost after the first year (Michael & Zumpe, 1976), a circannual rhythm in plasma testosterone persisted for over three years in intact males maintained in a constant photoperiod (Michael & Bonsall,

AGGRESSION AND PLASMA TESTOSTERONE

147

1977). The present study attempts to examine further the relations between plasma testosterone and both direct and redirected aggression in male rhesus monkeys, paired with the same four ovariectomized, estrogen-treated females, during 3 successive years in a constant photoperiod.

METHODS

Animals Eight fully mature male (weighing 7.6 - 12.3 kg) and four adult female (weighing 4.6 - 6 . 5 kg) rhesus monkeys were obtained through dealers directly from North India. After 4 - 10 m o n t h s quarantine in London, England, they were shipped to Atlanta, Georgia, 2 - 3 m o n t h s before the start of the study. Animals were housed in individual cages together in the same animal room from which natural daylight was excluded. Artificial lighting was rigorously controlled to give a 14 hr day between 0615 and 2015 hr. At the beginning o f June 1975, four males were moved into a room where they were exposed to the natural changes in the photoperiod (see below). Temperature was maintained between 20 and 24°C. Food consisted of Purina or Wayne Monkey Chow, supplemented with vitamins and fresh fruit or vegetables. Water was available ad libitum.

Operative procedures and hormone treatments All females were bilaterally ovariectomized through a mid-line, sub-umbilical incision 6 m o n t h s before the start of behavioral testing, and then received estradioi benzoate (Progynon, Schering Corporation), 10 lag in 0.2 ml sesame oil s.c. per day throughout the entire study. In February 1974, 14 m o n t h s after the start of behavioral testing, four males (A,B,C,D) were sham-castrated and given empty silastic implants s.c., while the remaining 4 males (E,F,G,H) were castrated and received subcutaneous silastic implants o f testosterone. O n the basis of their patterns of seasonal changes in ejaculatory activity during 1973, males were selected for either castration or sham-castration to give two groups that were well-matched in overall potency. Castration was performed via bilateral inguinal incisions, and silastic testicular prostheses were introduced into the empty scrotal sacs to maintain the external appearance of the genitalia. Sham-castration consisted of incising the scrotal and cremasteric sacs, mobilizing the testes, and closing the incisions. Testosterone implants were 1 0 0 - 1 2 0 m m effective lengths of Silastic Medical Grade tubing (3.35 m m i.d. × 4.65 m m o.d.) filled with 0 . 5 7 - 0 . 7 0 g crystalline testosterone (Steraloids). The estimated daily release rate was 39.4 lag/100 ram, and the lengths of the implants were designed to give plasma testosterone levels in the high physiological range. Immediately after surgery, each castrated male was given two testosterone implants s.c. into the subscapular region and each shamcastrated male was given two control implants of comparable length. At the beginning of June 1975, all implants were removed and replaced with fresh implants, and the castrated males were moved into a room with windows where they were exposed to changes in the photoperiod.

Testing procedure Observations were made between January 1973 and March 1976 on oppositely-sexed pairs of animals in quiet, isolated r o o m s from behind one-way vision mirrors angled in such a way that animals could not see their reflections. Tests were conducted in special observation cages, I. 19 m wide by 1.07 m deep by 1.14 m high, into which first the males and then the female were introduced at the start o f each test session (Michael, Saayman & Zumpe, 1968). At all other times, animals were caged singly. Tests were of 1 hr duration and were conducted 5 days a week. Each of the 8 males was tested on consecutive days with each of the 4 females in turn (32 pairs) so that all males were tested once and all females twice daily (6357 tests). After the first 8 months, during which each male received alternating morning and afternoon tests with a given female partner, the order in which animals were tested was kept constant. Few behavioral tests were available for February 1974 when all males underwent surgery.

Definitions and terminology The basic descriptions of the behavior observed during tests have been given elsewhere (Michael, Herbert & Welegalla, 1966, 1967). Particular attention is given here to the following patterns of behavior: (a) direct male aggression - the n u m b e r per test of aggressive gestures (jerks, open-jawed threats, hits, grabs, pulls and bites) that the male directed at the female partner (Michael & Zumpe, 1970); (b) redirected male aggression - the n u m b e r per test of aggressive gestures (jerks, open-jawed threats, hits, grabs and bites) that the male directed away from the female at nothing in particular (Zumpe & Michael, 1970). Many other behavior patterns were scored, including numbers of ejaculations, redirected aggression by females and female sexual invitations, but these do not receive detailed consideration here.

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Collection o f plasma samples and the androgen assay Between January 1973 and March 1976, 3 ml blood was obtained weekly at 0800 hr (1273 samples) from the saphenous veins of untranquillized males that had been adapted previously to the venipuncture procedure. Between January 1973 and April 1975, 3 ml blood was also obtained weekly at 1600 hr (861 samples) and once every two weeks at 2200 hr (496 samples) on the same day on which 0800 hr samples were obtained. Plasma androgen levels were estimated by a radioimmunoassay method on 100 ~tl plasma, without chromatography, using an antiserum to testosterone-3-carboxymethyl-oxime-bovine serum albumin. Cross-reactions of other 17-hydroxyandrogens relative to testosterone were as previously reported (Michael, Setchell & Plant, 1974) and the method, therefore, additionally estimated dihydrotestosterone at the ratio to testosterone of 1 : 11 (Bonsall, Baumgardner & Michael, 1976). The order in which samples and standards were assayed was randomized. Duplicate aliquots from a reference plasma pool and duplicate water blanks were assayed with each batch of unknowns, and all plasma androgen determinations were performed in duplicate. The mean recovery of ~H testosterone was 95 -+ 3.4 (S.D.)~/0. The coefficient of variation between duplicates (N = 54) over the range 1 5 6 - 2 9 8 7 ng/100 ml was 9.2°7o. The mean value of the blank (N = 16) (100 ~tl distilled water) was 4.7 __. 2.8 (S.D) pg: this was not substracted. The lower limit of the sensitivity of the assay (mean + 2 S.D. values) was therefore taken as 108.4 ng/100 ml.

Stat&tical treatment o f results The Spearman rank correlation coefficient was used to examine the relationship between monthly means for plasma hormone levels and both direct and redirected aggression. The chi-squared test was used to test the significance of relationships between hormone levels in individual samples and behavior from individual tests during each of the three successive years of study. For each of the 32 pairs of animals (8 males), male plasma hormone levels (at 0800, 1600 and 2200 hr or the combined daily means for those days when all three values were available) were categorized into either low (on or below the median value for the 12-month period) or high (above the median value). The behavioral data were partitioned into three categories: tests without the behavior, tests with values at or below the median for the year, and tests with values above the median. Two-by-three chisquared tests or, when there were empty cells, two-by-two chi-squared tests were then computed for the hormone data and the behavior from those tests nearest to the days of blood collection. The data were then analyzed again by off-setting the behavior from the hormone values by weekly increments from week - 8 (behavior test occurring 8 weeks before blood collection) to week + 8 (behavior test occurring 8 weeks after blood collection). This type of statistical analysis, used previously to relate h o r m o n e and behavior changes in females (Bonsall, Z u m p e & Michael, 1978), permits analyses by pair, by male, by female and by group. Group analyses were not considered significant unless the probability was less than 0.05 and, additionally, the expected trend occurred in half or more of the pairs. This type o f analysis eliminates problems associated with non-linearity and skew, and more nearly equalizes the contributions made to the combined data by individual pairs of animals and by individual animals. A total of 13,770 chi-squared tests was run on a P D P 8 / E computer. Analyses utilized data from three 12-month periods, January 1 9 7 3 - December 1973 (all 8 males intact), March 1 9 7 4 - F e b r u a r y 1975 (after castration and after sham-castration), and March 1 9 7 5 - F e b r u a r y 1976. For the off-set analyses, data were also generated from the three 8-week periods following those indicated above. RESULTS

Aggression and plasma testosterone in intact males Figure 1 shows the changes in the number (monthly means) of direct aggressive gestures by each of four intact males (A - D), together with the combined data for the group, for a 3-year period (39 months). Monthly mean 0800 hr plasma testosterone levels are also given in each case. The combined data (Fig. l, bottom) show a well-marked annual rhythm in the aggression directed by males towards their female partners, and the maxima corresponded to the timing of the changes that would be observed under free-ranging conditions. Peak aggression occurred in September 1973, September 1974 and October 1975. Although the timing of the maxima remained virtually unchanged, there was a progressive decline in levels from year to year, and the changes in the third year were less well-circumscribed. Three of the males contributed to these rhythmic changes while, in the fourth male (D), aggression was episodic and at rather low levels. Although animals were maintained in a constant photoperiod, the combined data for 0800 hr plasma

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t e s t o s t e r o n e also s h o w e d w e l l - m a r k e d seasonal ch an g es with m a x i m a l values o c c u r r i n g in S e p t e m b e r 1973, O c t o b e r 1974 a n d J a n u a r y 1976; a g r a d u a l d r i f t in the t i m i n g o f the m a x i m a to later in the season was a p p a r e n t . In the first year, the m a x i m a f o r aggression an d t e s t o s t e r o n e c o i n c i d e d in S e p t e m b e r , an d there was a close c o r r e l a t i o n b et w een h o r m o n e levels a n d b e h a v i o r ( J a n u a r y - D e c e m b e r 1973, r = 0.734, p < 0 . 0 1 , one-tailed).

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In the second year, the m a x i m u m for aggression was in September while that for plasma testosterone was in October, and the correlation was less close (March 1 9 7 4 - F e b r u a r y 1975, r = 0.615, p<0.025). In the final year of the study, the m a x i m u m for aggression occurred in October but that for testosterone was in January of the next calendar year, and the correlation was no longer significant (March 1 9 7 5 - F e b r u a r y 1976, r = 0.105, N.S.). In these four intact males, redirected aggression was intermittent and at low levels; there were minor peaks in October 1974 and July 1975 but no significant correlations. Data from individual males showed that seasonal changes in aggression could occur in the absence of seasonal changes in plasma testosterone and, conversely, that changes in plasma androgens could occur in the absence of seasonal changes in male aggression. For example, male C (Fig. 1) showed seasonal changes in aggression in each year of the study but plasma testosterone rhythms only during the first and third years. Male D showed testosterone rhythms in each year but no rhythmic changes in aggression in any year. It is clear that the timing of the maxima in both testosterone and aggression varied very considerably in different males.

Aggression and plasma testosterone in males studied while intact and after castration and testosterone replacement Figure 2 shows the changes in the number (monthly means) of direct and redirected aggressive gestures by each of four males (E - H), together with the combined data for the group, for a 3-year period (39 months). In these males there was enough redirected aggression to merit illustration. Monthly mean 0800 hr plasma testosterone levels are also given in each case. The combined data (Fig. 2 bottom) show that males E - H were more aggressive as a group than males A - D; they showed more direct and considerably more redirected aggression. Like the other group, these males also showed clear annual rhythms in both direct aggression and 0800 hr plasma testosterone during the first year while they were still intact. Plasma testosterone peaked in September and direct aggression peaked in October, and there was a significant positive correlation between monthly means ( J a n u a r y - D e c e m b e r 1973, r = 0.615 p<0.025). There was also a well-circumscribed increase in redirected aggression in September, coinciding with the peak in 0800 hr plasma testosterone. After castration and testosterone implantation in February 1974, plasma androgens remained consistently high and all signs of seasonal rhythmicity disappeared. However, both direct and redirected aggression continued to show marked changes. Direct aggression fluctuated considerably during the second year, but remained consistently higher between June 1 9 7 4 - F e b r u a r y 1975 than between either M a r c h - M a y 1974 or M a r c h - May 1975. Indeed, lowest levels of direct aggression occurred at the same time each year, namely, April 1973 (males intact), April 1974 and May 1975. After castration and testosterone replacement, redirected aggression showed a very large m a x i m u m during October, one month later than during the first year. There were no significant correlations between monthly means for aggression and 0800 hr plasma testosterone levels in the second year, and the data for individual males showed the same type of variability as described for males A - D.

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Because it was thought they were becoming exhausted, the testosterone implants were renewed in the first week of June 1975. At this time the males were moved into a room with windows where they were exposed to natural daylight and the changes in the photoperiod. Figure 2 shows that direct aggression reached a m a x i m u m in August and

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returned to very low levels in December 1975, when plasma androgens were rising. Redirected aggression increased to a maximum in July 1975 and declined to very low levels in January 1976. Data from individual animals showed that two males (E,G) contributed to the increase in direct aggression and one male (G) to the increase in redirected aggression. There were no significant correlations between monthly means for aggression and plasma androgens during the final year of the study.

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FIG. 3. Illustrating the confidence levels of significant temporal associations between aggression and plasma testosterone in eight intact male rhesus monkeys during the first year of study in a constant photoperiod. There were clear positive associations (histograms above abcissa) between 0800 hr plasma testosterone and direct aggression occurring in tests 1 - 4 weeks later. There were clear negative associations (histograms below abscissa) between 2200 hr plasma testosterone levels and redirected aggression occurring in tests I - 8 weeks earlier. There were no significant associations between behavior and 1600 hr levels and only one positive association between aggression and daily mean plasma testosterone. The horizontal interrupted lines give p = 0.05. The abscissa gives the offset intervals from - 8 (behavior test conducted eight weeks before plasma sampling) to week + 8 (behavior test conducted eight weeks after plasma sampling).

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Significant associations between plasma testosterone and aggression A. Direct aggression during the first year. Chi-squared tests matching hormone levels in individual plasma samples with direct aggression in individual tests during the first year in a constant photoperiod revealed significant temporal associations when all eight males were intact. Androgen levels were matched with behavior in the nearest test (within 24 hr), and with behavior in tests conducted either before or after the day of plasma sampling by offsets at weekly intervals of up to eight weeks. This type of analysis was performed for each plasma sample (and for the daily mean) collected during the 12-month period for all eight males (3043 chi-squared tests). The significance of these associations is illustrated in Fig. 3. The minus sign indicates that behavior tests were conducted before the day of plasma sampling, and the plus sign indicates that behavior tests were conducted after the day of sampling. The black histograms above the horizontal interrupted line show positive associations between plasma androgens and direct aggression, and the magnitudes of the histograms give the strengths of the associations. The horizontal interrupted lines set the 0.05 levels of significance. It can be seen that virtually all the positive associations that occurred were between 0800 hr plasma testosterone and direct aggression in tests conducted from one week before to four weeks after plasma sampling. The most highly significant association was between 0800 hr plasma testosterone and direct aggression 3 weeks later ( ~ = 16.92, d f 2 , p<0.0005; positive trends in 20 of 32 pairs involving all eight males). There were no significant associations of any kind between plasma testosterone in 1600 and 2200 hr samples and behavior, and a very weak association between mean plasma testosterone and direct aggression one week later ( ~ = 6.51, d f 2, p<0.05; positive trends in 16 of 32 pairs involving seven of eight males). There were no negative associations.

B. Redirected aggression during the first year. Identical analyses (3043 chi-squared tests) for plasma testosterone and redirected aggression also revealed significant associations, but they were of a strikingly different kind (Fig. 3). The hatched histograms below the horizontal interrupted line (p=0.05) show negative associations between plasma androgens and redirected aggression, and the magnitudes of the histograms give the strengths of the associations. Virtually all the negative associations occurred between 2200 hr plasma testosterone and redirected aggression in tests conducted from eight weeks before to one week after plasma sampling. The most highly significant association was between 2200 hr plasma testosterone and redirected aggression one week earlier (~2 = 18.50, d f 2, p<0.0005; negative trends in 19 of 32 pairs involving seven of eight males). There was also a weak negative association between 0800 hr plasma testosterone and redirected aggression 3 weeks earlier (~' = 8.52, df2, p < 0.02; negative trends in 18 of 32 pairs involving seven of eight males), but no significant associations of any kind between either 1600 hr or daily mean testosterone levels and behavior. There were no positive associations. C. Direct and redirected aggression during the second and third years. Identical analyses on plasma testosterone levels and direct and redirected aggression were carried out for males A - D and males E - H for each of the two remaining years of the study (7684 chi-squared tests). They revealed several associations, none of which reached a high level of significance but were in the p < 0.05 - 0.02 range. For males A - D, for example,

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there was a single weak negative association during the second year between 0800 hr plasma testosterone and redirected aggression seven weeks later (p < 0.05). There were no associations during the third year. For males E - H there were, for example, weak positive associations between 0800 hr plasma testosterone and direct aggression seven weeks earlier (p<0.05), and between 1600 hr plasma testosterone levels and direct aggression 2 - 3 weeks later (p<0.05). During the third year, there were no significant associations between androgens and behavior.

DISCUSSION

The present findings have demonstrated that the propensity to behave aggressively in the fall and winter months persisted at the appropriate time of the year in males maintained in a constant photoperiod under laboratory conditions when they were tested with the same four ovariectomized, estrogen-treated females. Despite the relative constancy of environmental and social stimulation, intact males continued to show annual rhythms in 0800 hr plasma testosterone levels for three years (Michael & Bonsall, 1977). The changes were smaller than those reported under outdoor colony conditions where androgens and sexual activity fall to very low levels during the spring and summer (Gordon, Bernstein & Rose, 1978). Moreover, there was a gradual drift in the timing of the maxima. During the first year, the changes in plasma androgens were significantly and positively correlated with the changes in aggression (p<0.01) (Fig. 1, bottom). In subsequent years, aggression tended to decline and the changes became less wellcircumscribed. The gradual dissociation of the androgen and aggression rhythms resulted in first a weakening and then the loss of a significant correlation. The View that the annual changes in plasma androgens were neither necessary nor sufficient to cause the changes in aggression was confirmed by data from individual males and by the results of castration and hormone replacement. Redirected aggression increased, and direct aggression decreased, at more or less the appropriate times of year, although the plasma testosterone levels remained remarkably constant. It is not known if annual changes in aggression would also be seen in castrated males without any testosterone replacement treatment. The aggression rhythms in both groups of males were of higher amplitude and better defined in the first year, when they coincided with the androgen rhythms, than during subsequent years when they became dissociated. This suggests that the changes in plasma testosterone under natural conditions might normally enhance, and influence the timing of, changes in male aggression. Our attempt to demonstrate an effect on behavior of changing the photoperiod in June 1975 basically failed. We had hoped that both sexual and aggressive activity would be stimulated, and they were not. Many variables were involved but it would be unprofitable to discuss them here at any length. There was marked variability in the data from individual males and individual pairs, not only in absolute levels but also in the patterns of change. As a consequence, the combined data are weighed by certain individuals, and the conclusions based on them must be regarded as somewhat suspect. Data of this type preclude the use of the cosinor method of rhythm analysis, which is not applicable when there are many zero values and

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where the periodicity of the rhythm is variable. The chi-squared test as employed here had the advantage of eliminating problems associated with non-linearity and skew. More importantly, it equalized the contributions made by each of the 32 pairs to the combined data and, by. means of the offset analyses, it facilitated an estimate of the significance of any temporal associations between hormones and behavior. In the first year, there were highly significant positive associations between 0800 hr plasma androgens and direct aggression 3 - 4 weeks later. This is in line both with the known latency for the effects of testosterone on behavior in castrated male rhesus monkeys (Michael & Wilson, 1974), and with the results obtained here from correlating monthly means. Much more difficult to conceptualize was the finding that increased levels of redirected aggression were associated with a decline in 2200 hr plasma testosterone levels 1 - 3 weeks later or vice versa (Fig. 3). Behavior could have been influencing hormone levels or the apparent relationship could have resulted from a chance association between two independent rhythms. The temporal relationships revealed by chi-square testing and offsetting were lost during the second and third years, which confirmed the results obtained by comparing monthly means. The analyses also draw one's attention to the question of what is meant by "the plasma androgen level" when an attempt is being made to relate behavior to plasma hormones in samples obtained at a given time of day. In the present study, although plasma testosterone in 0800 and 1600 hr samples showed quite similar patterns of seasonal change, the majority of the most significant associations with direct aggression were in 0800 hr samples. In the offset analyses in particular, virtually all the significant associations were with 0800 hr values while none of the associations with 1600 hr values were significant; a remarkable disjunction. The 0800 hr samples showed the least variability and were, of course, obtained before the daily behavioral test. The 1600 and 2200 hr samples were collected after the day's testing, and there are different views as to whether events occurring during the test may affect plasma androgens (Goldfoot, Slob, Scheffler, Robinson, Wiegand & Cords, 1975; Phoenix, Dixson & Resko, 1977; Gordon et al., 1978; Eberhart & Keverne, 1979). These males were able to direct aggression only towards their sexual partners or to redirect it in vacuo, rather than express it towards other members of the social group, particularly other males. This may have accounted for the flatter, sinusoidal pattern of direct aggression compared with the more sharply defined peaks in redirected aggression. One might speculate that the peaks in direct aggression were attenuated by the inhibition due to the female being also the sexual partner; in contrast, redirected aggression could be expressed fully without disrupting sexual interactions. Previous work in our laboratory has shown that both direct and redirected aggression by male rhesus monkeys change with changes in sexual interactions (Zumpe & Michael, 1970, 1979). Examinations of the present data on a test-by-test basis confirmed earlier findings that tests without sexual interactions were more likely to be associated with direct aggression whereas tests with high levels of sexual activity were more likely to be associated with redirected aggression. However, the seasonal changes in aggression could not be accounted for by seasonal changes in male sexual activity (mounting attempts, ejaculations); a distinct annual rhythm in the ejaculatory performance of intact males during the first year was lost during the second

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year (Michael & Z u m p e , 1976). Neither could the seasonal changes in male aggression be a c c o u n t e d for by changes in female sexual activity a n d redirected aggression. While female sexual i n v i t a t i o n s a n d redirected aggression showed a n n u a l fluctuations, the two r h y t h m s did not correlate either with each other or with the r h y t h m s in male direct a n d redirected aggression. However, in the second year, the m a x i m a in female sexual invitations a n d in male a n d female redirected aggression did indeed all coincide d u r i n g O c t o b e r 1974, a n d this m a y explain why male redirected aggression reached a much higher peak d u r i n g the second year t h a n d u r i n g the first a n d third years (Fig. 2). These results indicate that the a n n u a l changes in aggression depend o n a m e c h a n i s m with a large intrinsic c o m p o n e n t that seems to operate principally in the male. It does not depend o n such extrinsic factors as pituitary activation of the testes by the p h o t o p e r i o d a n d is i n d e p e n d e n t o f changes in the levels o f p l a s m a testosterone. If, as seems p r o b a b l e , the n o r m a l l y c h a n g i n g p h o t o p e r i o d entrains h o r m o n a l a n d behavioral r h y t h m s , then any effect o f testosterone in e n h a n c i n g male aggression would, u n d e r n a t u r a l conditions, occur a p p r o p r i a t e l y at the start of the m a t i n g season. These studies do not provide us with a n y insights into the m e c h a n i s m s u n d e r l y i n g these long-term changes in aggressive behavior, b u t several possibilities will spring to m i n d : for example, there m a y be changes in the sensitivity o f the neural substrate, or changes in n e u r o t r a n s m i t t e r a n d polypeptide c o n t e n t a n d , in these days, n o n e would overlook the possibility of changes in testosterone b i n d i n g a n d receptor properties. We are grateful for the assistance of Robert Walker in running the statistical analyses and of Judy Cain in conducting the radioimmunoassays. This work was supported by Grant No. MH 19506 from the U.S. Public Health Service and general research support was provided by the Georgia Department of Human Resources. This help is gratefully acknowledged. REFERENCES ALTMANN, S. A. (1962) A field study of the sociobiology of rhesus monkeys (Macaca mulaua). Ann. N.Y. Acad. Sci. 102, 338-435. ANDREW, R. J. 0957) The aggressive and courtship behavior of certain Emberizinae. Behaviour 10, 225 -308.

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