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Hormones and Behavior 30, 287–297 (1996) Article No. 0035

Hormonal Responses to Parental and Nonparental Conditions in Male Cotton-Top Tamarins, Saguinus oedipus, a New World Primate Toni E. Ziegler,*,† Frederick H. Wegner,† and Charles T. Snowdon* * Department of Psychology, University of Wisconsin, Madison, Wisconsin 53706; and † Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin 53715

The socially monogamous cotton-top tamarin (Saguinus oedipus) monkey is a cooperative breeder with the breeding male providing extensive parental care shortly after birth. We examined the relationship of urinary prolactin and cortisol excretion both to male parental care and as a stress response in the cotton-top tamarin monkey. First-morning urine samples were collected to determine hormonal concentrations. Hormonal and behavioral data were collected on 8 male cotton-top tamarins during the 2 weeks before and the 2 weeks following birth of infants to their mate, 11 nonparental males with exposure to females, and three eldest sons from large family groups. Prolactin levels were significantly higher in experienced fathers during the postpartum period than in the other males, while cortisol levels were significantly lower in experienced fathers and eldest sons. Prolactin levels in experienced fathers were consistently elevated before birth, following birth, and after infants were weaned; prolactin levels during times of infant independence were still significantly higher than those in nonfather males. First-time fathers exhibited prolactin levels that were significantly higher after the births of infants than these same males did when they were paired with nonpregnant females. Elevated prolactin concentrations also occurred prior to the first birth, suggesting that males may be receiving cues from their pregnant females. The elevated prolactin levels in parental males may be associated with the experience of the fathers. Correlation between prolactin levels and number of successful births, number of previous births, and age were high. The care of newborn infants did not appear to be a stressful event since cortisol levels were not elevated postpartum. Both cortisol and prolactin were elevated following capture and injection of saline or a dopaminergic receptor antagonist, indicating that pro-

lactin does respond to acute stress. Cortisol levels did not coincide with prolactin levels except under acute stress conditions, suggesting that different neural pathways are probably involved in prolactin release during parental care versus acute stress. These studies provide evidence that male urinary prolactin levels may be elevated due to cues from pregnant females and the constant exposure of males to the family environment. q 1996 Academic Press, Inc.

The physiology of maternal care for mammalian infants has been extensively studied (Rosenblatt, 1990). However, until recently there has been little emphasis placed on understanding the mechanisms of male parental care. Recent cultural trends in Western societies have led to an increased acceptance of a higher involvement of male parenting to infants and increased interest in the functional significance of male parenting (Yogman, 1990). Due to extensive variability in the level of mammalian male parenting, it is difficult to determine whether there is a biological basis to male parenting or if the variability is due to experience and social and ecological factors. Extensive male parental care of infants is relatively rare in mammals, with only approximately 10% of mammalian genera showing direct male parental care (Kleiman and Malcom, 1981). Changes in levels of LH, testosterone, and prolactin have been reported to occur with male parental behavior in some avian species with males fully participating in nest building, incubation, and feeding of infant birds (Wingfield and Goldsmith, 1990), and changes in prolactin and possibly testosterone levels occur in monogamous

0018-506X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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rodents with male involvement enhancing the infant survival rate (see Brown, 1993 for review). In rodents, species that demonstrate monogamous breeding systems are more likely to show higher levels of paternal care (Dewsbury, 1985). A model of male parental care has been proposed that considers the factors influencing the initiation and maintenance of male paternal behavior (Brown, 1993). The cotton-top tamarin, Saguinus oedipus, a small South American primate, exhibits many of the same factors that are associated with direct male parental investment in rodents. Cotton-top tamarins are members of the Callitrichidae family and most species live in primarily monogamous groups (Rylands, 1996). As in obligatory monogamy, it is necessary for male tamarins to participate in the rearing of offspring, thereby ensuring infant’s survival (Kleiman and Malcolm, 1981). Due to the occurrence of large, twin offspring at birth, multiple caretakers are required to ensure the survival of offspring. Not only does biparental care occur in tamarins, but all older tamarin offspring assist in rearing the newest offspring (Cleveland and Snowdon, 1984) and both male and female subadults delay their own reproductive potential (Ziegler, Savage, Scheffler, and Snowdon, 1987). The ecological conditions under which tamarins live require that the social system consists of more than two individuals and therefore promotes biparental care (Savage, Snowdon, Giraldo, and Soto, 1996). Several studies have shown that experience in caring for younger offspring is critical to parental competence with one’s own infants (Epple, 1978; Tardif, Richter, and Carson, 1984). In captivity, strong pair associations between a male and a female develop rapidly and it appears that males are especially responsive to guarding their mate when the female is pregnant (Porter, 1994). Male tamarins are highly responsive to infant stimuli and are known to begin carrying infants from the moment they are born (Hampton, Hampton, and Landwehr, 1966; Epple, 1975; Cleveland and Snowdon, 1984; Price, 1987). The adult breeding male of a group carries the infants as much or more than the mother in the first month after birth and is involved in weaning of infants by food sharing (Cleveland and Snowdon, 1984; Feistner and Chamove, 1986). Little is known about a male tamarin’s hormonal responses to infants, to their own mate’s pregnancy, or to the influences of the infant’s hormones. To date, only one study has implicated a role for prolactin in male parental care in a socially monogamous primate. Dixson and George (1982) found increased levels of prolactin in common marmoset males when the males had infants 10 to 30 days of age in the cage compared to males which lived with a cycling

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female or with a pregnant female. This effect was also thought to be related to infant contact since males which were carrying infants had higher prolactin levels than males which were not carrying infants prior to blood sampling. In contrast to the findings on prolactin and contact time in male marmosets, several studies in other nonhuman primates have indicated that prolactin elevations in males were reflective of acute ‘‘stress.’’ Studies performed on the rhesus macaques (Quadri, Pierson, and Spies, 1978; Puri, Puri, and Kumar, 1981), mangabeys, patas monkeys (Aidara, Tahiri-Zagret, and Robyn, 1981), and talapoin monkeys (Meller, Keverne, and Herbert, 1980) all suggested that prolactin levels were elevated in response to anesthetics, stress of immobilization, and agents that blocked adrenergic receptors, but in the talapoin, at least, not necessarily elevated by social stress (Eberhart, Keverne, and Meller, 1983). This information leads us to two hypothesis about why prolactin may be elevated in male cotton-top tamarins. One hypothesis would provide that an increase in prolactin following the birth of infants would be due to a prolactin release in response to increased stress following the changes in the social organization of the group brought on by the birth of the infants or the stress of infant caretaking. If prolactin is released in response to stress, then both prolactin and cortisol levels would increase shortly after birth and remain elevated through at least the first few weeks postpartum while the family is adjusting. Alternatively, if elevated prolactin is involved in facilitating male’s responsiveness to infants, then prolactin levels may be elevated prior to the birth of infants in response to cues from the pregnant female. An increase in parenting activities such as carrying should be correlated with increased prolactin levels. The following study was designed to determine if male cotton-top tamarins showed an increase in prolactin levels during the periparturitional period and if so, what social factors influenced prolactin release. A human prolactin assay was adapted for measuring tamarin prolactin from collecting daily first-morning void urine. Both urinary prolactin and cortisol were measured in samples collected from male cotton-top tamarins living under different social conditions and following stress-related manipulations.

MATERIALS AND METHODS Animals and Experimental Procedure A total of 21 male and 3 female cotton-top tamarins were used for the study. Males were housed in one of

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the following conditions: paired with a nonpregnant and nonlactating female, adjacent to a female in a single cage, or part of a family group. Cage sizes were large: cages for pairs and males residing alone, but adjacent to a female, measured 1.5 1 0.85 1 2.3 m, and family cages measured 3.0 1 1.8 1 2.3 m. Tamarins were housed indoors under a 12-hr day/night cycle and colony maintenance procedures were similar to those described in Snowdon, Savage, and McConnell (1985). First-morning void urine was collected from the animals for hormonal determinations. Males and females were studied under the following categories: Fathers. Urine was collected from eight expectant fathers for the 2 weeks prior to birth of infants to their mates and for the 6 weeks following birth. The fathers’ ages ranged from 3 to 14 years at the time of the birth. Four of the males (7.5 to 14 years of age) had experienced multiple births and had several offspring living in the cage. Four males (3 to 4 years of age) were experiencing their first successful birth after being paired with a female. Urine was collected three times per week prior to birth and daily during the 6 weeks postpartum. Additionally, urine was collected for 5 successive days 12 to 14 weeks pre- or postpartum for the four males with large families. Daily behavioral observations of infant care taking were performed during the 6 weeks postpartum. Eldest sons. Three of the four fathers with large families had an eldest son aged 20 months to 3 years. Physiologically, these males were considered to be postpubertal and behaviorally, these males were considered to be subordinate to their father. Urine was collected from the three sons while they were living in the natal family three times per week for the 2 weeks prior to the birth of their younger siblings and for the 6 weeks following the birth. Each eldest son had three to four younger siblings living in the family prior to the birth. Nonfather males: Paired or living adjacent to a female. Daily urine samples were collected for 5 to 10 consecutive days from seven males while they were living with females and from four males which were living adjacent to a female they would be paired with. The ages of these males ranged from 2 to 4 12 years. Acute stress response. Three male – female pairs were used to examine the release of prolactin following an injection with a dopamine antagonist, metaclopramide (Sigma Chemical Co., St. Louis, MO). First-morning void urine was collected from all six tamarins before and after injecting a control substance, saline (0.5 ml), im and a week later before and after the injection of

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metaclopramide (4 mg/0.5 ml sterile water) im per tamarin the following week. For each injection, urine was collected for 2 days. The tamarins were caught in the late afternoon (1600 – 1630) and injected im while the tamarin was still in the catching net. After injection, the tamarins were returned to their cages and lights were turned off so that the tamarins would retire for the night and be less likely to urinate. Urine was collected the following morning and for the next 2 days. All urine samples were collected as first-morning void following the procedures reported in Ziegler, Bridson, Snowdon, and Eman (1987). The collector entered the cage between 8 and 9 AM after turning on the lights to wake the tamarins. A container was held directly beneath the tamarin of interest until he or she urinated. This procedure allowed collection of urine without handling or relocation of the monkey and such samples are collected routinely from most tamarins in the colony. The procedure was not disruptive in large family groups. Collected urine was centrifuged and one aliquot was mixed with 0.52 M glycerol to stabilize the protein molecules during storage (Liversey, Roud, Metcalf, and Donald, 1983). Samples were stored frozen at 0207C until analysis.

Hormonal Assay Procedures All urine samples were assayed for prolactin, cortisol, and creatinine concentrations by the following procedures: Prolactin. Urine samples were concentrated 20fold prior to assay by the use of centrifuge concentrating tubes. These tubes allow the protein component of a sample to be concentrated while the lower molecular weight components filter through the membrane along with the majority of the sample volume. One-milliliter aliquots of urine in duplicate were pipetted into Centricon-10 tubes (10,000 MW cut-off, Amicon, Beverly, MA). Tubes were centrifuged at 5000 g in a fixed-angle rotor at 157C for 100 min. The samples, concentrated to 50 ml, were mixed with 250 ml of assay buffer (0.017 M sodium phosphate, 0.003 M potassium phosphate, 0.5% BSA, 9% sodium chloride, 0.02% sodium azide, pH 7.4) and inverted over 12 1 75 glass test tubes. The liquid was collected into the bottom of the tube by centrifuging at low speed for 2 min. The assay was performed by using a human prolactin 125 I liquid-phase double antibody RIA. Samples were mixed with 100 ml rabbit anti-human prolactin antibody (UCB-Bioproducts, Accurate, Westbury, NY) at 1:4000 dilution and 100 ml iodinated human prolactin at 1:22,000 cpm (Dupont, Wilmington, DE) and incubated

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FIG. 1. Dilution – response curves of cotton-top tamarin pooled urine (1 and 2) and cotton-top tamarin pituitary media in comparison to standard human prolactin (#Ho17/H, UCB Bioproducts), demonstrating parallelism.

at room temperature for 24 hr. The antigen – antibody reaction was precipitated with 500 ml of second antibody, sheep anti-rabbit (UCB-Bioproducts, Accurate), and after a 30-min incubation, 2 ml of assay buffer was added to each tube and then centrifuged at 1,948 g for 20 min at 207C. The supernatant was then aspirated and the bound fraction counted using a TM Analytic gamma counter (Model 1290). Purified human prolactin (UCB-Bioproducts, Accurate) was used as the standard in doses ranging from 0.1 to 33.3 ng/tube. The prolactin assay was validated for tamarin urine by determining parallelism and accuracy. Serially diluted pooled tamarin urine (n Å 4) and (n Å 3) paralleled the standard curve (n Å 6) with no difference in slope (t Å 0.83, df Å 26, P ú 0.05) (Fig. 1). The accuracy of the added tamarin urine pool to each standard curve dose was 114.29%, y Å 0.9031 / 0.646, r Å 0.994, and there was no difference between the means of the observed versus expected, t Å 01.39, P ú 0.05. Mean intraand interassay coefficients of variation for two tamarin pools n Å 10 and n Å 6 were 6.6 and 10.11%, respectively, for intraassay variation and 18.4 and 17.2%, respectively, for interassay variation. Biological validations for tamarin prolactin measurement by the human prolactin assay were performed. Serum and urine collected on the same day from a lactating female and serum from another female were analyzed. The urine value from a lactating female was only 16% of the value found in the serum

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(urine: 2.57 ng/ml versus serum: 15.17 ng/ml). The serum value for a nonlactating female was 5 ng/ml. Excretion of prolactin from cultured pituitary cells also provided evidence of tamarin prolactin crossreactivity with the human prolactin assay. The pituitary from a female cotton-top tamarin, taken at euthanasia by the methods reported in Matteri, Dierschke, Bridson, Rhutasel, and Robinson (1990) and Ziegler, Matteri, and Wegner (1993), was used to asses pituitary prolactin. The anti-human prolactin antibody cross-reacted with the prolactin from cultured pituitary cells and dilutions (n Å 5) were found to be parallel to the standard curve since there were no differences in slopes t Å 00.82, P ú 0.05 (Fig. 1). Biological validation was also provided by injection of metoclopramide. Urinary prolactin levels increased for all six tamarins the morning following metoclopramide injection and mean differences before and after injection were significant, paired t test, t Å 04.39, P õ 0.007 (Fig. 2). Cortisol. Urinary cortisol was measured by a cortisol ELISA as reported in Ziegler, Scheffler, and Snowdon (1995) for female tamarin urine. Intraassay coefficients of variation for a high and low pool for the male tamarin urine assays were 3.2 and 3.4%, respectively, and interassay coefficients of variation for the same pools were 11.0 and 11.7%, respectively. Creatinine. Creatinine concentration was measured for each urine sample as described in Ziegler et al.

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(1995). Creatinine concentration (mg/ml) was divided into the prolactin or cortisol levels to control for fluid variability. Intraassay coefficients of variation for pooled tamarin urine were 1.6 and 0.9% and interassay coefficients of variation were 5.1 and 5.4%.

Behavioral Measures Carrying behavior throughout the day was measured as scan samples (point observations) taken five times per day. During the first 6 weeks following birth at 0800, 1000, 1200, 1400 and 1600 hours, the tamarin carrying the infants was recorded for each tamarin family. In a previous study, these scan samples on infant carrying were found to be as reliable as focal animal sampling (Ziegler, Widowski, Larson, and Snowdon, 1990). Behavioral data were summarized as percentages of observed carrying for each individual tamarin carrier per week.

Analyses Prolactin and cortisol levels per milligram creatinine were averaged for the different categories and means are reported with the standard error of the mean, SEM. Comparisons between categories were made using oneway ANOVA with the post hoc Tukey test or repeated measures ANOVA for within-subjects comparisons and the paired sample t test. Correlations were computed by the Pearson product – moment correlation coefficient.

RESULTS Response to Acute Stress Mean responses for prolactin and cortisol levels to capture and injection of a saline control or to capture and metaclopramide injection are shown in Fig. 2. Prolactin concentration increased after capture and saline injection compared to prolactin levels prior to injection for each tamarin (paired t test, t Å 4.39, P Å 0.007) and also after the injection with metaclopramide (t Å 4.38, P Å 0.007). Cortisol levels increased after the saline control but not significantly (t Å 1.76, P Å 0.14) but did increase significantly following the metaclopramide injection (t Å 2.71, P Å 0.04).

Hormonal Response in Males A comparison of mean prolactin levels in fathers was performed during the 6 weeks following the birth and

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the initial 2 weeks postpartum. Prolactin levels were slightly higher during the first 2 weeks postpartum, 1.31 { 0.35 ng/mg Cr, versus the total 6 weeks, 1.22 { 0.34 ng/mg Cr, but this difference was not significant (t Å 2.24, df Å 5, P Å 0.075). The amount of time an individual male spent carrying his infants also was not significantly different between the first 2 weeks, 42.0% { 9.5, and the total 6 weeks, 40.6% { 8.3 (t Å 01.42, df Å 7, P Å 0.73). The following comparisons of fathers’ prolactin levels were made using mean prolactin levels for the first 2 weeks, when the infants were most dependent. Mean prolactin levels differed between tamarin males in different social conditions (ANOVA, F Å 6.63, P Å 0.007). Fathers had significantly higher prolactin levels than nonfathers (post hoc Tukey test, P Å 0.005) but not significantly higher than sons (P Å 0.27). When fathers were divided into experienced and first-time fathers and compared to nonfathers and sons, differences in mean prolactin levels for the different social conditions were found (Fig. 3; ANOVA, F Å 12.91, P õ 0.0001). Post hoc analyses by the Tukey test indicated that the mean prolactin levels from the experienced fathers were significantly higher than those from the firsttime fathers (P õ 0.005), nonfathers (P õ 0.0001), and eldest sons (P õ 0.01). Cortisol levels also differed significantly among the social conditions (ANOVA, F Å 4.95, P õ 0.01) (Fig. 3). Post hoc analyses by the Tukey test revealed that mean cortisol levels were significantly lower in experienced fathers (P õ 0.03) and eldest sons (P õ 0.04) than in first-time fathers and nonfathers. Mean cortisol levels in experienced fathers and sons were in the same range as basal cortisol levels reported for female tamarins (10.76 { 1.42 mg/mg Cr), (Ziegler et al., 1995). Mean prolactin levels were compared for the four experienced fathers during the 2 weeks prior to birth, following birth, and prolactin levels sampled during 12 to 14 weeks before (n Å 2) or after (n Å 2) parturition, when there were no dependent infants (Fig. 4). Mean prolactin levels were similar among the males during the three time frames although mean prolactin levels were lower during the noncarry time. The prolactin levels of experienced fathers during the noncarry time were significantly higher than mean prolactin levels for nonfather males (independent t test, t Å 4.12, P õ 0.03). Comparisons of mean prolactin levels from tamarin males when they were paired compared with the 2 weeks prior to birth of their first infants and the 2 weeks following birth are shown in Fig. 5. Significant differences were found in prolactin concentrations; mean prolactin levels were significantly lower when males were paired than following the first birth (P Å 0.03) but

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FIG. 2. The mean levels of prolactin and cortisol per milligram of creatinine in the urine of male and female cotton-top tamarins in response to capture, restraint, and control injection or capture, restraint, and metaclopramide injection (dopaminergic receptor blocker). Hormonal levels were averaged from urine samples collected for the 2 days prior to injection (preinjection). Postinjection levels represent the hormonal response from the urine collected the day following injection. All injections occurred in the late afternoon so that the following morning urine sample would provide for the hormonal output. Asterisks indicate significant differences between the measurements before and after.

not for the 2 weeks prior to the first birth (P Å 0.12). Three of the four first-time fathers showed a step-wise increase in their prolactin levels from preconception to the 2 weeks prior to their first birth to the 2 weeks postpartum.

births with the male carrying infants with his mate (r Å 0.85, P Å 0.04), and the age of the male (r Å 0.93, P Å 0.008).

DISCUSSION Between Father Variation The amount of time a father spent carrying infants depended on whether there were helpers in addition to the mother available for carrying. For males with more than one helper, they spend 10 to 30% of observed time carrying infants while the males with no helpers except the mother spend 60 to 80% of their time carrying infants. There was considerable variability in mean prolactin levels among fathers postpartum. To account for this variation among the fathers, prolactin levels were compared with several variables. Correlation between mean prolactin levels and the number of helpers present in the family, representing group size, was nonsignificant (y Å 1.37x / 1.4, r Å 0.46, P Å 0.50). The percentage of time that a male was observed carrying infants during the first 2 weeks or for the entire 6 weeks were not significantly correlated with prolactin levels (2 weeks: r Å 00.14, P Å 0.79; 6 weeks: r Å 00.16, P Å 0.76). However, mean prolactin levels during the first 2 weeks postpartum were significantly correlated with the number of previous births the male had been exposed to regardless of whether the infants survived (r Å 0.92, P Å 0.01), the number of previous successful

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The data reported in this study provide evidence that prolactin levels are associated with male parental care in the cotton-top tamarin. All fathers had higher prolactin levels than nonfather tamarins. Prolactin levels were elevated in fathers postpartum whether they were experienced fathers or first-time fathers. Cortisol levels were elevated only in the first-time fathers. Prolactin levels were also elevated before the birth in all fathers. Neither one of our original hypothesis fit the data presented. Birth of infants was not accompanied with increased cortisol levels as expected if the elevated prolactin was due to a stress response, except in first-time fathers. Additionally, there was no correlation of the percentage of infant carrying with levels of prolactin. An alternative hypothesis would be that there are two different factors influencing prolactin release in male cotton-top tamarins associated with parental care. First, chemical or social cues from the pregnant female may stimulate prolactin release in socially bonded male tamarins prior to the birth of infants. Gestation length in cotton-top tamarins is 6 months (Ziegler et al., 1987a). All of the first-time fathers in the present study demonstrated elevated prolactin levels during at least the last

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FIG. 4. Mean prolactin levels per milligram of creatinine in four experienced father tamarins during the 2 weeks prior to birth, the 2 weeks following birth, and for 5 successive days during 12 to 14 weeks before (two males) or following (two males) birth when infants were not being carried in the cage. Mean prolactin levels per milligram of creatinine for nonfather males are also shown and the noncarry prolactin levels for experienced males (a) was significantly different from the prolactin levels in nonfathers (b) by t test.

known to form pair-bonds with their females (French, 1982; Porter, 1994). Copulations occur throughout ovarian cycling and pregnancy in callitrichid monkeys

FIG. 3. Mean urinary prolactin levels per milligram of creatinine in the upper graph and mean urinary cortisol levels per milligram of creatinine in the lower graph for male cotton-top tamarins under different social conditions. Seven to ten consecutive urine samples were collected on paired males and adjacent males. Asterisks indicate a significant difference between experienced fathers and eldest sons and first-time fathers and nonfathers.

2 weeks of pregnancy. Second, cues from the infants may stimulate prolactin release and long-term exposure to infants may provide for continued prolactin elevation. Experienced males in the present study had elevated prolactin levels even when the infants were independent and no longer being carried. From studies performed in rodents, there appears to be a male hormonal response to copulation, cohabitation with females, and female odors (for review, see Brown, 1993; Brown, Murdoch, Murphy, and Moger, 1995; Winslow, Hastings, Carter, Harbaugh, and Insel, 1993). Responsiveness of the male to the female may facilitate pair-bonding and reduce aggressive tendencies in monogamous males. Captive tamarin males are

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FIG. 5. Mean levels of urinary prolactin (nanogram/milligram creatinine) in four first-time fathers at preconception, 2 weeks prior to birth of the first infants, and 2 weeks postpartum. Prolactin levels were significantly higher in the 2 weeks following birth (a) than during pairing with a female preconception (b).

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(Kleiman, 1977; Epple and Katz, 1982; French, 1982), which may stimulate neuroendocrine changes such as those occurring in rodents (Dluzen and Ramirez, 1989). Paired tamarin males increase their aggression toward intruders over time as they continue to cohabitate with the same female (Porter, 1994). The timing of the elevation of prolactin levels prior to the birth of first-time infants is unknown, except that elevated prolactin levels are found in the last 2 weeks of the female’s pregnancy. Male tamarins may be receiving chemical cues from the female’s pregnancy that stimulate prolactin release. It is hypothesized in rodents that urinary odors during the last half of pregnancy suppress infanticidal behavior in male rodents and the odors may act as pheromones which facilitate male paternal behavior (Jemiolo et al., 1987; Brown, 1985). The elevated prolactin levels found in the common marmoset (Dixson and George, 1982) were primarily associated with infant contact since males carrying infants at the time of sampling had significantly higher levels of prolactin than fathers not carrying at the time of blood sampling. In the present study, it appeared that continued association with infants was associated with elevated prolactin levels in the male tamarins. Male tamarins living with infants and juveniles in their family cages, regardless of whether they were fathers or eldest sons, had higher levels of prolactin than males living with nonpregnant females. Prolactin levels in older siblings carrying infants in large families may only show a decline in prolactin levels when they are removed from their families and living without infant exposure. In contrast to the data on the common marmoset, we found no correlation between the amount of time a father tamarin spent carrying infants and the levels of prolactin in the male. Unfortunately, a direct correlation between contact time and prolactin levels may not be testable given our first-morning daily urine sampling procedure that measures accumulated prolactin rather than measuring the immediate changes that occur in the blood. Hormonal and neurochemical factors play an important role in regulating maternal behavior for many mammalian species. Hormonal and neurochemical signals are involved in regulating maternal behavior in rats (Bridges, 1990). In the closely related red-bellied tamarin (Saguinus labiatus) prepartum estradiol levels have been linked to the amounts of maternal behavior (Pryce, Abbott, Hodges, and Martin, 1988; Pryce, Doebli, and Martin, 1993). Prolactin has been linked to maternal behavior in rats (Bridges, DiBiase, Loundes, and Doherty, 1985) and rabbits (Rosenblatt, 1994). In these species and others, mothering behavior depends

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on the sensitizing action of hormones secreted periparturitionally and continued by the suckling stimulus (Fleming, Corter, and Steiner, 1995). Human prepartum hormones may not activate maternal behavior directly but may exert many diffuse effects which together increase the probability of positive maternal behavior (Fleming, 1990). Pseudopregnancy in women has been associated with unusually elevated prolactin levels along with elevated LH and an increase in maternal responsiveness. Additionally, prolactin levels seem to rise with each successive birth in normal women (Warren and Shortle, 1990). Most likely, male parental behavior is initiated and continued by different factors than is female maternal behavior. Certainly learning and experience would play a key role in a primate male’s interest towards infants, although there may be a facilitation of paternal behavior with an increase in certain hormones in males. In male tamarins, a female’s prepartum hormones may provide chemical cues which stimulates a male’s physiological response. Also, the level of experience and age could influence prolactin levels since experience with births was highly correlated with prolactin. Although tamarin males with higher prolactin levels were not observed carrying infants more, it may be that prolactin acts as a mechanism to provide an increased tolerance of the presence of infants. Infant care appears to be a learned response in many primates. Experience with infants may be important for ensuring the survival of one’s own infants in species with flexible behavioral responses. Prior experience with infant care of siblings in both marmosets and tamarins is known to affect the outcome of primiparous births for both mothers and fathers (Epple, 1978). More females and males successfully raise their first set of infants when they have had experience carrying their siblings. Prolactin levels were elevated in eldest cottontop tamarin sons compared with paired and adjacent males during the postpartum period and may be a reflection of the experience of sibling caretaking. All of the paired and adjacent males had prior sibling experience. Since males with early family experience, but removed from the family and paired, showed low prolactin levels, this may indicate that prolactin levels decline after leaving the group. Sustained prolactin elevation may be a consequence of infant care but may need constant infant exposure to remain elevated. Group size or the number of helpers did not relate to prolactin levels, per se, but exposure to new infants at least once a year may provide a renewed stimulus to sustain prolactin elevations. Whether prolactin has a role in mediating the behavioral responses to infants in males or is more

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a consequence of the learning process is unknown at present. Prolactin levels also responded to acute stress. As was found for talapoin males (Eberhart et al., 1983), cortisol and prolactin responded simultaneously to the acute stress of handling, but not under social conditions. Cortisol and prolactin levels were elevated in both male and female cotton-top tamarins either due to handling and saline injections or after injection with a dopaminergic receptor blocker known to release prolactin by inhibiting dopamine release. The acute stress effect of capture and injection was short term. Elevated levels of both cortisol and prolactin were found in the urine only on the morning following the capture, while elevated prolactin levels were found in fathers continued for the entire 6 weeks postpartum. Prolactin levels were independent of cortisol elevations in the experienced fathers postpartum. Cortisol levels were at basal levels in experienced fathers (Ziegler et al., 1995). Firsttime fathers had higher levels of cortisol than experienced fathers but lower levels of prolactin. Therefore, simultaneous but short-term elevated prolactin and cortisol appears to indicate a stress response in cotton-top tamarins and may be mediated by different neuroendocrine pathways than the sustained elevations of prolactin, but not of cortisol, associated with parental care. In this paper, we described the use of urine sampling and measuring urinary prolactin by RIA. Our technique allowed us to examine once a day hormonal levels of prolactin without any disruption to the tamarin families. We could collect on several individuals in the same family without any disturbance to their normal routine. Since prolactin levels were increased upon handling cotton-top tamarins, it is important to prevent any stress-induced elevation of prolactin when studying parental care. The human assay system detected pituitary, blood, and urinary prolactin and metoclopramide injection indicated a prolactin response providing evidence that prolactin is being measured. Also, prolactin levels are known to vary considerably throughout the day in humans due to such factors as sleep, exercise, stress, and sexual interactions and therefore, accumulative measurements of prolactin through urine may provide less variable data than serum measurements (Keely and Faiman, 1994). Using first-morning urine collection allowed us to provide a more accumulative assessment of prolactin levels than using a single daily blood sample. Alternatively, human and rat prolactin levels in the blood are elevated during sleep due to the circadian release of prolactin (Stern and Reichlin, 1990) and it may be that overnight urine collected as first-morning void may only repre-

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sent night-time levels of prolactin, a time when the amount of infant contact is unknown. Tamarin fathers exhibited a high correlation between prolactin levels and age. Since the ages of fathers, 3 to 14 years, overlapped the ages of the paired males, 2 to 4.5 years, with the levels of prolactin higher in the fathers, the age effect is confounded with experience. In our colony, most males are paired for breeding between 2 and 3 years of age and therefore will have an increasing experience with births as they age. In another nonhuman primate, the rhesus monkey, old males showed no significant differences in prolactin levels compared to young males (Chambers and Phoenix, 1992), but these males were singularly housed and the authors did not mention the infant experience of those males. However, in men, there is conflicting evidence. One study indicated that there was no significant change in the levels of prolactin with age (Davidson, Chen, Crapo, Gray, Greenleaf, and Catania, 1983) while another study on old male rats and men indicated that prolactin level did increase with age (Meites, Steger, and Huang, 1980). Hyperprolactinemia is usually associated with lowered fertility (Donovan, 1985). Our study cannot discount age effects on prolactin levels although no decrease in conception rates were seen in our oldest males. Thus, although age is a possible influence on prolactin levels, cumulative experience may be an important influence. Male parental investment varies as much in nonhuman primates as it does in humans. Some species of nonhuman primates, such as male chimpanzees (Redican and Taub, 1981), show little interest in infants. An example of a species with highly involved males are the Barbary macaques. Adult males assist in several aspects of learning in infants (Burton, 1972). Many monogamous species have extensive male parental involvement. Male siamangs (Chivers, 1972), marmosets (Locke-Haydon and Chalmers, 1983), tamarins (Cleveland and Snowdon, 1984), owl monkeys (Dixson, 1983), Goeldi’s monkeys (Heltne, Turner and Wolhandler, 1973), and titi monkeys (Mendoza and Mason, 1986) all show a high level of parental involvement and generally live in monogamous groups. The level of male parental involvement, however, varies even between these species as to when the male begins to care for the infant (Ingram, 1977; Cleveland and Snowdon, 1984; Mendoza and Mason, 1986). Tamarin males begin participation with neonates from the moment they are born (Hampton, Hampton, and Landwehr, 1966; Epple, 1975; Cleveland and Snowdon, 1984; Price, 1987). The adult breeding male of a group carries the infants as much or more than the mother in the first month and

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is involved in weaning of infants by food sharing (Cleveland and Snowdon, 1984; Feistner and Chamove, 1986). We might expect covariation of prolactin with parental investment if prolactin is a parental response. Whether sustained elevations in prolactin occur in response to parental care in other primates or whether it occurs only in those exhibiting monogamous breeding systems is unknown. In rodents, monogamous and polygynous species show species differences in sexual dimorphic areas of the brain along with differences in male parental care. Neuroendocrine organization differences in sexually dimorphic areas of the brain have been found in the hypothalamus between monogamous and polygynous species of voles (Sapiro, Leonard, Sessions, Dewsbury, and Insel, 1991) and in the amount of oxytocin receptor sites in the brains of monogamous and polygynous mice (Insel, Gelhard, and Shapiro, 1991). Mammalian species exhibiting monogamous social systems may be genetically predisposed to exhibiting neuroendocrine changes associated with male parental care. What consequences a physiological response to parental care in primates has on the quality of care that infants received is also unknown.

ACKNOWLEDGMENTS We thank Anne A. Carlson and Cecily Mui for their help in sample analysis and Guenther Scheffler for the cortisol ELISA set up. We also thank the members of the Psychology Department Callitrichid Research Laboratory for collecting samples, catching tamarins, and helping organize the study, especially Anita Ginther. Earlier drafts of the manuscript were reviewed by Anne Carlson and Fred Bercovitch. We also thank anonymous reviewers for their helpful comments. This research was supported by Grants MH35215 to C.T.S. and T.E.Z. and RR00167 to the Wisconsin Regional Primate Research Center (WRPRC). This is Publication No. 36-025 of the WRPRC.

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